A protocol to investigate the effects of lncRNAs on in vivo protein-protein interactions using proximity ligation assay

Summary A large variety of cellular signals are triggered and transmitted by protein-protein interactions (PPIs). Long noncoding RNAs regulate PPIs by enhancing or destabilizing these interactions. Here, we use the proximity ligation assay technique to determine PPIs between p53 and SET regulated by long intergenic noncoding RNA 324 (LINC00324). We detail procedures for establishing LINC00324 knockdown and overexpression U2OS and HepG2 cells followed by in situ PLA protocol. This approach has many potential applications for the study of cellular factors that regulate PPIs. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2023).1


BEFORE YOU BEGIN
3][4][5] Duolink PLA technology allows one to visually recognize PPIs at single cell resolution with high sensitivity and specificity. 6Here, we describe a protocol for visualization of p53-SET interaction regulated by LINC00324 (Figure 1).

Timing: 2 weeks
The following steps describe the procedure to establish LINC00324 knockdown and overexpression stable cell lines.The knockdown cells were generated using shRNA expression lentivirus with puromycin resistance.The control and LINC00324 shRNA sequences (Table 1) were cloned into lentivirus vector LV2 (U6/Puro).The lentivirus constructs were co-transfected with helper vectors, pGag/Pol, pRev and pVSV-G into 293T packaging cells (GenePharma, Suzhou, China).The cell supernatants were collected into 50 mL centrifuge tubes and centrifuged at a low speed of 400 g for 4 min at 4 C. Then the supernatants were filtered through a 0.45 mm filter.The filtrates were collected by ultra-centrifuging for 2 h at 3000 g at 4 C.For stable ectopic LINC00324 expression, LINC00324 complementary DNA (cDNA) was inserted into the pcDNA3.1/myc-His-A(+) vector to generate LINC00324 overexpression constructs.The empty vector was used as a control (CON).These constructs were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen).The stable cells overexpressing LINC00324 were screened by G418 resistance selection.
a.The day before infection, seed U2OS and HepG2 cells in a 6-well plate at a density sufficient to reach 50%-60% confluence on the day of infection.b.Incubate the cells in incubator at 37 C, 5% CO 2 for 16-24 h.c.Mix the lentivirus expressing control and LINC00324 shRNAs with culture medium in total volume of 2 mL to achieve a multiplicity of infection (MOI) of 50-100 for both cells, add polybrene to a final concentration of 5 mg/mL.d.Move out the medium from the 6-well plate, replace with the virus diluent and incubate at 37 C in the CO 2 incubator for 24 h.e. Replace with 2 mL fresh medium and incubate at 37 C in the CO2 incubator for 24-48 h.f.Select cells with 2 mg/mL puromycin for 2 weeks.During this process, replace with fresh medium containing 2 mg/mL puromycin every 2-3 days.Step by step representation of PLA detection of p53-SET interaction.LINC00324 disrupts the p53-SET interaction.
Table 1.The sequences of shRNAs and primers transfection reagent with a regular culture medium.e. Incubate cells at 37 C in the CO 2 incubator for 24 h.f.After digestion, seed cells at 1:50, 1:100, 1:200, and 1:500 into fresh culture medium.g.Next day, add 1 mg/mL G418 for stable cell line selection.h.Select cells with 1 mg/mL G418 for 2 weeks.During this process, replace with fresh selection medium containing 1 mg/mL G418 every 2-3 days.i. Select single colonies containing more than 50 cells to the 24-well plate to continuous expansion (Figures 2C and 2D, left panels).j.Collect (0.5-1)310 6 cells from each colony, extract cellular total RNAs and reverse-transcribed into cDNAs.k.Test for transgene expression using qRT-PCR to select positive colonies (Figures 2C and 2D, right panels).

Choice of primary antibodies
Timing: 2 days The two primary antibodies must be raised in two different species and must bind with high specificity for the protein of interest.This section describes the procedure to identify the specificity of the SET and p53 antibodies considered to be used for PLA experiments.
3. Separate protein from U2OS and HepG2 cells extracts by SDS-PAGE and transfer onto the nitrocellulose membrane.4. Block the membrane in 5% skim milk for 1 h at room temperature (22 C-26 C). 5. Remove the milk and incubate the membranes in antibody solutions of rabbit SET antibody (1:1000), mouse SET antibody (1:1000), or rabbit p53 antibody (1:1000) at 4 C for 12-18 h. 6. Wash the membrane in 13 TBST 3 times for 10 min each at room temperature (22 C-26 C). 7. Incubate the membrane with corresponding secondary antibodies for 1 h at room temperature (22 C-26 C). 8. Wash the membrane in 13 TBST 3 times for 10 min each at room temperature (22 C-26 C). 9. Develop the target band signals using an ECL solution and a ChemiDoc XRS detection system.CRITICAL: To assess p53-SET interaction, we tested the specificity of rabbit and mouse SET antibodies, and p53 antibodies.The mouse SET antibody and rabbit p53 antibody showed optimal specificity while rabbit SET antibody showed non-specific bands (Figure 3).We chose mouse SET antibody and rabbit p53 antibody for PLA experiment.

Reagents preparation
Timing: 30 min 10. 13 PBS: Dilute 103 PBS in a 1:10 ratio in Milli-Q water.11. 0.3% Triton X-100: Mix 13 PBS with 0.3% (vol/vol) Triton X-100.12. 13 Wash buffer (Wash buffer A and B): Dissolve the content of 1 pouch of powdered buffer in Milli-Q water to a final volume of 1 L. 13. 0.013 Wash buffer B: Dilute 13 Wash buffer B in a 1:100 ratio in Milli-Q water.
Note: Keep the solutions at room temperature (22 C-26 C) for short-term storage (one week or less).For long-term storage, store at 4 C.This section describes the procedure to prepare cells for proximity ligation assay (PLA).

KEY RESOURCES
1. Place the 14 mm round glass coverslips into a 24-well plate.
Note: Coverslips should be clean and sterile.
2. Seed cells onto the coverslips in the 24-well plate.
3. Let cells stand for 15 min for adhesion and then incubate at 37 C in the CO 2 incubator for 24 h. 4. For p53 knockdown, transfect 10 pmol siRNA for p53 into LINC00324 knockdown and overexpression U2OS cells using the Lipofectamine RNAiMAX transfection reagent according to manufacturer's instructions (https://www.thermofisher.com/us/en/home/references/protocols/rnai-epigenetics-and-gene-regulation/rnai-protocol/lipofectamine-rnaimax.html).Add 10 pmol si-p53 to 50 mL Opti-MEM medium and 1.5 mL Lipofectamine RNAiMAX reagent to 50 mL Opti-MEM medium.Combine both solutions and incubate for 10-20 min at room temperature (22 C-26 C).Add the complexes to each well containing cells.Mix gently by rocking the plate back and forth.Incubate the cells at 37 C in a CO 2 incubator.After 48 h growing, subject the cells to fixation.

Note:
The optimal cell confluence is 50%-70% at the time of fixation.

Timing: 2.5 h
This section describes the steps for cell fixation, permeabilization and blocking.Pause point: Fixed cells can be stored in 13 PBS at 4 C for several days.The plates should be prevented from drying out.
Note: The fixation time may vary according to the cell type.Methanol is an alternative fixing reagent.The best choice of fixation may vary depending on your sample type, antibodies, and the protein target you want to study.
13 Wash buffer B: It can also be prepared in the lab as follows instead of which is provided as a powder from Sigma-Aldrich

Reagent
Final concentration Amount CRITICAL: Be sure to cover the entire sample with all solutions.Do not allow the cells to dry before adding the antibody.

Timing: 18 h
This section describes the steps for primary antibodies incubation.
Note: Dilute your primary antibody to a suitable concentration in your custom antibody diluent.If using two primary antibodies, dilute the two antibodies in the same diluent.9. Remove the blocking solution from the coverslips and add 60 mL of the primary antibodies solution to each sample.
Note: Aspirate off the blocking solution as much as possible to avoid dilution of the primary antibodies.
10. Incubate the slides in a humidity chamber for 12-18 h at 4 C.
CRITICAL: Do not allow the cells to dry.Make sufficient solutions for all samples.
The following PLA steps are referred to the Duolink PLA Fluorescence Protocol from Sigma-Aldrich with minor modifications.

Duolink PLA probe incubation
Timing: 1.5 h This section describes the steps for probe hybridization.
11. Dilute the two 53 Duolink PLA probe and mix Anti-Mouse PLUS and Anti-Rabbit MINUS PLA probes at 1:5 dilution in the Duolink Antibody Diluent.12. Remove the primary antibody solution and wash the slides with 50 mL 13 Wash Buffer A 2 times for 5 min each on a shaker at room temperature (22 C-26 C).
Note: Bring the wash buffer to room temperature (22 C-26 C) before use.
13. Remove the excess wash buffer and add 60 mL of the PLA probe mixture solution to each sample.14.Incubate the slides in a pre-heated humidity chamber for 1 h at 37 C.
Note: Do not allow the cells to dry.Make sufficient solution for all samples.

Ligation
Timing: 1 h This section describes the steps for oligonucleotide ligation.
15. Remove the PLA probe solution, wash the slides with 50 mL 13 Wash Buffer A 2 times for 5 min each at room temperature (22 C-26 C). 16.Dilute the 53 Duolink Ligation buffer in a 1:5 ratio in Milli-Q water.

Note:
The buffer contains DTT that may precipitate during freezing.Gently pipette to dissolve.17.Add ligase to the 13 Ligation buffer from step 16 in a 1:40 ratio and mix.
Note: Ligase should stay on ice.Add the enzyme to the reaction mix immediately before use.
18. Remove the excess wash buffer and add 60 mL of the Ligase-Ligation solution to each sample.19.Incubate the slides in a humidity chamber for 30 min at 37 C.

Rolling circle amplification (RCA)
Timing: 2 h This section describes the steps for rolling circle amplification.20.Remove the Ligase-Ligation solution, wash the slides with 50 mL 13 Wash Buffer A 2 times for 5 min each at room temperature (22 C-26 C). 21.Dilute the 53 Amplification Far Red buffer in a 1:5 ratio in Milli-Q water.
Note: Amplification buffer is light-sensitive.Make sure to protect from light from this step.
22. Add Polymerase to the 13 Amplification buffer from step 21 in a 1:80 ratio and mix.
Note: Polymerase should stay on ice.Add the enzyme to the reaction mix immediately before use.
23. Remove the excess wash buffer and add 60 mL of the Polymerase-Amplification solution to each sample.24.Incubate the slides in a humidity chamber for 100 min at 37 C.

Timing: 25 min
This section describes the steps for washing after amplification.Protocol 25.Remove the Polymerase-Amplification solution, wash the slides with 50 mL 13 Wash Buffer B 2 times for 10 min each at room temperature (22 C-26 C). 26.Wash the slides with 50 mL 0.013 Wash Buffer B for 1 min at room temperature (22 C-26 C).
Note: Make sure the slides are protected from light during the procedure.

Timing: 30 min to 3 h
This section describes the steps for coverslips mounting and image acquisition.
27. Remove the excess wash buffer, air dry in the dark.
Note: a paper towel can be used to carefully absorb the excess buffer.
28. Drop 5 mL DAPI Fluoromount G on a microscope glass slide.Put coverslips upside down on the slide carefully to make sure the mounting solution is spread all over.
Note: Avoid air bubbles during the mounting process.You can also use the Duolink In Situ Mounting Medium with DAPI from Sigma-Aldrich as an alternative.
29. Place the slides in the dark for 15 min at room temperature (22 C-26 C) to dry before viewing using a confocal microscope (LEICA TCS SP8).
Note: Use clear nail polish or neutral balsam to seal all sides of coverslips for long-time storage.
30.Acquire images using at least a 203 objective.
Pause point: The slides can be stored at 4 C for 3-4 days or at -20 C for longer time before imaging.
CRITICAL: Make sure the images in the experimental and control groups acquired in the same parameters for proper comparison.

EXPECTED OUTCOMES
The PLA signal recognized as fluorescent spots should be produced by successful PLA if the two proteins are closer than 40 nm. 7,8In this protocol we performed PLA in U2OS and HepG2 cells to determine the effects of LINC00324 on p53-SET interaction by comparing fluorescent spots between the controls and LINC00324 knockdown or overexpression cells (Figures 4A-4D).PLA signals should not be detected in the sample in which the single antibody was added to (Negative control).The results demonstrated that the PLA spots were increased in LINC00324 knockdown U2OS and HepG2 cells (Figures 4A and 4C) but were suppressed in LINC00324 overexpression cells (Figures 4B and 4D).These results indicate that LINC00324 interrupts the p53-SET interaction.

QUANTIFICATION AND STATISTICAL ANALYSIS
Open the images using Image J software.Count the number of PLA spots and nuclei manually using ''Multi-points'' tool.The average PLA spots of each cell were calculated by dividing the number of PLA spots of all cells by the number of nuclei in the visual field.Take the average of the three random fields.Perform student's t-test analysis using GraphPad Prism 8.0 (Figures 4A-4D).

LIMITATIONS
First, there exist off-target effects for the shRNA-mediated knockdown.One should choose at least two shRNA sequences targeting the different locations of an lncRNA.Second, it is difficult to get effective shRNAs to knock down a target lncRNA in some conditions.More shRNAs sequences should be designed in order to select effective fragments.Third, the long-term antibiotic selection may affect cell growth or even lead to cell differentiation resulting from shRNA-mediated knockdown or other indirect effects.One should be careful that the observed effects of an lncRNA on PPIs were caused from an indirect effect.Similar conditions may appear for the overexpression stable cells.Transient siRNA-mediated knockdown or overexpression experiments can be used to confirm the results, albeit at a lower expression efficiency compared with stable cells.Finally, an unspecific signal may appear if the primary antibody recognizes non-specific proteins.Try several antibodies from different source to determine which one is fit for the experiment.If the specificity of signals was suspicious, one may perform siRNA-mediated target protein knockdown to confirm the specificity.

Figure 1 .
Figure 1.Workflow overview of the Duolink proximity ligation assay (PLA)Step by step representation of PLA detection of p53-SET interaction.LINC00324 disrupts the p53-SET interaction.
p53 siRNA (si-p53)Sense: 5 0 -GACUCCAGUGGUAAUCUACdtdt-3'; anti-sense: 5 0 -GUAGAUUACCACUGGAGUCdtdt-3 0 LINC00324 Forward primer CTACGGTTTCTGGTCAGCGT LINC00324 Reverse primer ACGACGGCAGCCATTACTTT GAPDH Forward primer ATCAATGGAAATCCCATCACCA GAPDH Reverse primer GACTCCACGACGTACTCAGCG g.Collect (0.5-1)310 6 cells from control and knockdown groups, extract cellular total RNAs using TRIzol reagent and then reverse-transcribed into cDNAs.h.Verify interference efficiency using quantitative real-time PCR (qRT-PCR) (Figures2A and 2B). 2. Preparation of LINC00324 overexpression stable cell lines.a.One day before transfection, plate U2OS and HepG2 cells in a 6-well plate at a density sufficient to reach 70%-90% confluence at the time of transfection.b.For each well to be transfected, prepare mixture as follows.i. Dilute 5 mg plasmids in 250 mL of Opti-MEM I Reduced Serum Medium.ii.Dilute 10 mL Lipofectamine 2000 in 250 mL of Opti-MEM medium.Incubate for 5 min at room temperature (22 C-26 C). iii.Gently mix the diluted plasmids with diluted Lipofectamine 2000.Incubate for 20 min at room temperature (22 C-26 C). c. Replace the medium in the 6-well plate with 1.5 mL Opti-MEM medium.Add the 500 mL of transfection complexes into each well.Mix gently by rocking the plate back and forth.d.Incubate cells at 37 C in the CO 2 incubator for 4-6 h.Replace the medium containing the

Figure 2 .
Figure 2. Preparation of LINC00324 knockdown and overexpression U2OS and HepG2 cells (A and B) The expression of LINC00324 was detected by qRT-PCR in LINC00324 knockdown U2OS (A) and HepG2 cells (B).(C and D) Colonies were selected after G418 selection for 2 weeks.qRT-PCR was used to determine the expression of LINC00324 to select positive over-expression cells in U2OS (C) and HepG2 cells (D).Two positive stable cell lines for each cell type were selected for subsequent experiments.Scare bar, 100 mm.

Figure 3 .
Figure 3. Identification of primary antibodies specificity Western blot analysis of SET rabbit antibody, SET mouse antibody and p53 rabbit antibody using cell extracts from U2OS and HepG2 cells.Rb, rabbit; Ms, mouse.These data are from the original Figure S5A in Zhang et al. (2023).1

1
Figure 3. Identification of primary antibodies specificity Western blot analysis of SET rabbit antibody, SET mouse antibody and p53 rabbit antibody using cell extracts from U2OS and HepG2 cells.Rb, rabbit; Ms, mouse.These data are from the original Figure S5A in Zhang et al. (2023).1
Adjust the pH to 7.5.Filter the solution through a 0.22 mm filter.Store at 4 C for up to 1 year.Protocol 6. Permeabilization.a. Wash cells with 13 PBS 3 times for 5 min each at room temperature (22 C-26 C). b.Permeabilize with 13 PBS with 0.3% Triton X-100 for 20 min at room temperature (22 C-26 C). 7. Blocking.a. Wash cells with 13 PBS 3 times for 5 min each at room temperature (22 C-26 C). b.Put the coverslips on slides.Draw a hydrophobic circle along the coverslips' edges with a hydrophobic PAP pen to delimit the reaction area.c.Add 60 mL of Duolink Blocking Solution to each sample.d.Incubate the slides in a humidity chamber for 1 h at 37 C. Pause point: Blocking can be run for 12-18 h at 4 C.

Figure 4 .
Figure 4. Confocal images of in situ PLA show that LINC00324 disrupts the p53-SET interaction The data of Figure 4C and 4D are from the original Figures S5B and S5C in Zhang et al. (2023). 1 (A and B) Duolink PLA detected the interaction between p53 and SET in U2OS cells stably expressing the shRNAs lv-NC, lv-324-1, and lv-324-2 (A) and in U2OS control cells transfected with empty vector (CON) and U2OS cells stably expressing ectopic LINC00324 (U1 and U2) (B).siRNA against p53 and single antibody p53 or SET were performed as negative controls.(C and D) Duolink PLA detected the interaction between p53 and SET in HepG2 cells stably expressing the shRNAs lv-NC, lv-324-1, and lv-324-2 (C) and in HepG2 control cells transfected with empty vector (CON) and HepG2 cells stably expressing ectopic LINC00324 (H1 and H2) (D).The average number of PLA dots from three random fields per coverslip was quantitated and presented in the bar diagram.Scale bar, 50 mm *p < 0.05, **p < 0.01, which were calculated using two-tailed Student's t test.The data are presented as mean G SD.

TABLE
Store the culture medium at 4 C for 1 month.Bring the culture medium to room temperature (22 C-26 C) for 1 h or warm the medium at 37 C water bath for 30 min before use.
(Continued on next page)MATERIALS AND EQUIPMENTNote: Adjust the pH to 7.4.Filter the solution through a 0.22 mm filter.Store at 4 C for up to 1 year.STAR Protocols 4, 102757, December 15, 2023