Fura 3 am

Ca²⁺ levels were determined by the Ca²⁺ indicator Fura 3 AM (Thermo Scientific) using confocal microscopy imaging as described previously. Briefly, the cells were seeded to sterile coverslips and incubated overnight in a humidified chamber to adhere. Cells were or were not treated with Salubrinal (25 µM), Rapamycin (100 nM), and Bafilomycin A1 (100 nM) for 2 h. Cells were then thrice washed with DPBS and exposed to UV-B treatment at 30 mJ/cm² as described earlier and supplemented with fresh DMEM media with indicated concentrations of Salubrinal, Rapamycin, and Bafilomycin as required and incubated further for 6 h post-UV-B irradiation. HDFs were loaded with fluorescent Ca²⁺ indicator dye Fura 3 AM at 5 µM for 45 min before imaging post 6 h UV-B irradiation. Cells were washed three times with live cell imaging solution for imaging using a laser scanning confocal microscope (OLYMPUS FUOVIEW FV1000) by using a 40× objective lens. Five random microscopic fields were selected, and the intensity of fluorescence was quantified using the ImageJ software.

Human primary dermal fibroblast cell line from juvenile foreskin (HDF) and primary fibroblast expansion media was obtained from HiMedia, Mumbai, India. Fetal bovine serum (FBS), penicillin–streptomycin, trypsin–EDTA, 3-(4, 5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT), phosphatase-protease cocktail, RIPA buffer, and H₂DCFDA dye were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). Antibodies against P62, BECN1, phospho-ATM, phospho-ATR, phospho-mTOR, phospho-p53, phospho-Chk1, phospho-Chk2, Bcl-2, phospho-eIF₂α, eIF₂α, LC3B, phospho-AMPKα, AMPK, and phospho-χH₂AX were purchased from Cell Signaling Technology, Danvers, MA. Antibodies against GRP78, PTEN, CHOP/GAD153, DDB2, phospho-AKT, and siRNA P62/Atg7 and secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Fura 3 AM, DAPI, ER tracker, Acridine Orange, Ethidium Bromide, Salubrinal, Rapamycin, Chloroquine, Bafilomycin A1, Everolimus, and GFP-RFP-LC3B puncta assay kit were purchased from Thermo Scientific. Antibodies against P21 and P27, TUNEL assay, and CPD ELISA kits were procured from Abcam. Bradford reagent was obtained from Sigma-Aldrich. PVDF membrane was purchased from Bio-Rad, Hercules, CA. Antibody against β-actin was purchased from Sigma-Aldrich.

CD4 T cells (1 × 10⁷) were loaded with a mixture of 4 μM Fura-3AM (Molecular Probes, USA) and 0.7 mg/ml of Probenecid (Sigma-Aldrich) at 37°C for 30 min. After washing, cells (1 × 10⁵) were analyzed in a fluorescence microscope (Nikon Inverted Microscope, Japan) to determine the fluorescence intensity. Intracellular calcium was determined by calculating the corrected total cell fluorescence (CTCF). CTCF = integrated density—(area of selected cell × mean fluorescence of background readings).