Glass bottom cell culture dishes

Cells were cultured on Glass Bottom Cell Culture Dishes (NEST Biotechnology, Wuxi, China) for 12 h, fixed using Immunol Staining Fix Solution (Beyotime Institute of Biotechnology, China) for 10 min at room temperature, and blocked using Immunol Staining Blocking Buffer (Beyotime Institute of Biotechnology, China) for 1 h at room temperature. The cells were incubated in presence of pSmad3 primary antibody (1:100, Ser^423/425, clone C25A9, Cell Signaling Technology, Danvers, USA) and Flag primary antibody (1:10000, Medical & Biological Laboratories, Co., Ltd. Nagoya, Japan) overnight at 4 ^°C and then were washed three times with PBS followed by a 1 h incubation in the dark with the secondary antibodies coupled to Alexa Fluor 647 (Abcam, Cambridge, UK) or Alexa Fluor 594 (Abcam, Cambridge, UK), respectively. After another three washes with PBS, nuclei were stained by ProLong® Gold Antifade Mountant with DAPI (P36941, life technologies corporation, USA). The cells were observed under a confocal laser microscopy (LSM780, Carl Zeiss, Oberkochen, Germany).

RPMI-1640, MEM, phosphate-buffered saline, RIPA buffer and the penicillin-streptomycin antibody cocktail solution were obtained from Welgene, Korea. FBS and trypsin were procured from Hyclone (GE Healthcare Life Sciences), and the antibody cocktail used in this study (penicillin and streptomycin) was procured from Gibco, Korea. Nitric oxide and peroxide determination kit were procured from Bioassay Systems, USA. FGM-2, bulletkit was obtained from Lonza, NJ, USA. RNAiso plus (Trizol) was purchased from Takara, Japan. Glass bottom cell culture dishes were purchased from NEST, New Jersey, USA. Nitrocellulose MEMbrane, Isopropanol and Triton X-100 were purchased from Thermofisher, Korea. Diethyl pyrocarbonate (DEPC) and 4% Paraformaldehyde were obtained from Biosesang, Korea. The nucleus staining DAPI dye was procured from Sigma Aldrich, Korea. Anti-rabbit monoclonal cleaved caspase-3 primary antibody was purchased from cell signalling technologies, USA and anti-mouse- GAPDH antibody was procured from Bio-Rad, USA. The ReverTra Ace^® qPCR RT Master Mix cDNA synthesis kit was purchased from Toyobo, Japan. The SYBR Green Master mix was procured from Bio-Rad, Korea. All the primers for real-time polymerase chain reaction (q-PCR) analysis were obtained from Searchbio, Korea.

Intracellular Ca²⁺ was measured using the fluorescence dye Fluo-3/AM according to a modified procedure described in a previous report [41 (link)]. The conjunctival epithelium from the rabbits was digested and harvested with 1 mg/mL dispase-II solution for approximately 1 h, seeded in DMEM/F12 medium on glass-bottom cell culture dishes (20 mm, NEST, Wuxi, China), and cultured at 37 °C. After 10 min of cell adhesion, the cells were prepared and loaded with the fluorescent dye Fluo-3/AM (Molecular Probes, Eugene, OR, USA). The fluorescence signal was monitored and recorded using a laser-scanning confocal imaging system (TCS SP2; Leica Microsystems, Wetzlar, Germany). The change in fluorescence intensity after ATP treatment was normalized to the initial intensity.

RSV, PA, Protein Carbonyl Content Assay Kit, NAC and MitoTEMPO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan), and glass-bottom cell culture dishes were obtained from Nest Biotechnology (Wuxi, China). MitoTracker Deep Red and MitoSOX Red were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies against MHC (ab 24642), PGC-1α (ab 106814), TFAM (ab 131607), mfn2 (ab 50838), FoxO3a (ab 23683) and VDAC1 (ab 15895) were purchased from Abcam (Cambridge, UK). Antibodies against LKB1 (3047), p-LKB1 (3482), AMPK (2532), p-AMPK (2531), p-mTOR (2971), mTOR (2972), p-S6K (9205), S6K (9202) and GAPDH (5174) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against PKA (sc-903), p-PKA (sc-12905), atrogin-1 (sc-33782) and MuRF1 (sc-27642) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against drp1 (BD611113) was obtained from BD Biosciences (San Jose, CA, USA).

HaCaT cells were incubated with HBLV-mRFP-GFP-LC3 adenovirus (Hanhbio, Shanghai, China) for 24 h and screened with puromycin. The transduced cells were cultured in glass bottom cell culture dishes (NEST, Wuxi, China) and treated with 100 nM of sodium arsenite for 4 h or 30 μM of CQ for 6 h. Pictures were acquired with AI⁺ confocal microscopy (Nikon, Tokyo, Japan).