P16 ink4a

Cells were fixed using 4% paraformaldehyde, permeabilized with 0.4% TritonX100, blocked with 1% BSA, and incubated with rhodamine phalloidin (Thermo Fisher, Waltham, MA, USA), anti-GATA6 (Abcam, Cambridge, UK), anti-p53 (Cell Signaling Technology, Beverly, MA, USA), p21(Abcam, Cambridge, UK), p16-INK4A (Proteintech, Rosemont, IL, USA), and ZEB1 (Cell Signaling Technology, Beverly, MA, USA) for 4 h at room temperature. After washing, cells were incubated with an Alexa Fluor 488 (Abcam, Cambridge, UK)-conjugated secondary antibody. Images were analyzed using EVOS M7000 (Invitrogen, Waltham, MA, USA).

Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad). GAPDH antibody was used as control. p16^INK4A, p14^ARF, p21, p53, and GAPDH antibody were purchased from Proteintech (Wuhan, China).

Mice were sacrificed by inhalation of CO2, and eyes were removed to obtain the whole retina. Retinas (n = 6 in each group) were ground in RIPA lysis buffer (Thermo scientific) with 1 mM PMSF and 2 mM EDTA. The lysate was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected. Protein in per sample was 2.5–2.7 mg added with 5× loading buffer for 8 min boiling, loaded on 4–12% SDS-polyacrylamide gels (Beyotime), transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk dissolved in Tris-buffered saline for 2 h at room temperature. Then the membranes were sequentially probed overnight at 4°C with primary antibodies diluted with blocking solution. The primary antibodies were arranged for the following molecules: IRF3 (1:1000, Abcam), P53 (1:1000, Santa Cruz Biotechnology), P16^INK4a (1:1000, Proteintech), PML (1:1000, Santa Cruz Biotechnology), P-H2A.X (1:1000, Cell Signaling Technology), IREα (1:1000, Cell Signaling Technology), GAPDH (1:1,000, Invitrogen). Membranes were washed with TBS-T (pH = 7.5) containing 0.2% Tween for three times, and incubated with appropriate secondary antibody for 90 min at room temperature. The band intensities were measured using exposure machine (Omega Lum^TM G). Quantification of band intensity was analyzed by using ImageJ and GraphPad Prism 6.0.

Cells were lysed in modified RIPA buffer supplemented with protease and phosphatase cocktail inhibitors (Sigma). Whole cell lysate were separated via SDS-PAGE electrophoresis and transferred to 0.2 μM PVDF Western blotting membrane. Membranes were then probed with mouse anti-Yap/Taz (Santa Cruz, 101199, 1:100), rabbit anti-phospho Yap Ser127 (Cell Signaling, #9411, 1:1000) (this antibody can detect murine pYap S112 and pTaz S89^{25 (link)}), pan-Ras (BD Bioscience, #610001, 1:1000), p53 (Cell Signaling, #2524, 1:1000), p16-INK4A (Proteintech, #10883-1-AP, 1:500), small-T-antigen (Millipore, #D01, 1:1000), tubulin (Sigma, #B512, 1:2000), actin (Sigma, #A5060, 1:2000) and Gapdh (Abcam, #Ab8245, 1:10000). Primary antibody binding was then visualised using species-specific conjugated secondary antibodies (Invitrogen) and digital imaging.

The antibodies used for experiments included antibodies against GFP (1:1000; sc-9996; Santa Cruz Biotechnology, Dallas, TX, USA); GST (1:5000; sc-138; Santa Cruz Biotechnology), actin (1:10,000; sc-47778; Santa Cruz Biotechnology), WRN (1:300; GTX101081; GeneTex and 1:300; sc-376182; Santa Cruz Biotechnology), lamin A/C (1:10,000; sc-376248; Santa Cruz Biotechnology), progerin (1:300; sc-81611; Santa Cruz Biotechnology and ab66587; Abcam, Cambridge, UK), Ki67 (1:400; ab15580; Abcam), H3K9me3 (1:400 and 1:2000; ab8898; Abcam), p16-INK4A (1:300; 10,883–1-AP; Proteintech, Rosemont, IL, USA), cyclin B1 (1:200; sc-594; Santa Cruz), cdc25C (1:500; #4688; Cell Signaling technology) and Rad51 (1:200; #05-530; Millipore).

The proteins isolated from exosomes or cells were homogenized in RIPA buffer (Solarbio) and the protein concentration was measured using a BCA protein assay kit (Beyotime, China). The proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, United States). The membranes were incubated in BSA (Solarbio) and then with the antibody TSG101 (67381-1-lg, Proteintech), CD63 (67605-1-lg, Proteintech), Calnexin (Cat No. 10427-2-AP, Proteintech), PCNA (610664, BD, United States), VEGF (2E2H9, Proteintech), p16^INK4A (10883-1-AP, Proteintech), or NRF2 (66504-1-lg, Proteintech) overnight. Secondary antibodies (5230-0336 and 5230-0341, KPL, IN, United States) were added 1 h after washing the PVDF membranes with TBST (Solarbio). Bands were developed using ECL solution (Solarbio) and imaged using a ChemiDoc MP (Bio-Rad).