SUrface SEnsing of Translation (SUnSET), a Method Based on Western Blot Assessing Protein Synthesis Rates in vitro (2024)

Abstract

Keywords: SUnSET, Protein synthesis, Translation rates, Puromycin, Alternative to radioactivity

Background

As a structural analogue of aminoacyl-tRNA, the aminonucleoside antibiotic puromycinis incorporated through non-hydrolysable peptide bounding into the growing peptidechain along the elongation process (Nathans, 1964).While high concentrations block the elongation phase and hence translation, at lowdoses the overall translation rate of protein synthesis remains unchanged.Consequently, the rate of formation of puromycin-labeled peptides mirrors the rateof protein translation. Taking advantage of this property, 3H-puromycin labeling wasfirst used in 1979 to assess the rate of protein synthesis in various tissues invivo under nutrient- and protein-poor diets (Nakanoand Hara, 1979). The SUnSET (SUrface SEnsing of Translation) technique wasdeveloped 30 years later by Schmidt and colleagues to detect variations in proteinsynthesis rates in cultured cells by western blotting (Schmidt et al., 2009). This method was then coupled with othertechniques (fluorescence-activated cell sorting or immunohistochemistry) to assessprotein synthesis at different scales. Further developments, notably based on theClik-it technology combined with O-propargyl puromycin (OP-Puro), a puromycinanalogue, enable visualization of puromycilated proteins and assessment ofelongation rates in tissues (Morral et al., 2020).The main advantage of using puromycin and its analogues is that it does not requireradioactive isotopes such as 35S-methionine, historically used to measure proteinsynthesis rates, with comparable analytical performance (Schmidt et al., 2009). Because proteostasis defects areassociated with a variety of chronic diseases (cancer, tissue fibrosis, inflammatorysyndromes, etc.) or aging, it is necessary to monitor the changes in proteinsynthesis rates in response to a variety of stressors in order to better understandthe translational reprogramming underlying cell adaptation. The following protocoldescribes a SUnSET method (Figure1) suitable for assessing protein synthesis rates in lysates from cells grownin vitro by conventional western blot analysis.

Materials and reagents

Biological materials

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

Reagents

1. Puromycin (Sigma-Aldrich, catalog number: P9620)

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2. PBS (Sigma-Aldrich, catalog number: D1408)

3. Complete anti-protease (Roche, catalog number: 11836145001)

4. Dry milk (Régilait, catalog number: 304934416704)

5. Bovine serum albumin (BSA) (Roche, catalog number: 10735094001)

6. DC Protein Assay kit (Bio-Rad, catalog number: 5000111)

7. Prestained protein ladder (Euromedex, catalog number: 06P-0111)

8. Immobilion Forte western substrate (Merck Millipore, catalog number:WBLUF0500)

9. Ponceau red solution (Sigma-Aldrich, catalog number: 141194)

10. Mouse anti-puromycin antibody (clone 12D10) (Merck Millipore, catalog number:MABE343)

11. Mouse anti-tubulin antibody (clone DM1A) (Sigma, catalog number: T6199)

12. HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology,catalog number: 7076)

13. Stripping buffer (Thermo Scientific, catalog number: 21059)

Solutions

1. RIPA protein lysis buffer 2× (see Recipes)

2. Tris-buffered saline-Tween (TBS-T) (see Recipes)

   1. TBS-T 5% BSA (see Recipes)

   2. TBS-T 5% dry milk (see Recipes)

3. Laemmli 6× (see Recipes)

Recipes

1. RIPA protein lysis buffer 2×

   Reagent	Finalconcentration
   Tris-HClpH 7.2	100mM
   NaCl	300mM
   EDTA	10mM
   Sodiumdeoxycholate	2%
   SDS20%	0.1%
   Triton100×	2×
   Na3VO4	4mM
   βglycerophosphate	20mM
   NaF	20mM
   Completeanti-protease	2×

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   Dilute to 1× in H₂O.

2. Tris-buffered saline-Tween (TBS-T)

   Reagent	Finalconcentration
   Tris-HClpH 7.5	50mM
   NaCl Tween	150 mM 0.1%

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   Add 5% of BSA or dry milk for obtaining TBS-T 5% BSA or 5% dry milk,respectively.

3. Laemmli 6×

   Reagent	Finalconcentration
   Tris-HClpH 6.8	0.5M
   Glycerol	1%
   SDS20%	2%
   DTT	0.6M
   Bromophenolblue	0.4%

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Laboratory supplies

1. 6-well or 10 cm diameter tissue culture plates

2. Sterile scrapers

3. Microcentrifuge tubes

4. Plastic wrap

Equipment

1. Tissue culture apparatus (tissue culture hood, CO₂ incubator,etc.)

2. Pipettes and micropipettes

3. Vacuum pump

4. Centrifuge (4 °C)

5. Cold room

6. Heat block

7. Western blotting apparatus (SDS-PAGE running cassette, power supply, shaker,transfer cassette, nitrocellulose membrane, etc.)

8. ChemiDoc imaging system (Bio-Rad, catalog number: 12003153)

Software and datasets

1. Fiji (National Institutes of Health)

Procedure

1. Puromycin incorporation in vitro

   1. Seed HTC116 cells one day before the experiment and maintain at 37°C and 5% CO₂ to allow attachment to the tissueculture plate. In the experiment presented in Figure 2, 200,000 cellsper well were seeded in a 6-well plate.

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      Figure 2.

      SUnSET assay demonstrating a repression of proteinsynthesis upon treatment with GCN2 kinase inhibitor.

      HCT116 cells were treated or not for 24 h with a GCN2kinase inhibitor (GCN2i also named TAP20) and exposed topuromycin (5 μg/mL) 15 min before protein extraction.Protein translation rates were assessed by western blotanalysis using an anti-puromycin antibody (Puro).Tubulin is presented here as a loading control. Thecorresponding molecular weights are indicated on theright of the western blots. Results extracted fromPiecyk et al. (2023). Quantification of the data on theleft represents the mean ± SEM (n = 3). Unpairedtwo-tailed t-test with p-value (** p < 0.01).

   2. On the day of the experiment, apply the studied treatment on cellsfor the desired time.

   3. Before harvesting and 15 min before the end of the treatment, exposecells to 5 μg/mL puromycin directly diluted in the media. Allsamples must be incubated with the same concentration of puromycinfor an equal period.

2. Protein extracts

   1. At the end of the 15-min incubation with puromycin, remove media andrinse cells once with cold PBS.

   2. Put the tissue culture plate on ice and incubate with 1× RIPAprotein lysis buffer (see Recipes) containing proteases andphosphatases inhibitors (1 volume of 1× RIPA for 1 volume ofcell 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 °Cto get rid of cellular debris.

   5. Collect the supernatant in a new microcentrifuge tube and add theappropriate volume of Laemmli 6× (see Recipes).

   6. Dose the amounts of extracted proteins using the DC Protein Assay kitaccording to manufacturer’s instructions.

   7. Add the appropriate volume of Laemmli 1× to normalize proteinconcentrations in all samples and denaturate by heating at 95 °Cfor 5 min.

3. 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 toestimate the molecular weight of visualized proteins.

   2. When separated, transfer proteins onto nitrocellulose membranes as astandard western blot protocol. Stain with Ponceau red solution tocheck equal protein amount loading before electrophoresis.

   3. Block the membrane with TBS-T 5% dry milk for 1 h at room temperaturewith gentle shaking.

   4. Wash with TBS-T for 5 min on a shaker and repeat the operation twotimes.

   5. Incubate overnight at 4 °C on a gentle shaker with puromycinantibody 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 twotimes.

   7. Incubate at room temperature for 1 h with the HRP-conjugatedanti-mouse secondary antibody (1/10,000 dilution) into TBS-T 5% drymilk.

   8. Wash with TBS-T for 5 min on a shaker and repeat the operation twotimes.

   9. Gently dry the membrane using paper towels and place it face up andflat on a sheet of plastic wrap.

   10. Directly add the Immobilion Forte western substrate onto the wholemembrane.

   11. Detect chemiluminescence with the ChemiDoc imaging system (Figure 2).Intensity of the smear depends on cell ability to incorporatepuromycin 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 thatno chemiluminescent signal is detected on the ChemiDoc imagingsystem.

   14. Block the membrane with TBS-T 5% dry milk for 30 min at roomtemperature on gentle shaking.

   15. Incubate at room temperature for 1 h with the anti-tubulin antibodydiluted into TBS-T 5% dry milk.

   16. Wash with TBS-T for 5 min on a shaker and repeat the operation twotimes.

   17. Incubate at room temperature for 1 h with the HRP-conjugatedanti-mouse secondary antibody (1/10,000 dilution) into TBS-T 5% drymilk.

   18. Wash with TBS-T for 5 minutes on a shaker and repeat the operationtwo times.

   19. Gently dry the membrane using paper towels and place it face up andflat on a sheet of plastic wrap.

   20. Directly add 1 mL of the Immobilion Forte western substrate in orderto cover the whole membrane.

   21. Detect chemiluminescence with the ChemiDoc imaging system (Figure 2).Intensity of the bands is proportional to protein loading beforeelectrophoresis.

   22. Quantification of the puromycin and tubulin signals can be performedusing the Fiji software (refer to Data analysis section).

Data analysis

1. Open the Fiji software.

2. Click on the File menu to seek for the ChemiDoc images ofthe SUnSET experiment.

3. Use the Rectangular tool to graph a frame around the first smear to quantifyand define the region of interest.

4. In the Analyze menu, select Gels and SelectFirst Lane.

5. Move the section to the next lane and select Gels and SelectNext Lane in the Analyze menu.

6. Repeat the operation for each lane.

7. Select Plot Lanes in the Analyze/Gels menuto create each lane profile plot.

8. Define a closed area for each lane plot using the Straight LineSelection Tool to draw a baseline for each peak.

9. Measure each peak area clicking inside with the Wand tool.

10. Report area measurements in a Results sheet.

11. Repeat this process for tubulin signal quantification.

12. Normalize: for each lane, calculate the ratio SUnSET area/Tubulin area.

13. Compare the ratio observed in each lane to assess the impact of studiedconditions on protein synthesis rate.

Validation of protocol

This protocol was adapted from the original article: Schmidt et al. (2009). TheSUnSET method was performed and validated for in vitro applications in severalarticles 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 principlehas been adapted for in vivo and ex vivo applications (Goodman et al., 2011; Morral etal., 2020) and at the single-cell level coupled to flow cytometry for energymetabolism assessment by Dr. Pierre’s group (Argüelloet 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 wascreated with BioRender.com. This protocol was adapted from the original article:Schmidt et al., 2009.

Competing interests

The authors declare no competing interests.

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