Materials and reagents

Biological materials

Cell lines of interest obtained from the American Type Culture Collection (ATCC). In this study, we used the colon cancer cell line HCT116.

Reagents

Puromycin (Sigma-Aldrich, catalog number: P9620)
PBS (Sigma-Aldrich, catalog number: D1408)
Complete anti-protease (Roche, catalog number: 11836145001)
Dry milk (Régilait, catalog number: 304934416704)
Bovine serum albumin (BSA) (Roche, catalog number: 10735094001)
DC Protein Assay kit (Bio-Rad, catalog number: 5000111)
Prestained protein ladder (Euromedex, catalog number: 06P-0111)
Immobilion Forte western substrate (Merck Millipore, catalog number: WBLUF0500)
Ponceau red solution (Sigma-Aldrich, catalog number: 141194)
Mouse anti-puromycin antibody (clone 12D10) (Merck Millipore, catalog number: MABE343)
Mouse anti-tubulin antibody (clone DM1A) (Sigma, catalog number: T6199)
HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology, catalog number: 7076)
Stripping buffer (Thermo Scientific, catalog number: 21059)

Solutions

RIPA protein lysis buffer 2× (see Recipes)
Tris-buffered saline-Tween (TBS-T) (see Recipes)
1. TBS-T 5% BSA (see Recipes)
2. TBS-T 5% dry milk (see Recipes)
Laemmli 6× (see Recipes)

Recipes

RIPA protein lysis buffer 2×
Reagent Final concentration
Tris-HCl pH 7.2 100 mM
NaCl 300 mM
EDTA 10 mM
Sodium deoxycholate 2%
SDS 20% 0.1%
Triton 100× 2×
Na₃VO₄ 4 mM
β glycerophosphate 20 mM
NaF 20 mM
Complete anti-protease 2×
Dilute to 1× in H₂O.

Tris-buffered saline-Tween (TBS-T)
Reagent Final concentration
Tris-HCl pH 7.5 50 mM
NaCl
Tween
150 mM
0.1%
Add 5% of BSA or dry milk for obtaining TBS-T 5% BSA or 5% dry milk, respectively.

Laemmli 6×
Reagent Final concentration
Tris-HCl pH 6.8 0.5 M
Glycerol 1%
SDS 20% 2%
DTT 0.6 M
Bromophenol blue 0.4%

Laboratory supplies

6-well or 10 cm diameter tissue culture plates
Sterile scrapers
Microcentrifuge tubes
Plastic wrap

Equipment

Tissue culture apparatus (tissue culture hood, CO₂ incubator, etc.)
Pipettes and micropipettes
Vacuum pump
Centrifuge (4 °C)
Cold room
Heat block
Western blotting apparatus (SDS-PAGE running cassette, power supply, shaker, transfer cassette, nitrocellulose membrane, etc.)
ChemiDoc imaging system (Bio-Rad, catalog number: 12003153)

Software and datasets

Fiji (National Institutes of Health)

Procedure

Puromycin incorporation in vitro
1. Seed HTC116 cells one day before the experiment and maintain at 37 °C and 5% CO₂to allow attachment to the tissue culture plate. In the experiment presented in Figure 2, 200,000 cells per well were seeded in a 6-well plate.
2. On the day of the experiment, apply the studied treatment on cells for the desired time.
3. Before harvesting and 15 min before the end of the treatment, expose cells to 5 μg/mL puromycin directly diluted in the media. All samples must be incubated with the same concentration of puromycin for an equal period.

Protein extracts
1. At the end of the 15-min incubation with puromycin, remove media and rinse cells once with cold PBS.
2. Put the tissue culture plate on ice and incubate with 1× RIPA protein lysis buffer (see Recipes) containing proteases and phosphatases inhibitors (1 volume of 1× RIPA for 1 volume of cell pellet) for 20 min.
3. Scrape the cells and collect in a microcentrifuge tube.
4. Centrifuge at 13,000× g for 20 min at 4 °C to get rid of cellular debris.
5. Collect the supernatant in a new microcentrifuge tube and add the appropriate volume of Laemmli 6× (see Recipes).
6. Dose the amounts of extracted proteins using the DC Protein Assay kit according to manufacturer’s instructions.
7. Add the appropriate volume of Laemmli 1× to normalize protein concentrations in all samples and denaturate by heating at 95 °C for 5 min.

Western blotting
1. Load 20 μg of proteins for all studied conditions on a 10% SDS-PAGE gel. We recommend adding 5 μL of protein ladder to estimate the molecular weight of visualized proteins.
2. When separated, transfer proteins onto nitrocellulose membranes as a standard western blot protocol. Stain with Ponceau red solution to check equal protein amount loading before electrophoresis.
3. Block the membrane with TBS-T 5% dry milk for 1 h at room temperature with gentle shaking.
4. Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
5. Incubate overnight at 4 °C on a gentle shaker with puromycin antibody diluted at 1/10,000 into TBS-T 5% BSA.
6. Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
7. Incubate at room temperature for 1 h with the HRP-conjugated anti-mouse secondary antibody (1/10,000 dilution) into TBS-T 5% dry milk.
8. Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
9. Gently dry the membrane using paper towels and place it face up and flat on a sheet of plastic wrap.
10. Directly add the Immobilion Forte western substrate onto the whole membrane.
11. Detect chemiluminescence with the ChemiDoc imaging system (Figure 2). Intensity of the smear depends on cell ability to incorporate puromycin and is thus representative of protein synthesis rate.
12. Rinse the membrane in TBS-T and transfer in 5 mL of stripping buffer.
13. Protect from the light and put on thorough agitation for 15 min. Check that all bound antibodies have been detached by verifying that no chemiluminescent signal is detected on the ChemiDoc imaging system.
14. Block the membrane with TBS-T 5% dry milk for 30 min at room temperature on gentle shaking.
15. Incubate at room temperature for 1 h with the anti-tubulin antibody diluted into TBS-T 5% dry milk.
16. Wash with TBS-T for 5 min on a shaker and repeat the operation two times.
17. Incubate at room temperature for 1 h with the HRP-conjugated anti-mouse secondary antibody (1/10,000 dilution) into TBS-T 5% dry milk.
18. Wash with TBS-T for 5 minutes on a shaker and repeat the operation two times.
19. Gently dry the membrane using paper towels and place it face up and flat on a sheet of plastic wrap.
20. Directly add 1 mL of the Immobilion Forte western substrate in order to cover the whole membrane.
21. Detect chemiluminescence with the ChemiDoc imaging system (Figure 2). Intensity of the bands is proportional to protein loading before electrophoresis.
22. Quantification of the puromycin and tubulin signals can be performed using the Fiji software (refer to Data analysis section).
  
  Figure 2. SUnSET assay demonstrating a repression of protein synthesis upon treatment with GCN2 kinase inhibitor. HCT116 cells were treated or not for 24 h with a GCN2 kinase inhibitor (GCN2i also named TAP20) and exposed to puromycin (5 μg/mL) 15 min before protein extraction. Protein translation rates were assessed by western blot analysis using an anti-puromycin antibody (Puro). Tubulin is presented here as a loading control. The corresponding molecular weights are indicated on the right of the western blots. Results extracted from Piecyk et al. (2023). Quantification of the data on the left represents the mean ± SEM (n = 3). Unpaired two-tailed t-test with p-value (** p < 0.01).

Data analysis

Open the Fiji software.
Click on the File menu to seek for the ChemiDoc images of the SUnSET experiment.
Use the Rectangular tool to graph a frame around the first smear to quantify and define the region of interest.
In the Analyze menu, select Gels and Select First Lane.
Move the section to the next lane and select Gels and Select Next Lane in the Analyze menu.
Repeat the operation for each lane.
Select Plot Lanes in the Analyze/Gels menu to create each lane profile plot.
Define a closed area for each lane plot using the Straight Line Selection Tool to draw a baseline for each peak.
Measure each peak area clicking inside with the Wand tool.
Report area measurements in a Results sheet.
Repeat this process for tubulin signal quantification.
Normalize: for each lane, calculate the ratio SUnSET area/Tubulin area.
Compare the ratio observed in each lane to assess the impact of studied conditions on protein synthesis rate.

Validation of protocol

This protocol was adapted from the original article: Schmidt et al. (2009). The SUnSET method was performed and validated for in vitro applications in several articles from Dr. Chaveroux’s group (Sarcinelli et al., 2020; Piecyk et al., 2021 and 2023) and other teams in the literature (e.g., Martineau et al., 2014; Mesclon et al., 2017; Arioka et al., 2020; Fong et al., 2021). The SUnSET principle has been adapted for in vivo and ex vivo applications (Goodman et al., 2011; Morral et al., 2020) and at the single-cell level coupled to flow cytometry for energy metabolism assessment by Dr. Pierre’s group (Argüello et al., 2020).

Acknowledgments

This work was supported by the Cancéropôle CLARA (CVPPRCAN000174, CVPPRCAB000180 and CV-2021-039), Region Auvergne Rhone-Alpes (19-010898-01), Institut National Du Cancer (PLBIO22-227), Projets Fondation and Aide doctorale (R16173CC, ARCMD-Doc22021020003295) from ARC, Ligue Nationale contre le Cancer (R17167CC, R19007CC), Institut Convergence François Rabelais (17IA66ANR-PLASCAN-MEHLEN), and the IPR (Innovation Pharmaceutique et Recherche) program. Figure 1 was created with BioRender.com. This protocol was adapted from the original article: Schmidt et al., 2009.

Competing interests

The authors declare no competing interests.

References

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