Casp3

Passage 2–3 chondrocytes were used for protein extraction. The method for collagen type II (COL2A1) extraction was as described previously [27 (link)]. Briefly, chondrocytes were washed with PBS three times and lysed with RIPA lysis buffer (Beyotime, China) supplemented with 1 mM protease inhibitor cocktail and 1 mM phosphatase inhibitor cocktail (Thermo, USA). The mixture was homogenized and lysed on ice and centrifuged at 12000 r/min for 30 min, 4 °C. The resulting supernatant was collected. Protein concentration was determined by the BCA method. The same amount of protein sample was electrophoresed and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with 5% skim milk for 1 h at room temperature, and incubated at 4 °C overnight with the primary antibody against COL2A1 (Abcam, UK), MMP-13 (Abcam, UK), CGRP (GeneTex, USA), iNOS (GeneTex, USA), COL1A1 (Abcam, UK), TGFβ1 (Abcam, UK), PCNA (Abcam, UK), Casp3 (Abcam, UK), and GAPDH (Abclonal, USA). After washing, the membrane was incubated with the secondary antibodies (Abcam, UK) at room temperature for 1 h. Integrated density for protein bands was determined using Tanon 5200 (Tanon Science &Technology, China).

ExoQuick-TC exosome-isolation reagent (EXOTC50A-1), SeraMir exosome RNA-isolation kit (RA808A-1), exosome-specific marker anti-αCD63 antibody (EXOABCD63A-1), and florescence exosome-preparation reagent (EXDC300A-1) were from System Biosciences (SBI, Palo Alto, CA, USA). Penicillin-streptomycin (PS) solution, trypsin-EDTA solution, RPMI medium, and α-MEM media were obtained from Life Technologies GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used for immunoblotting were against phosphorylated p53 (Cell Signaling Technology, Beverly, MA, USA), BAX, BCL2, CASP9, CASP3, β-actin (Abcam, Cambridge, MA), and cytochrome c (ENZO Diagnostics Inc., Farmingdale, NY, USA).

After incubating with 10% FBS, the sections were incubated with primary antibodies overnight at 4°C, then, the primary antibodies were replaced by secondary antibody labels and cell nuclei were stained with DAPI. Primary antibodies: α-SMA (cat. no. Ab5694; dilution: 1:50; Abcam), CASP3 (cat. no. ab32351; dilution: 1:50; Abcam), IL-1β (cat. no. ab254360; dilution: 1:50; Abcam). Secondary antibodies: Alexa Fluor^® 568-conjugated goat anti-rabbit immunoglobulin G (cat. no. A-11034; dilution, 1:250; Thermo Fisher Scientific), and Alexa Fluor^® 488-conjugated goat anti-mouse immunoglobulin G (cat. no. A32727; dilution, 1:250; Thermo Fisher Scientific). Images were obtained by E2000U microscope (Nikon Corporation, Tokyo, Japan).

The proteins lysed from left ventricle were separated by 10% SDS-polyacrylamide gel and transferred to PVDF membranes using a semidry transferring method. The membranes were sharked for 1 h and incubated with specific primary antibodies at 4°C overnight. The next day, the membranes were incubated in horseradish peroxidase- (HRP-) labeled secondary antibodies (Beyotime, Nanjing, China), colored with fluorescent substrates and captured on the Bio-Rad system. Antibodies against collagen I, collagen III, p-P65, Bcl-2, Bax, CASP-3, GAPDH, and histone H3 were purchased from Abcam (Cambridge, MA, USA). Antibodies against p-ERK, p-JNK, p-P38, TGF-β1, α-SMA, MMP9, and TIMP-1 were obtained from Cell Signaling Technology (Danvers, MA, USA). GAPDH and histone H3 were used as internal controls.

For histological analysis, the spinal cord specimens were managed the same as above reported. Immunofluorescent staining was performed on 20‐μm sections, which were stained with the following antibodies: green fluorescence protein (GFP, 1:200, A10262, Molecular probes); neurofilament (NF, 1:200, Ab8135, Abcam); caspase 3 (Casp3, 1:200, Abcam); neuron‐specific marker (NeuN, 1:200, Ab104224, Abcam), ß‐tubulin III (Tubulin; 1:100, T8578, Sigma); glial fibrillary acidic protein (GFAP, 1:400, Ab10062, Abcam); oligodendrocyte‐specific antibody (O4, 1:200, Ab7474, Abcam); and 4′,6‐diamidino‐2‐phenylindole (DAPI, 1:1000, D1306, Invitrogen). Sections of spinal cord were imaged at 10×, 20×, and 40× magnifications using a confocal microscope (Leica SP5, Germany). GFP staining was performed to check the survival of the BMSCs. NF staining was used to detect axon growth in the injured area. NeuN staining was performed to detect the spared neurons in the area adjacent to the lesion. Double immunofluorescent assay (GFP/Tubulin, GFP/GFAP, GFP/O4) was performed to determine whether the GFP⁺ cells (include the BMSCs) differentiate into other types of cell.

Primary keratinocytes were seeded in a 12-well plate at 100,000 cells/well in LabTek Chambered -Microscopic slides (Thermofisher, Australia) and exposed to UV and/or heat stress. For immunocytochemistry, cells were fixed with 4% paraformaldehyde, washed twice with TBS. Skin tissues were processed and stained according to previous protocols [14 (link)]. Primary NHEK and skin sections were incubated with antibodies to either thymine dimer (CPD) (mouse monoclonal, 1:500 dilution; Kamiya Biomedical, USA), p53 antibody (rabbit monoclonal 1:50 dilution; Abcam, USA), p53 acetyl K382 (rabbit monoclonal 1:200, Abcam USA), SIRT1, SIRT1-p, Casp-3 (cleaved), Survivin or anti-pan Cytokeratin and ki67 (rabbit polyclonal, 1:50 dilution; Abcam, USA). The mouse monoclonal antibody to pan-cytokeratin (1:50; Abcam, USA) was used to label keratinocytes. The secondary antibodies anti-mouse Alexa Fluor 488 (for keratin and CPD) and anti-rabbit Alexa Fluor 550 (for caspase 3, p53 and ki67) were used for detection. Three sections were analysed per exposure replicate and five images of randomly selected fields-of-view were captured from each section. To determine percentages of keratinocytes positive for each individual antibody, positive cells were quantified within 5 images randomly chosen per section.