Measurement of autophagy via LC3 western blotting following DNA-damage-induced senescence

Summary Senescent cells accumulation is associated with aging and age-related diseases, and recent findings suggest that autophagy, the activity of the intracellular degradation system, decreases during senescence. In this protocol, we detail steps to induce cellular senescence in response to DNA damage, evaluate the senescent state using SA-β-gal staining and western blot for p21, LAMP1, and Lamin B1, and detect autophagy via LC3 western blotting. This protocol can be used in most cell lines and for various types of senescent cells. For complete details on the use and execution of this protocol, please refer to Yamamoto-Imoto et al. (2022).

SUMMARY Senescent cells accumulation is associated with aging and age-related diseases, and recent findings suggest that autophagy, the activity of the intracellular degradation system, decreases during senescence. In this protocol, we detail steps to induce cellular senescence in response to DNA damage, evaluate the senescent state using SA-b-gal staining and western blot for p21, LAMP1, and Lamin B1, and detect autophagy via LC3 western blotting. This protocol can be used in most cell lines and for various types of senescent cells. For complete details on the use and execution of this protocol, please refer to Yamamoto-Imoto et al. (2022).

BEFORE YOU BEGIN
CRITICAL: All materials for cell culture need to be sterile, and all sterile procedures handling cells need to be performed in a Class II biological safety cabinet under standard aseptic techniques.

Cell preparation
Timing: 1 day 1. Passage the proliferative human retinal pigment epithelial cell line (hRPE1 cells) using Trypsin-EDTA solution. 2. Plate the cells to evaluate senescent state.
ii. Put micro cover glass (0.16-0.19 mm thickness) in 35 mm culture dish (Thermo Fisher Scientific, 150460). iii. Coat the dish with 2 mL of Cellmatrix Type I-C solution. iv. Incubate the dish for 10 min at room temperature. v. Wash the dish twice with 2 mL of PBS, and remove the wash solution.
vi. Plate the cells on the dish at the density of 0.5 3 10 5 cells per dish. b. For SDS-PAGE and Western blot.
i. Plate the cells on 6-well dish (Nuncä Cell-Culture Treated Multidishes, Thermo Fisher Scientific, 140675) at the density of 0.5-1.0 3 10 5 cells per well. 3. Incubate the cells for 24 h at 37 C with 5% CO 2 in a humidified chamber.
Note: Nomal cells need to be seeded at the density of 0.5 3 10 5 cells per dish or well 3 days before sample preparation to evaluate the senescent state (step 4) or detection of autophagic activity (step 19) in step-by-step method details.
Note: Bring PBS and culture medium to room temperature (20 C-22 C) before starting cell culture. Note: Protease inhibitors including cOmplete and PMSF should be added just before use.

Reagent preparation
Alternatives: Other protease inhibitors and detergents can be used. PhosSTOP TM (04906845001; Roche) can be added to the lysis buffer to assess the expression levels of phosphorylated protein such as Rb, NF-kB, and AMP-activated protein kinase as senescence markers.
Note: Bring this buffer to room temperature (20 C-22 C) before mixing with samples.

STEP-BY-STEP METHOD DETAILS
Induction of cellular senescence as response to DNA damage Timing: 5 or 10 days This step should be carried out at 40%-50% confluency. 3. Add 2 mL of the doxorubicin solution per well, and maintained at 37 C with 5% CO 2 in a humidified chamber for 5 or 10 days. No need to change the culture medium during the treatment. Troubleshooting 1.
CRITICAL: Although cellular senescence is induced in cells treated with doxorubicin for 5 days as seen in the senescence markers including cyclin-dependent kinase inhibitor p21, lysosome-associated membrane protein 1 (LAMP1), and Lamin B1 in Figure 2, SAb-gal positivity is still not fully increased compared with cells treated for 10 days (Figure 1) (Yamamoto-Imoto et al., 2022). Therefore, we recommend to investigate in both situations to decide which is suitable state for your experiments. Duration of doxorubicin treatment can be changed if the possibility remains that alternative time points could lead to differences in your data.

Sample preparation to evaluate the senescent state
Timing: 1 h Before exploring autophagic activity, senescent state should be determined by SA-b-gal staining and Western blot for p21, LAMP1, and Lamin B1.
Note: Since senescent cells show enlarged cell size (Bent et al., 2016;Druelle et al., 2016), observing using normal microscopy can easily help to determine when to evaluate senescent state. These morphological changes are seen in Figure 1  i. Wash each well twice with 2 mL of cold-PBS, then remove the wash solution.
ii. Add 25 mL of lysis buffer and scrape each well on ice.
Note: The amount of lysis buffer depends on the cell number. A suggested amount of lysis buffer is 25-100 mL.
iii. Pipette 7 times to lyse well, and then transfer to a microcentrifuge tube. iv. Centrifugation at 20,000 3 g for 15 min at 4 C. v. Transfer the supernatant to a new microcentrifuge tube. vi. Measure the protein concentration using Pierce BCA Protein Assay Kit as described in https://www.thermofisher.com/order/catalog/product/23225. vii. Adjust the protein concentration among all samples using lysis buffer and mix well with 53 SDS-sample buffer. viii. Heat them at 95 C for 7 min, and then place the tube at room temperature for 5 min.
Pause point: Samples at steps 4.b.v. and 4.b.viii. can be stored at %À80 C and %À20 C until use, respectively.

Evaluation of the senescent state SA-b-gal staining
Timing: 2 days 5. Transfer the micro cover glass with the fixed cells from step 4.a.iii. into 24-well plate filled with PBS. 6. Remove the PBS. 7. Add 1 mL of Staining mixture including X-gal Solution (Sigma, CS00030). 8. Seal the plate with Parafilm. 9. Incubate the cells at 37 C for 16-20 h without CO 2 .
Note: Appropriate staining time should to be optimized in each types of cell using microscope.
10. Wash the cells three times with 1 mL of PBS. 11. Cover with Mounting Medium with DAPI (VECTASHIELD, H-1200) and observe the cells on DIC and DAPI images with Olympus cellSens Standard Imaging Software using microscope (1003 magnification, BX53, Olympus). Count more than 200 DAPI-positive cells manually, and calculate the percentage of SA-b-gal-positive cells per condition in each experiment. Please refer to (Yamamoto-Imoto et al., 2022). Troubleshooting 2.

SDS-PAGE and western blot
Timing: 2 days For more details, please see Yamamoto-Imoto et al. (2022).
12. Lysates are run on reducing SDS-PAGE gels.

OPEN ACCESS
Note: Load 10-20 mg of total protein, but less amount of total protein can be used when senescence is markedly induced.
13. Blot the protein to PVDF membrane and stained with Ponceau S to confirm the proteins are equally blotted among samples. 14. Blocking with 1% skim milk for 30 min at room temperature. 15. Incubated overnight at 4 C in primary antibody. 16. Incubated with appropriate HRP-conjugated secondary antibody at room temperature for an hour. 17. Blots are developed with Immobilon Forte Western HRP Substrate or ImmunoStar LD. 18. Signals are detected in ChemiDocTM Touch Imaging System (Bio-Rad). Troubleshooting 3.
Note: After washing the membrane with TBST and ddH 2 O, dried membrane can be stored at room temperature to reuse for detecting other senescence markers.

Detection of autophagic activity
Timing: 3 h Although this protocol describes the specific steps to assess autophagy in DNA damage-induced senescent cells, we have also used this protocol in the replicative senescence model. It is definitely important to compare the expression of LC3-II between the cells treated with or without bafilomycin A1, which is the inhibitor of the fusion of autophagosome and lysosome as described in (Mizushima et al., 2010). Although confirming the LC3 expression level using microscope might be one of the indicators related to the autophagic activity, it still remain unclear that increased levels of LC3-II is due to the upregulation of autophagosome formation or inhibition of autophagic degradation of LC3-II.
19. Dilute the bafilomycin A1 stock solution or DMSO 1:1250 in culture medium to obtain the bafilomycin A1 (the final concentration is 200 nM) or DMSO solution. 20. Wash each well twice with 2 mL of PBS, then remove the wash solution. 21. Add 2 mL of culture medium with bafilomycin A1 or DMSO solution. 22. Incubate the cells for 2 h at 37 C with 5% CO 2 in a humidified chamber. 23. Prepare the samples by the same method as step. 4.b. 24. Perform SDS-PAGE and Western blot for LC3 as described above. Troubleshooting 4.
Note: Performing SDS-PAGE in 15% gel is better to detect LC3-II.
Optional: Increased levels of Rubicon can be the useful indicator of decreased autophagic activity (Yamamoto-Imoto et al., 2022;Matsunaga et al., 2009).
Optional: Measuring the expression levels of p62, which interact with autophagic substrates, with or without bafilomycin A1 is also used to explore autophagic activity.

EXPECTED OUTCOMES
SA-b-gal positivity is increased in DNA damage-induced senescent cells induced by doxorubicin treatment for 5 or 10 days (Figure 1). In addition, increased levels of p21 and LAMP1, and decreased expression of Lamin B1 can be detected in senescent cells (Figure 2). LC3 autophagic flux, determined by subtracting LC3-II expression in the samples treated with bafilomycin A1 from the ones without the inhibitor, can be decreased in senescent cells compared with non-induced cells ( Figure 3).

LIMITATIONS
Since the long-term treatment of bafilomycin A1 triggers premature senescence as seen in the upregulated levels of p21, p53, and SA-b-gal positivity, and mitochondrial dysfunction determined by increased mitochondrial superoxide (Takenaka et al., 2022), treatment duration of bafilomycin A1 needs to be shorter as possible.

TROUBLESHOOTING Problem 1
Poor cells after the doxorubicin treatment for 10 days (step 3).

Potential solution
Spread cells to be able to maintain a confluence of 80% after the senescence induction. Please note that since DNA-damage drives drastic changes in cell size and morphology, it is not recommended to spread too many cells before the induction of senescence.

Potential solution
Optimize the concentration and duration of the doxorubicin treatment in each types of cell. Use the appropriate Fixation Buffer, do not use 4% paraformaldehyde to fix the cell. If cells were fixed with 4% paraformaldehyde, it may be hard to see the cells coloring blue even after incubation for 20 h. Incubate the cells longer with Staining mixture at 37 C without CO 2 and observe well using microscope.

Problem 3
Expression levels of senescent markers determined by Western blot are not altered (steps 4b, 12-18).

Potential solution
Confirm the concentration of loading protein and primary antibody in SDS-PAGE and Western blot. Other hallmarks of senescent state, such as p16 and senescence-associated secretory phenotype (SASP) (Gorgoulis et al., 2019), need to be addressed. Strongly recommend to optimize the concentration and duration of the doxorubicin treatment if both Problem 2 and 3 occurred.

Potential solution
Concentration and appropriate duration of the bafilomycin A1 treatment need to be optimized. Please note that the treatment duration of bafilomycin A1 should be shorter as described in Limitation above. Other lysosomal inhibitors such as chloroquine can be used. Performing SDS-PAGE in 15% gel is better to detect LC3-II. Tandem fluorescent-tagged LC3 is the useful alternative to determine the autophagic activity. Please refer to (Kimura et al., 2007) for complete details.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shuhei Nakamura (shuhei.nakamura@fbs.osaka-u.ac.jp).

Materials availability
This study did not generate new unique reagents. Relative intensity to non-induced cells are presented as the mean G SD. Statistical analyses were performed using an unpaired t-test.

Data and code availability
This study did not generate and analyze any original datasets/codes.