Identification of phosphorylation site on PARP1 mediating its cytosolic translocation in virus-infected HeLa cells

Summary Poly (ADP-ribose) polymerase 1 (PARP1) localization is controlled by its phosphorylation state. Here, we describe a protocol to monitor PARP1 subcellular localization in HSV-1-infected HeLa cells using immunofluorescence microscopy and cytoplasmic/nuclear fractionation. We detail steps to identify phosphorylation sites on PARP1 using conserved motif analysis and mass spectrometry. This protocol can be applied to the study of other protein phosphorylation events in other cell types. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).


SUMMARY
Poly (ADP-ribose) polymerase 1 (PARP1) localization is controlled by its phosphorylation state. Here, we describe a protocol to monitor PARP1 subcellular localization in HSV-1-infected HeLa cells using immunofluorescence microscopy and cytoplasmic/nuclear fractionation. We detail steps to identify phosphorylation sites on PARP1 using conserved motif analysis and mass spectrometry. This protocol can be applied to the study of other protein phosphorylation events in other cell types. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).
Note: All cells were free of mycoplasma confirmed by the LookOut Mycoplasma PCR Detection Kit (MP0035, Sigma-Aldrich).

Timing: 7 days
Culture Vero cells in a 500 cm 2 culture flask. When the cell confluency reaches 80%-90%, replace the complete DMEM medium with serum-free DMEM medium, and infect the cells with HSV-1 (MOI = 0.001). After 1 h, replace it with complete DMEM medium (without Penicillin-Streptomycin Solution). 3-4 days later, 90% of the cells are diseased and then collected in a 50 mL centrifuge tube. Freeze cells at À80 C for 1 h and thaw at 25 C for 3 times, collect the supernatant and centrifuge at 120 3 g for 5 min. Measure the titer of HSV-1 in the supernatant through means of 50% of the tissue cultures infectious dose (TCID50) on Vero cells (Chen et al., 2013;Zhou et al., 2016).

Timing: 14 days
Use LentiCRISPRv2 vectors to generate knockout (KO) cells (Sanjana et al., 2014). Transfect the HEK293T cells with pMD2.G, pSPAX2 and lentiCRISPRv2 harboring the guide (g)RNA that target human PARP1 (5 0 -CGAGTCGAGTACGCCAAGAG-3 0 ) at a ratio of 1:3:4. Harvest the lentiviruses after 48 h post transfection, collect and store the cell culture supernatant at 4 C. Collect cell supernatant again after adding complete DMEM medium (without Penicillin-Streptomycin Solution) for 24 h. Mix the two supernatants, centrifuge at 120 3 g for 5 min to remove cell debris, and store at À80 C. Mix the virus-containing medium with an equal volume of complete DMEM medium (without Penicillin-Streptomycin Solution) and add to HeLa cells. 48 h later, select the infected cells with puromycin (4 mg/mL) for 48 h-72 h and acquire single-cell cloning through serial dilutions in a 96-well plate. Confirm the deletion of target gene in KO cells by western blot using anti-PARP (46D11) (CST, Danvers, MA) at 1:1000 dilution. See also troubleshooting 1, 2.
Note: Before selection, the optimal concentration of puromycin could be determined by culturing HeLa cells in medium with increasing concentration of puromycin at a range. During transfection, an empty lentiCRISPRv2 control should be designed.

Virus infection
Timing: 7 days for step 1

Virus infection.
Propagate HSV-1 viruses and measure the titers of virus by TCID50 assay on Vero cells (Chen et al., 2013;Zhou et al., 2016). Store the virus at À80 C for 2-4 years. Infect HeLa cells with HSV-1 (MOI = 1) for indicated time.

Detection of the localization of PARP1 after virus infection
Timing: 3 days for step 2 Timing: 5 days for step 3 Buffer B (stocks can be kept at À20 C for 1-3 months) Note: Light should be avoided during the incubation of secondary antibody to prevent fluorescence quenching.

Reagents
i. Incubate cells with 200 mL DAPI Staining Solution (Beyotime, Shanghai, China) at 25 C for 5-10 min in the dark.
Note: DAPI incubation time should not be too long to avoid high fluorescent background.
j. After 3 washes with 13PBS, add 1 mL of antifade mounting medium. Collect images using a Leica TCS SP8 confocal laser microscopy system (Leica Microsystems, Buffalo Grove, IL) with 633 oil immersion.
Note: It is best to observe dish with antifade mounting medium immediately. If not immediately, it is recommended to store the sample at 4 C in dark for no more than 1 week.
k. Determine PARP1 localization in cells from different dishes. The photos of ten different microscopic fields were taken. The DAPI signal corresponds to the nucleus. The localization of PARP can be analyzed by ImageJ software (1.8.0.172) according to signal segmentation and further data analysis can be carried out using the GraphPad Prism 8 software. Data were representative of three independent experiments with similar results can be found in Figure 1A.
Note: Avoid dish drying when washing with PBS or adding reagents in all the steps to prevent failure in experiment or collection of misleading information.
3. Detection of PARP1 localization by cytoplasmic and nuclear extraction. a. Seed HeLa cells in 6-well-plates (about 1 3 10 6 cells/well), and culture for 24 h to reach 70%-80% confluency. Collect cells from two wells as one sample for cytoplasmic and nuclear fraction.
Note: In our experience, to ensure sample quantity, cells from at least two wells were needed for one group.

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Alternatives: If you want to improve the sample quantity, you can choose one 10 cm dish cells as a sample for cytoplasmic and nuclear extraction.
b. Collect cells and wash twice with 13PBS in the 1.5 mL reaction tube; c. Centrifuge for 5 min at 120 3 g and remove the supernatant completely; d. Add 40 mL Protease inhibitor cocktail (1 tablet in 2 mL PBS), 1 mL 1 M DTT to every 1 mL Buffer A (stocks can be kept at À20 C for 1-3 months) before use. Add 150-200 mL Buffer A supplemented with protease inhibitor cocktail and DTT to 20 mL cell pellet, place the tube on ice for 30 min, and centrifuge at 15,000 3 g for 6 min. The supernatant is cytoplasmic extract, which is transferred to a pre-cooled 1.5 mL reaction tube.
Note: Buffer A is best to be prepared freshly and pre-cooled before use. The nucleoplasm must be separated cleanly, and the residual liquid should be aspirated with a pipette.
e. Wash the pellets 5 times with 500 mL wash buffer (10 mM HEPES (pH 7.9), 2 mM MgCl 2 , 20 mM KCl, 100 mM EDTA (pH 8.0)) (stocks can be kept at À20 C for 1-3 months) supplemented with protease inhibitor cocktail and DTT. Avoid pipetting the pellet, centrifuge at 1,000 3 g for 5 min. f. Add Protease inhibitor cocktail 40 mL (1 tablet in 2 mL PBS) and 1 M DTT 1 mL for every 1 mL Buffer B (stocks can be kept at À20 C for 1-3 months) before use, add 50-80 mL Buffer B to the pellet, vortex the tube at high speed for 10-15 s every 10 min for a total of 30 min, and place it on ice during the period. Centrifuge the mixture at 15,000 3 g for 15-30 min and collect the supernatant as the nuclear extract.
Note: Buffer B is best prepared freshly and pre-cooled before adding.
g. Denature both the cytoplasmic and nuclear fractions in 13 SDS protein loading buffer (stocks can be kept at À20 C for 3 months) at 95 C for 8 min for the following western blot analysis.
Note: Collected samples should be stored at À20 C, if they are not tested immediately.
h. Configure and run 10% SDS-PAGE regular gels (https://www.bio-rad.com/) (stocks can be kept at 4 C for 3 days). Electrophoresis was performed in 13 running buffer (https://www. bio-rad.com/) (stocks can be kept at 25 C for 1 week). Condensing gel electrophoresis conditions: 80 V, 30 min; separating gel electrophoresis conditions: 100 V, until bromophenol blue runs out of the separating gel; i. Membrane transfer: wet transfer. Transfer the protein to a 0.22 mm pore size nitrocellulose (NC) membrane in 13 transfer buffer (https://www.bio-rad.com/) (stocks can be kept at 25 C for 1 week) at 100 V for 1.5 h. j. Blocking: 5% skim milk (in 13TBST) or 5% BSA (in 13TBST) for 1 h at 25 C. k. Dilute the control Primary antibody (anti-IgG Rabbit antibody), Primary antibody (anti-PARP1 Rabbit antibody, anti-tubulin Rabbit antibody and Histone H3 Rabbit antibody) in Blocking buffer (stocks can be kept at 4 C for 1 day) at 1:1,000 dilution. Incubate the NC membrane with the antibody for 12 h at 4 C on a rotator. l. Recover the primary antibody and wash 3 times with 13TBST (stocks can be kept at 25 C for 1 week) on a shaker; 10 min each time. m. Dilute the secondary antibody (Goat Anti-Rabbit IgG Antibody, HRP conjugate) in 13TBST at 1:5,000 and incubate the membrane for 1 h at 25 C followed by washing three times with 13TBST. n. Expose the gel to Supersignal West Pico developer, and then collect the image data by Amersham Imager 600 (General Electric Company, GE). The corresponding protein was detected as marker proteins to indicate the cytoplasmic and nuclear fractions, respectively ( Figure 1B). See also troubleshooting 4, 5, 6.

Validation of the phosphorylation site(s) responsible for PARP1 cytosolic translocation
Timing: 1 h for step 4 Timing: 2 weeks for step 5 4. Prediction of PARP1 phosphorylation sites.
The human PARP1 protein sequence can be downloaded from the database UniProtKB (http://www. uniprot.org/): P09874. The sites recognized in vitro by DNA-PK are reported to be D/E/Q-S/T-Q, with a glutamine residue on the C-terminal side being important (Lees-Miller and Anderson, 1991;Watanabe et al., 1994). We predict T325 residue of PARP1 as a potential phosphorylation site according to the conserved substrate motif of DNA-PK. 5. Mass spectrometry detection of PARP1 phosphorylation sites. a. Seed HeLa cells in 10 cm cell culture dish (about 1 3 10 7 cells/well) and culture for 24 h to reach 70%-80% confluency and infect with HSV-1 (MOI = 1) for 4 h. b. Wash cells twice with 13PBS and lyse using 200 mL RIPA Lysis Buffer (Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail (P8340, Sigma-Aldrich), 1 mM of PMSF and phosphatase inhibitor cocktail (P5726, Sigma-Aldrich). Centrifuge the lysate with tabletop centrifuge at 15,000 3 g for 10 min and the cellular debris was discarded.
Note: After the cells were mixed with lysis buffer, they were immediately placed on a shaker at 4 C for 30 min to thoroughly lyse the cells.
c. Wash Protein A/G agarose twice with 1 mL 13PBS and prepared for a 50% protein A/G agarose working solution (in 13PBS). Incubate the cell lysates with protein A/G agarose (50 mL) and anti-PARP1 antibody (5 mL) at 4 C for 12 h. d. Wash the beads with 1 mL 13 PBST (0.5% triton X-100 in 13PBS) five times, discard the supernatant completely.
Note: 13PBST should be pre-cooled before use.

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e. Add 30-50 mL 13 protein loading buffer to the beads, 95 C, 10 min, and subject the boiled protein sample to 10% SDS-PAGE gel electrophoresis. f. Wash the gel (the purpose of this step is to wash off interfering substances such as SDS in the gel, reduce the staining background, and improve the staining sensitivity). i. Put the gel into a sterile 10 cm dish.
ii. Add 50 mL of deionized water.
iii. Heat it in the microwave for 3 min on high heat. iv. Shake for 5 min on a side-swing shaker or a horizontal shaker. g. Stain the gel.
i. Carefully pour off the liquid, remove the residual liquid.
ii. Add an appropriate amount of Coomassie brilliant blue fast staining solution (about 20 mL for each gel with a size of about 8 cm) to ensure that the staining solution can cover the gel and the liquid level is at least three gels thick. iii. Stain the gel on a side or horizontal shaker at 25 C. The staining time at 25 C should be 10-30 min. iv. Discard the staining solution until clear target protein bands appear. v. Add an appropriate amount of deionized water, wash off the residual staining solution, stop the staining reaction. vi. Take pictures and record the results. h. Elute the gel (This step can be performed if a background-free picture of the gel is desired).
i. Add about 100 mL of deionized water, shake the gel on a shaker to decolorize.
ii. Every 5-15 min, carefully pour off the liquid, add 100 mL of deionized water, and continue to decolorize on the shaker. Usually, de-staining for 30-120 min can highly reduce the background staining. See also troubleshooting 7, 8. i. After Coomassie brilliant blue staining and elution, the PARP1 band was cut, decolorized and subjected to mass spectrometry (MS).
Note: The area of the cleaved band should be less than or equal to 1 cm 3 1 cm (the maximum area does not exceed 1.5 cm 3 1.5 cm), the target band should be clear, clearly visible to naked eyes, and there are no excess bands in the remaining molecular weight range; the cleaved band should be located in separating gel rather than concentrating gel; store in a 1.5 mL tube and keep the isolated gel moist by adding a small amount of deionized water.
Note: If the gel concentration is relatively low and the gel is prone to be damaged due to heating, you can put the gel after electrophoresis into a container of appropriate size, add 100 mL of deionized water so that the gel is completely covered with water. Shake and wash the gel on a rotator for a total of 5 min. Washing 3-5 times can also achieve the effect of removing contaminants such as SDS in the gel. If you want to speed up the washing, you can add 100 mL of boiled deionized water, shake it on a shaker for 5 min, and wash 2-3 times in total. The purpose of microwave heating or washing with heated deionized water is to speed up the washing process and shorten the washing time. If the time is sufficient, it can be washed at 25 C.
j. Using Proteome Discoverer 1.4 (Thermo Fisher Scientific) to obtain the resulting MS/MS data. i. Load the sample onto an HPLC chromatography system named Thermo Fisher Easy-nLC 1000 equipped with a C18 column (1.8 mm, 0.15 3 100 mm). ii. Solvent A contained 0.1% formic acid and solvent B contained 100% acetonitrile. The elution gradient was from 4% to 18% in 182 min, 18%-90% in 13 min solvent B at a flow rate of 300 nL/min. iii. Mass spectrometry analysis were carried out at the PTMBio Co., Ltd. (Shanghai, China) in the positive-ion mode with an automated data-dependent MS/MS analysis with full scans (350-1,600 m/z) acquired using FTMS at a mass resolution of 30,000 and the ten most intense precursor ions were selected for MS/MS. iv. Use higher-energy collision dissociation to acquire the MS/MS at 35% collision energy at a mass resolution of 15,000. v. Tandem mass spectra were searched against human PARP1 protein sequence. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. vi. Mass error was set to 20 ppm for precursor ions and 0.05 Da for fragment ions. vii. Carbamidomethyl on Cys were specified as fixed modification, oxidation on Met and phosphorylation on Ser, Thr, Tyr were specified as variable modifications. Peptide confidence was set at high, and peptide ion score was set > 20 ( Figure 2A). See also troubleshooting 9, 10.

EXPECTED OUTCOMES
Detect and quantify the localization of PARP1 and corresponding PARP1 mutants with immunofluorescence microscopy and cytoplasmic/nuclear fraction. The results were expected to demonstrate that PARP1 is able to translocate from the nucleus to the cytoplasm in response to HSV-1 infection. Based on the results from mass spectrometry analysis, we generate PARP1 phosphodead mutants HA-PARP1(T594A) plasmid and reconstitute PARP1 KO HeLa cells with HA-PARP1, or HA-PARP1(T594A) plasmids, respectively. We then analyze the localization of PARP1 WT vs PARP1(T594A). The results were expected to determine T594 as the phosphorylation site of PARP1 responsible for its cytosolic translocation.

LIMITATIONS
In this protocol, only HeLa cells was employed to quantitatively analyze the cytoplasm/nucleus of PARP1. Meanwhile, confocal microscopy and cytoplasmic/nuclear extraction can only reflect a rough distribution of PARP1, and a more accurate subcellular localization requires more precise experimental methods to detect. In order to better study the function to cytosolic PARP1, fusion of PARP1 with nuclear export sequence (NES), LEKLKL sequence (Fu et al., 2013;Huang et al., 2021;Kosugi et al., 2014;Sun et al., 2021) would be helpful to confine PARP1 in the cytosol (Wang et al., 2022).

Problem 1
In the experimental process of constructing PARP1 KO cells, no positive cells could be obtained.

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Potential solution gRNA might not be efficient. It is recommended to check whether gRNA design has a problem and design several pairs of gRNAs.

Problem 2
In the experimental process of constructing knockout cells, the cell state is poor.

Potential solution
During selecting positive knockout cells with puromycin or G418, the dead cells should be removed in time, and the fresh medium or new cell culture dish should be replaced.

Problem 3
The knockout cells or reconstituted HeLa cells grow slowly.

Potential solution
Increase the amount of FBS in the medium. The concentration of FBS can be increased to 20%.

Problem 4
The unspecific background signals are too high in obtaining image of immunoblotting at step 3.

Potential solution
Proper extension of blocking time; wash the nitrocellulose filter membrane thoroughly; use appropriate concentration of primary and secondary antibody; the incubation time of the antibody should not be more than 24 h.

Problem 5
Incomplete separation in step 3 of nucleus and cytoplasm.

Potential solution
The cytoplasm contains nuclear components. When lysing cells, try to pipette the precipitated cells evenly. At the same time, the cleavage time is needed to be optimized, too short cleavage time would lead to incomplete cleavage, and too long cleavage time would lead to increased content of nucleus in cytoplasm.

Problem 6
No obvious PARP1 band in step 3 of nucleus or cytoplasm fractionation.

Potential solution
On the basis of the initial cell amount of 1 3 10 7 , increase the number of cells. Increase the anti-PARP1 antibody concentration and incubation time.

Problem 7
Unstained band in the process of coomassie brilliant blue staining and elution at step 5.

Potential solution
Increase the amount of loaded sample. It is recommended to load 30-50 mg of BSA as a positive control during electrophoresis.

Problem 8
The sensitivity of the stained band is not ideal in the process of coomassie brilliant blue staining and elution at step 5.

Potential solution
Coomassie brilliant blue fast staining solution can be used for the second staining to improve the staining effect. Prolonging the staining time can improve the detection sensitivity, and fully de-staining with deionized water can also reduce the staining background and significantly improve the staining sensitivity.

Problem 9
There is only solvent peak in the spectrum at step 5.

Potential solution
The injection needle is damaged; The carrier gas flow rate is too low; The sample concentration is too low; The sample is adsorbed by the column or injector bushing.

Problem 10
Mass deviation of mass spectra at step 5.

Potential solution
If the mass number of the mass spectrometer is off, it is time to calibrate your instrument.

RESOURCE AVAILABILITY
Lead contact Further information and requests for reagents may be directed to, and will be fulfilled by the lead contact, Prof. Haipeng Liu (haipengliu@tongji.edu.cn).

Materials availability
Requests for the plasmids and cell lines generated in this study should be directed to the lead contact with a completed Materials Transfer Agreement.