Effects of 5G-modulated 3.5 GHz radiofrequency field exposures on HSF1, RAS, ERK, and PML activation in live fibroblasts and keratinocytes cells

The potential health risks of exposure to radiofrequency electromagnetic fields from mobile communications technologies have raised societal concerns. Guidelines have been set to protect the population (e.g. non-specific heating above 1 °C under exposure to radiofrequency fields), but questions remain regarding the potential biological effects of non-thermal exposures. With the advent of the fifth generation (5G) of mobile communication, assessing whether exposure to this new signal induces a cellular stress response is one of the mandatory steps on the roadmap for a safe deployment and health risk evaluation. Using the BRET (Bioluminescence Resonance Energy-Transfer) technique, we assessed whether continuous or intermittent (5 min ON/ 10 min OFF) exposure of live human keratinocytes and fibroblasts cells to 5G 3.5 GHz signals at specific absorption rate (SAR) up to 4 W/kg for 24 h impact basal or chemically-induced activity of Heat Shock Factor (HSF), RAt Sarcoma virus (RAS) and Extracellular signal-Regulated Kinases (ERK) kinases, and Promyelocytic Leukemia Protein (PML), that are all molecular pathways involved in environmental cell-stress responses. The main results are (i), a decrease of the HSF1 basal BRET signal when fibroblasts cells were exposed at the lower SARs tested (0.25 and 1 W/kg), but not at the highest one (4 W/kg), and (ii) a slight decrease of As2O3 maximal efficacy to trigger PML SUMOylation when fibroblasts cells, but not keratinocytes, were continuously exposed to the 5G RF-EMF signal. Nevertheless, given the inconsistency of these effects in terms of impacted cell type, effective SAR, exposure mode, and molecular cell stress response, we concluded that our study show no conclusive evidence that molecular effects can arise when skin cells are exposed to the 5G RF-EMF alone or with a chemical stressor.

BRET measurements. Cells were sham-exposed for 24 h (i.e. the cells were cultivated in absence of RF-EMF) or exposed to the indicated RF-EMF exposure conditions for 24 h. During the last 18 h, 4 h or 15 min of either sham or RF-exposure, the cells were incubated with the indicated concentration of MG132, As 2 O 3 or PMA respectively (Fig. 1A). Only one chemical was tested in each 96-well plate; however various concentrations of the same chemical compound were tested simultaneously in a single plate by injecting 10X stock-solutions pre-heated at 37 °C using a multi channel pipette. When indicated, mock-treatment were performed by injecting only the solvating agent into the cell culture medium (DMSO for MG132 and PMA or water for As 2 O 3 ).To perform the chemical treatment, the plates were removed from the reverberating chamber (see below "Cells exposure, exposure set-up, and dosimetry" section of the Material and methods) and immediately docked on a Thermostat Plus microplate Peltier heater (Eppendorf, Hamburg, Germany) to keep the cells at 37 °C. The shamexposed plates were treated the same way. Removing the plate from the reverberating chamber, dispatching the chemicals at the various concentrations in the cell culture media using a multi-channel pipette and replacing the cells into the reverberating chamber took less than 1 min. After completion of the remaining RF exposure, 5 µM Coelenterazine H was added to the cell culture medium and the BRET signal was immediately acquired using a TriStar2 LB942 microplate reader (Berthold Technologies, Bad Wildbad, Germany) pre-heated at 37 °C and equipped with emission filters centered at 515 ± 20 nm for mNeonG (I acceptor ) and 460 ± 20 nm for nLuc (I donor ). Alternatively, for real-time BRET measurement under RF-EMF exposure, 5 µM Coelenterazine H was added to the cell culture medium 10 min before the end of the RF-EMF exposure and the full BRET spectra were recorded remotely for the last 5 min of RF-EMF exposure and the 5 next min in absence of RF-EMF. Full BRET spectra were acquired using an optical fiber linked to an IsoPlane SCT-320 Imaging Spectrograph equipped with a BLAZE:400B back illuminated CCD camera system camera for recording the full visible spectrum (Teledyne France-Princeton Instruments, Lisses, France).
The BRET signal was determined by calculating the ratio of the emission intensity measured in the acceptor window (I mNeonG ) over the emission intensity measured in the donor window (I nLuc ), according to Eq. (1) Due to the overlapping emission spectra of nLuc and mNeonG, a fraction of the light detected in the mNeonG filter originates from the luciferase emission, resulting in a contaminating signal 33 . In that configuration, the net BRET was therefore defined as the BRET ratio of cells co-expressing nLuc and mNeonG constructs minus the BRET ratio of cells expressing only the nLuc construct in the same experiment.
BRET data processing and statistical analysis. The GraphPad Prism v8.00 for Mac software (Graph-Pad Software, La Jolla, CA, USA) was used for plotting dose-response curves and performing statistical analyses. where X is the logarithm of agonist concentration and Y the response; Bottom is the Y value at the bottom plateau and is taken as the measure of basal level of activation of the various probes; Top is the Y value at the top plateau and Top-Bottom is taken as the measure of the maximal efficacy of a given chemical treatment on each BRET probe; Log EC50 is the X value when the response is halfway between Bottom and Top (Supp. Fig. 1). The EC50 value represents therefore a measure of the apparent potency of the various chemical compounds to trigger the activation of their cognate BRET probe (Supp. Fig. 1). Potencies of chemicals to activate or inhibit the different probes are expressed as pEC50 ± S.E.M (standard error of the mean), that is equal to -log EC50. The one sample Wilcoxon signed-rank test was used to assess the statistical significance against the null hypothesis of the differences calculated in each independent experiment between sham (no RF EMF condition) and RF-EMF exposure conditions for basal BRET, chemicals' potencies and efficacies (hereafter denominated ΔBasal BRET, ΔpEC50, and ΔMax efficacy, respectively). The total number of independent experiment (n) performed for each experimental condition is indicated. One sham exposure was performed for each RF-EMF exposure condition. P-values less than 0.05 were considered as statistically significant.
Cells exposure, exposure set-up, and dosimetry. Cells were exposed for 24 h in 96-well tissue culture plates (TCP) at SAR levels of 0.25, 1, and 4 W/kg. Intermittent exposure (5 min ON/10 min OFF) at the same average SAR level as with the continuous wave (CW) mode was implemented to mimic actual real-life exposure and help detect potential nonthermal bioeffects. RF EMF sham exposures were also performed under identical experimental conditions but with the generator turned off, i.e. at SAR equal to 0 W/kg. A novel exposure system, recently designed and characterized, was used for the first time for cells exposures to 5G-modulated 3.5 GHz signals 34 . The system was based on a cell culture incubator that allowed maintaining the desired biological conditions of 37 °C and 5% CO 2. Comprehensive characterization of the system, through experimental measurements and numerical simulations has been described in detail elsewhere 34 . Briefly, a 150-l incubator (BINDER Gmbh, Tullingen, Germany), made of stainless-steel walls, was used as a reverberation chamber, i.e. a metallic large, closed cavity, with a high Q-factor, where a statistically homogeneous, randomly polarized, and isotropic field distribution was achieved via mechanical stirring of the field components 35 . Electromagnetic signal at 3.5 GHz was delivered to the biological samples through a printed patch antenna. A plastic holder with five levels was RF-EMF (or sham RF exposure) is applied for 24 h whatever the drug considered, that is injected for the indicated period. (B,C) Temperature variation in plates exposed to a continuous (B) or intermittent (C) 5G-modulated 3.5 GHz RF-EMF signal at 4W/kg. The temperature in the incubator was set to ensure cellular exposure at 37 °C and to compensate for the RF EMF-induced temperature increase at the onset of the exposure period. Drug injection induces a less than 0.5 °C transient drop in the cell culture temperature (B). www.nature.com/scientificreports/ used to accommodate and simultaneously expose ten TCPs of 6-or 96-wells i.e. two per holder level. Each well of the 6-and 96-well TCPs was filled with 2 ml and 200 µl of cell culture medium, respectively. To ensure experimental reproducibility during exposure, the incubator was loaded with the same configuration used for the electromagnetic characterization, i.e. four and six 6 -and 96-well TCPs, respectively, due to the high SAR dependence on the chamber load. The signal generation unit, composed of a RF signal generator (SMBV100A, Rohde & Schwarz, Munich, Germany), a 45-dB gain amplifier (Mini-circuits, ZHL-16W-43 + , NY, USA), a power circulator (Pasternack, PE83CR1005, CA, USA), and a bidirectional coupler (Mini-circuits, ZGBDC30-372HP + , NY, USA), was located outside the incubator. In addition, to ensure the continuous monitoring of the desired input power into the chamber, incident and reflected powers were monitored with a power meter (Agilent N1912A, USA) connected to the bidirectional coupler.
Local SAR was experimentally retrieved through temperature measurements of the RF-EMF induced heating recorded with a fiber-optic probe (Luxtron One, Lumasense Technologies, CA, USA). Measured SAR in the 96-well TCP was around 1 W/kg per watt antenna input power. To validate our systems, numerical simulations were performed using the finite difference time domain (FDTD)-based electromagnetic methodology 36 . The results of simulations were averaged over 50 positions of the stirrer, corresponding to a complete rotation. Although numerical simulations might not guarantee the absence of hotspots at specific locations of the exposed wells, the continuous stirring of the field components via the mechanical rotation of the metallic stirrer ensured the achievement of a good SAR homogeneity with variation within 30%. Overall, we showed that experimental and numerical SARs were in good agreement with differences < 30% considering the standard deviation that is compliant with ICNIRP guidelines 3 . According to measured and simulated values normalized to 1 W, incident power during biological exposure was adjusted to obtain required exposure levels of 0.25, 1, and 4 W/kg in a 96-well tissue culture plate. Measurements of the induced temperature elevation of the exposed medium were also performed using the Luxtron probe (Lumasense) under the specific cellular exposure condition of the study, showing a temperature increase of 1.7 °C at 4 W/kg, 0.7 °C at 1 W/kg and a negligeable temperature increase below 0.1 °C at 0.25W/kg using a continuous RF exposure, and a temperature increase of 0.8 °C at 4 W/kg, 0.3 °C at 1 W/kg and less than 0.1 °C at 0.25 W/kg using an intermittent (5 min ON, 10 min OFF) RF exposure. The temperature of the incubator was decreased accordingly to maintain the biological samples at 37 °C. Temperature stability of cell cultures at the bottom of the culture wells during the whole RF sessions at the various SAR levels was carefully assessed in a set of separate plates (See Fig. 1B,C for typical temperature traces obtained at 4 W/kg under continuous and intermittent exposure conditions, respectively). As expected, the temperature of the cell culture exposed to the intermittent signal is slightly waving (Fig. 1C). Of note, injection of the chemical triggered a transient drop in cell culture temperature by less than 0.5 °C as exemplified in Fig. 1B.

Results
The hypothesis that 5G-modulated 3.5 GHz RF-EMF can impact HSF1, RAS, ERK, or PML basal or chemicallyinduced activity was tested using BRET-based assays previously described by our team 13,14,26 . BRET is a cell-based assay for studying protein-protein interactions and protein conformational changes in real-time and live cells. This technique relies on Forster resonance energy transfer from a bioluminescent donor to a fluorescent acceptor, both fused to the proteins of interest. In our experiment design, BRET probes were expressed in live cells. Since the fibroblast and keratinocyte cell lines used in this study are more difficult to transfect than the HEK293T cells used in our former studies, we systematically replaced the rLuc2 protein in our BRET assays with the brighter nanoLuciferase (nLuc) 28 . We also replaced the fluorescent acceptor sYFP2 with mNeonGreen (mNeonG) in all our assays because of the greater overlap between nLuc emission spectra and mNeonG excitation spectra 37 .

Impact of 5G-modulated 3.5 GHz RF-EMF exposure on basal or chemically-induced HSF1 trimerization in XP6BE fibroblasts cells.
To monitor HSF1 activity, we previously designed an intermolecular BRET test allowing the follow-up of HSF1 trimerization 13 , a key event on the roadmap of HSF1 activation in response to stress conditions such as heat-stress, oxidative-stress or proteotoxic-stress 38 . In this assay, N-terminally nLuc-tagged HSF1 is co-expressed with mNeonG-tagged HSF1 into a given cell line. Given that HSF1 trimerizes upon activation, the resulting BRET signal increases following HSF1 activation since trimerization brings donor and acceptor groups in close proximity 13 (Fig. 2A).
To assess the functionality of this assay in skin fibroblasts, we co-expressed nLuc-HSF1 with mNeonG-HSF1 in the XP6BE fibroblast cell line 39 , and challenged the transfected cells with increasing concentrations of the proteasome inhibitor MG132 to trigger a proteotoxic stress 40 . As expected, MG132 induced a concentrationdependent increase in the basal BRET signal with an EC 50 in the hundred nanomolar range (Fig. 2B), showing the assay's effectiveness.
We then assessed whether continuous or intermittent (5 min ON/10 min OFF) cell exposure to 5G-modulated 3.5 GHz RF-EMF signal at 0.25, 1, and 4 W/kg under isothermal conditions for 24 h impacted the nLuc-HSF1/ mNeonG-HSF1 basal BRET signal, or either the MG132 potency or efficacy to activate HSF1 in the transfected XP6BE fibroblast cell line. Interestingly, we measured few but significant and reproducible decrease of the HSF1 basal BRET when the XP6BE fibroblast cell line was continuously exposed at 0.25 W/kg for 24 h or intermittently exposed at 0.25 and 1 W/kg for 24 h (Fig. 2C). While this BRET signal decrease represents only ~ 6 to 10% of the basal BRET measured, it proportionally corresponds to ~ 37 to 61% (in absolute terms) of the effect triggered by MG132 (Tables 1 and 2). No effect was measured at 4 W/kg whether the signal was emitted continuously or intermittently. Also, exposure to the RF-EMF signal changed neither the MG132 potency nor its efficacy to activate HSF1, whatever the SAR and the continuous or intermittent emission mode of the signal (Fig. 2D  The pEC50 of MG132 was 6.83 ± 0.25 while the maximal efficacy of MG132 was 0.106 ± 0.018. (C-E) XP6BE skin fibroblasts transfected with the nLuc-HSF1/mNeonG-HSF1 BRET probe were sham-exposed or exposed to 5G-modulated 3.5 GHz at 0.25, 1 or 4 W/kg for 24 h either continuously or intermittently (5 min ON/10 min OFF). Cells were activated using increasing concentrations of MG132 under sham or RF-EMF exposure for the last 18 h of the RF-EMF exposure period before BRET measurement were performed. The results in panels C-E represent the Box and whisker plots of the basal BRET variation (C), the MG132-potency variation (D) and the MG132-maximal efficacy variation (E) between the 5G RF-EMF exposed-(Expo) and sham-conditions in both continuous and intermittent (on/off) exposure mode. Statistical significance of the derivation from the null hypothesis (no difference between sham and RF-EMF exposure) was assessed using the one-Sample Wilcoxon Signed Rank Test. n = 6-12 depending on the experimental condition. n.s. not significant; *p < 0.05; **p < 0.01.

Impact of 5G-modulated 3.5 GHz RF-EMF exposure on basal or chemically-induced RAS and ERK activities in XP6BE fibroblasts cells.
To assess the potential impact of 5G-modulated 3.5 GHz RF-EMF exposures on basal-or chemically-induced RAS activity, XP6BE fibroblast cells were transfected with a BRET probe consisting in sandwiching the H-Ras and the Ras-Binding Domain of Raf (Raf RBD) in-between nLuc and mNeonGreen. This molecular BRET probe is derived from the Fluorescence Resonance Energy Transfer (FRET)-probe described by the Matsuda laboratory 27,41 , and relies on the rapprochement of nLuc and mNeonG following the binding of GTP-Ras to Raf RBD (Fig. 3A). Similarly, the potential impact of 5G-modulated 3.5 GHz RF-EMF signal exposure on basal-or chemicallyinduced ERK activity was assessed in XP6BE fibroblast cells transfected with a BRET probe comprising a ERK sensor domain and a ERK ligand domain connected by a flexible linker, but sandwiched by a mNeonG and nLuc instead of two fluorescent energy acceptor and donor as initially described 27 (Fig. 4A). Once activated, endogenous ERK proteins phosphorylate the sensor part of this BRET probe. This triggers the sensor domain interaction with the ligand domain, thereby inducing the biosensor closure onto itself. Such conformational change brings nLuc in close proximity to mNeonGreen, thereby increasing the BRET efficiency.

Impact of 5G-modulated 3.5 GHz RF-EMF exposure on basal or chemically-induced PML SUMOylation in XP6BE fibroblasts cells.
Finally, knowing that post-translational covalent addition of SUMO to PML, a process known as SUMOylation, is a key-event leading to PML activation and the formation of PML NBs, we used an intermolecular BRET assay 26 to assess whether continuous or intermittent 5G-modulated 3.5 GHz RF-EMF signal exposure may affect PML activity. In this assay, we measured the BRET signal between a SUMO1 protein N-terminally tagged with mNeonGreen and a PMLIII protein C-terminally tagged to nLuc (Fig. 5A). XP6BE fibroblasts transiently transfected with PMLIII-nLuc/mNeonG-SUMO1 expression vectors were therefore challenged with arsenic trioxide (As 2 O 3 ) , a well-known oxidative-stress inducer in cells 43 that efficiently triggers PML SUMOylation 26,44 . As expected, As 2 O 3 dose-dependently increased the BRET signal between PMLIII-nLuc and mNeonG-SUMO1 in XP6BE fibroblasts cells with an EC 50 in the tens of nanomo- pRaichuEV-Ras BRET probe were sham-exposed or exposed to 5G-modulated 3.5 GHz at 0.25, 1 or 4 W/kg for 24 h, either continuously or intermittently (5 min ON/10 min OFF). Cells were activated using increasing concentrations of PMA under sham or RF-EMF exposure for the last 15 m before BRET measurement were performed. The results in panels C-E represent the Box and whisker plots of the basal BRET variation (C), the PMA-potency variation (D) and the PMA-maximal efficacy variation (E) between the 5G RF-EMF exposed-(Expo) and sham-conditions in both continuous or intermittent exposure mode. Statistical significance of the derivation from the null hypothesis (no difference between sham and RF-EMF exposure) was assessed using the one-Sample Wilcoxon Signed Rank Test. n = 6-12 depending on the experimental condition. n.s. not significant; *p < 0.05; **p < 0.01.  (Fig. 5B), indicating an efficient PML SUMOylation in these cells. Furthermore, neither basal PML SUMOylation nor As 2 O 3 potency or maximal efficacy to trigger PML SUMOylation was affected following intermittent exposure of transiently transfected XP6BE fibroblasts to 5G-modulated 3.5 GHz RF-EMF signal for 24 h, whatever the SAR tested ( Fig. 5C-E). We, however, measured a slight but reproductive decrease in the basal PML SUMOylation when XP6BE fibroblasts were continuously exposed for 24 h at 4 W/kg (Fig. 5C). This variation stood for less than 3.5% of the PML basal BRET signal (Table 1) and for 14.9% of the As 2 O 3 maximal efficacy (in absolute terms) ( Table 2). Also, continuous exposure for 24 h did not change As 2 O 3 potency to trigger PML SUMOylation (Fig. 5D, Table 3), but led to a slight decrease in As 2 O 3 maximal efficacy when compared to the condition (Fig. 5E, Table 4).

Scientific Reports
Checking the absence of BRET signals variation following the interruption of exposure to RF-EMF. We next assessed wether we had not missed a potential effect of RF exposure that might have disappeared during the short time laps between the end of the exposure and the completion of the BRET reading (less than 5 min). To verify this hypothesis, coelenterazine H was added to the cell culture wells to start the bioluminescent reaction 10 min before the end of cells exposure to 5G-modulated 3.5 GHz signal emitted continuously at 4 W/kg. Then, using an optical fiber, we remotely measured the BRET ratio in real-time during the 5 last min of the 24 h RF-EMF exposure period and kept measuring during the 5 next min without RF exposure. As indicated in Fig. 6, whatever BRET probe considered, and in the presence or absence of chemical activation, we did not detect any variation of the BRET signal after the end of RF exposure, thereby validating the results previously obtained. pEKAREV BRET probe were sham-exposed or exposed to 5G-modulated 3.5 GHz at 0.25, 1 or 4 W/kg for 24 h, either continuously or intermittently (5 min ON/10 min OFF). Cells were activated using increasing concentrations of PMA under sham or RF-EMF exposure for the last 15 m before BRET measurement were performed. The results in panels C-E represent the Box and whisker plots of the basal BRET variation (C), the PMA-potency variation (D) and the PMA-maximal efficacy variation (E) between the 5G RF-EMF exposed-(Expo) and sham-conditions in both continuous or intermittent exposure mode. Statistical significance of the derivation from the null hypothesis (no difference between sham and RF-EMF exposure) was assessed using the one-Sample Wilcoxon Signed Rank Test. n = 6-12 depending on the experimental condition. n.s. not significant; *p < 0.05; **p < 0.01.

Impact of 5G-modulated 3.5 GHz RF-EMF exposure on basal or chemically-induced HSF1, RAS/ERK and PML activities in HaCAT keratinocyte cells.
In a further effort to study the potential effect of 5G signal on skin cells and given that the upper layer of the skin (epidermis) is primarily composed of keratinocytes, we performed a new set of experiments using the HaCaT keratinocyte cell line to assess whether a 24 h continuous exposure to 5G-modulated 3.5 GHz RF-EMF may impact HSF1, RAS, ERK, and PML activities.
HaCaT cells transiently transfected with the BRET probes probing HSF1, RAS, ERK, and PMLIII were respectively challenged with increasing concentration of MG132 (for HSF1), PMA (for RAS and ERK), and As 2 O 3 (for PML) following sham-or continuous-exposure to 5G-modulated 3.5 GHz RF-EMF for 24 h at three different SAR of 0.25, 1, and 4 W/kg. Dose-response curves were generated for each experimental condition (See Supp. Fig. 2 for the curves derived from the sham-exposed condition). Basal BRET of each probe and the potency and maximal efficacy of each chemical agent were therefore calculated, and the variation of each parameter between sham-exposed and RF-EMF exposed conditions was reported in Fig. 7. We found no difference between sham and exposed conditions, whatever the molecular target, the SAR, or the metric considered. The only exception was a slight increase in the maximal PMA efficacy to activate ERK when HaCaT cells were exposed at 1 W/kg. fibroblasts co-transfected with the mammalian expression vector encoding PML-nLuc and mNeonGreen-SUMO1 were sham-exposed or exposed to 5G modulated 3.5 GHz at 0.25, 1 or 4 W/ kg for 24 h, either continuously or intermittently (5 min ON/10 min OFF). Cells were activated using increasing concentrations of As 2 O 3 under sham or RF-EMF exposure for the last 4 h before BRET measurement were performed. The results in panels C-E represent the Box and whisker plots of the basal BRET variation (C), the As 2 O 3 -potency variation (D) and the As 2 O 3 -maximal efficacy variation (E) between the 5G RF-EMF exposed-(Expo) and sham-conditions in both continuous or intermittent exposure mode. Statistical significance of the derivation from the null hypothesis (no difference between sham and RF-EMF exposure) was assessed using the one-Sample Wilcoxon Signed Rank Test. n = 6-12 depending on the experimental condition. n.s. not significant; *p < 0.05; **p < 0.01.

Discussion
In this study, we investigated whether continuous or intermittent (5 min ON / 10 min OFF) exposure to 5G-modulated 3.5 GHz RF-EMF signal at 0.25, 1, and 4 W/kg for 24 h under isothermal conditions impacted human skin fibroblast and keratinocyte cell stress response at the molecular level. Using our own existing BRET-based molecular probes, we focused on HSF1, RAS/ERK, and PML proteins which are at the crossroad of various molecular pathways, ensuring proper cell response to a wide array of environmental stress, including thermal injury, oxidative stress, and proteotoxic stress 38 . We assessed whether 5G-signal exposure affected either basal or chemically-activated activity for each of these cellular stress molecular markers. We found that the HSF1 basal BRET level was reduced when XP6BE fibroblasts cells were respectively exposed to continuous and intermittent 5G signal for 24 h at 0.25 W/kg and to intermittent 5G signal for 24 h at 1 W/kg. No change in HSF1 basal BRET signal was detected at 1 W/kg when a continuous signal was used or when cells were continuously or intermittently exposed at 4 W/kg. At first glance, the RF-EMF induced decrease of the HSF1 basal BRET signal may appear relatively small since it represents only 6-11% of the HSF1 basal BRET signal in the sham experiment (Fig. 2B). However, since chemical activation with MG132 only increased the basal BRET by 0.1 BRET unit (i.e. a 14% increase of the basal BRET) (Fig. 2B), the effect of 5G RF-EMF exposure appear important in term of absolute values. Interestingly, these results parallel the ones previously obtained by us with HEK293T cells exposed for 24 h to either unmodulated (continuous wave, CW) or GSM-modulated 1.8 GHz signal 13 . In this former work, RF-EMF exposure slightly decreased the HSF1 basal activity at the lower SAR tested (1.5 W/kg) but not at higher SAR (4 W/kg). Whether the effect of RF-EMF on basal HSF1 may impact the physiological HSF1-dependent stress response in RF-EMF exposed organisms remains to be determined. In this previous in-vitro study, while we detected that MG132 maximal efficacy to trigger HSF1 trimerization in HEK293T cells was increased following 24 h exposure to CW or GSM signals at 1.5 W/kg and to CW signal at 6 W/kg 13 , we did not detect any variation of the MG132 potency or efficacy to trigger HSF1 activation following 5G RF-EMF exposure (Fig. 2).
Considering the other BRET assays performed on skin fibroblasts, we only detected a slight leftward shift, less than a quarter of a log, of PMA potency to activate ERK at 0.25 W/kg and a small reduction in As 2 O 3 efficacy to trigger PML SUMOylation, but only when cells were continuously exposed. No other RF-induced effect could be detected on 5G RF-EMF exposed fibroblasts, whatever the molecular probe considered and whatever the SAR Figure 6. Real-time monitoring of the BRET variation after RF-EMF shutdown. HEK293T cells that were transfected with either the HSF1 (A), RAS (B), ERK (C) or PML (D) BRET probes were exposed to a 5G-modulated 3.5 GHz at 4 W/kg for 24 h and either mock-treated or treated with 1 µM of PMA, 10 µM of As 2 O 3 or 10 µM of MG132 according to the timeline given in Fig. 1A. Coelenterazine H was injected into the cell culture media 10 min before the end of the RF-EMF exposure. BRET signals were remotely measured for the last 5 min before the end of RF-EMF exposure and during the next 5 min after the end of RF-EMF exposure. For each BRET probe, the kinetic of the BRET signal evolution is shown in mock-treated and drug-treated cell, and represents the average of 3-4 independent experiments. www.nature.com/scientificreports/ or the signal envelope used. Finally, all over the assays performed on keratinocytes, we only detected a ~ 37% increase in PMA efficacy to activate ERK at 1 W/kg but not at 0.25 or 4 W/kg. Importantly, no changes in BRET measurement were detected following the end of RF-EMF exposure when the BRET signal was read in real-time in fibroblasts cells that were continuously exposed for 24 h to the 5G modulated 3.5 GHz at 4 W/kg, ruling-out a potential bias due to the time needed to read our BRET signal in 96-well plates after the end of RF-EMF exposure. Altogether, these results can appear puzzling at several levels. Considering the cell types used, it is remarkable that RF-EMF exposure with two different carrier waves (1.8 and 3.5 GHz) and with different signal modulation (CW or GSM-modulation in our previous study and 5G-modulation in the present study) can consistently decrease HSF1 basal activity in HEK293T embryonic kidney cells 13 and XP6BE skin fibroblasts (Fig. 2) but not in HaCaT keratinocytes (Fig. 7). Similarly, we detected that PMA maximal efficacy to activate ERK (i) decreased when HEK293T cells were exposed to a CW or a GSM-modulated 1.8 GHz signal, (ii) increased in keratinocytes, but (iii) was not impacted in 5G RF-EMF exposed skin fibroblasts. Finally, As 2 O 3 maximal efficacy to trigger PML SUMOylation was slightly decreased in skin fibroblasts when exposed continuously but was not modified in keratinocytes.

Figure 7.
Effect of continuous or intermittent 5G RF-EMF exposure on HSF1, ERK, RAS and PML activities in HaCaT keratinocytes. HaCaT keratinocytes cells transiently expressing HSF1, ERK, RAS and PML constructs were sham-exposed or exposed to 5G-modulated 3.5 GHz at 0.25, 1 or 4 W/kg for 24 h, either continuously or intermittently (5 min ON/10 min OFF). Cells were activated using increasing concentrations of either MG132 (HSF1), PMA (RAS and ERK) or As 2 O 3 (PML) under sham or RF-EMF exposure for the last 18 h (HSF1), 15 min (RAS and ERK) or 4 h (PML) of the RF-EMF exposure period. RF-EMF exposure was then shut-down and BRET signals were measured immediately. For each dose response curve, basal BRET signal, potency and maximal efficacy of the activating chemical agent were derived and reported as boxes and whisker plots of the variation between the 5G RF-EMF exposed-(Expo) and sham-conditions in both continuous or intermittent exposure mode. Statistical significance of the derivation from the null hypothesis (no difference between sham and RF-EMF exposure) was assessed using the one-Sample Wilcoxon Signed Rank Test. n = 6-13 depending on the experimental condition. n.s. not significant; *p < 0.05; **p < 0.01. www.nature.com/scientificreports/ Several authors have already reported qualitative or quantitative variations in some biomolecular effects induced in different cell lines following their exposure to RF-EMF signals in the GHz range under identical experimental conditions [45][46][47] . While it is fully expected that different cell types can respond differently to the same stimulus, to our knowledge, no research team has yet elucidated why and how low-level RF-EMF may impact living matter. Beside the lack of non-thermal mechanisms, it is also intriguing that the few RF-EMF induced effects herein detected on HSF1, ERK, and PML activities do not follow a classical dose-response profile. For example, the RF-EMF induced inhibition of HSF basal activity in human skin fibroblasts could only be detected at 0.25 W/kg but not at 4 W/kg, under both intermittent and continuous exposures. Strikingly, at 1 W/kg, there was no change in the HSF1 basal BRET when the cells were continuously exposed, while the HSF1 basal BRET was further decreased when the cells were intermittently exposed. Similarly, PMA maximal efficacy to activate ERK in keratinocytes was increased at 1 W/kg but neither at 0.25 W/kg nor 4 W/kg (Fig. 5). Only As 2 O 3 maximal efficacy to trigger PML SUMOylation seemed to decrease dose-dependently with the SAR under continuous exposure, but the magnitude of this effect was small (Table 4).

Scientific
May RF-EMF exposure at low levels elicit a non-thermal biological effect while RF-EMF exposure at higher levels may not? Several authors in this research field have already reported on the so-called "window" effect, where EMF exposure at specific intensities produces a given biological effect that could not be detected using EMF exposure to lower or higher intensities 48,49 . Unfortunately, no further experimental proofs were later published concerning such window effect and, accordingly, no explanation concerning the underlying molecular mechanism was provided by the authors.
More recently, Pooam et al. invoked a hormetic dose-response effect to explain that ROS production in HEK293T cells exposed to 1.8 GHz RF-EMF is maximal at an intermediate signal amplitude 50 . Hormesis is a toxicological concept characterized by a stimulated biological response when exposed to a low, subtoxic amount of stressor and by detrimental effects of high, toxic levels of the same stressor 51 . Furthermore, an impressive array of cytoprotective molecular mechanisms and signal transduction pathways have been shown to respond in a hormetic way, including the activation of HSF1 and ERK 52,53 . Therefore, whether RF-EMF exposure, alone or in combination with a chemical activating agent, could trigger or magnify a hormetic response on HSF1 or ERK activity needs to be studied more carefully. Interestingly, hormesis is also a time-dependent process 54 . Of note, a time-dependent hormetic effect has been proposed to explain that short exposure (1 h) to 1.8 GHz RF-EMF at an average SAR of 4.0 W/kg induces DNA fragmentation in mouse embryonic fibroblasts while more prolonged exposure (36 h) decreased DNA fragmentation to a lower level than for the sham condition 55 . Accordingly, since we only tested one exposure time (24 h), it will be interesting to repeat these experiments to assess potential time-dependent variation of the herein detected effects.
In conclusion, our BRET study shows no conclusive evidence that molecular effects can arise when skin cells are exposed to a 5G RF-EMF signal (3.5 GHz) for 24 h, even at levels above the ICNIRP guidelines for farfields public exposure (0.08 W/Kg). Only a few statistically-significant changes were detected that depend on (i) the molecular probe used, (ii) the type of cells used, (iii) the presence of an activating chemical agent, with a most than probable time-and dose-dependency, and (iv) the characteristics of the RF-EMF exposure such as SAR, frequency, or modulation of the carrier wave. Given that we used more than 100 different experimental parameters and that all but one of the detected effects were within a risk of 5% error, it was statistically expected to have some false positive data. We reached a similar conclusion when we studied the effect of various 1.8 GHz signals on primary brain cell cultures and neuroblastoma cell lines using label-free techniques 56 . Therefore, apart from the effect of RF-EMF on HSF1 basal activity that may deserve further investigations, we found no sufficient evidence toward physiological effect of the tested RF-EMF alone or in combination with chemicals.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.