Lc3 2775

At indicated times after PDT, cells were lysed in a lysis buffer (50 mM HEPES pH 7.4, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 5 mM EDTA) supplemented with protease inhibitors (Roche, Mannheim, Germany). After measuring protein concentration using Protein Assay (Bio-Rad, Hercules, CA, USA), equal amounts of proteins were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane (Schleicher and Schuell BioScience, Dassel, Germany). Membranes were incubated with following primary antibodies: LC3–2775, ATG5–9980, caspase 3–9665, PARP-9542 (Cell Signalling, Beverly, MA, USA) according to the manufacturer’s recommendations. After TBST washing, membranes were probed with HRP-linked secondary antibodies (Cell Signaling), developed with self-made ECL (50 mM Tris-HCl pH 8.5, 0.2 mM coumaric acid, 1.25 mM luminol, 0.006% hydrogen peroxide) or Supersignal West Femto ECL (Thermo Scientific, Waltham, MA, USA) and visualized with Stella 8300 bio-imager (Raytest, Straubenhardt, Germany) or ChemiDoc Touch (Bio-Rad). To ensure equal protein loading, the membranes were reprobed with anti-β-actin antibody (Sigma-Aldrich, Saint Louis, MO, USA: A2228).

HLE cells were adhered overnight in 24-well plates to grow to 80% cell density, then transfection with or without 1.0 μg/well peGFP-C1-GJA8 plasmid for 24 h, and washed with PBS for twice, replaced with no fetal calf serum media for 2 h or 4 h (Sta2h/Sta4h group) or complete medium (NC group) for 2 h. Then the cells were washed with PBS for twice, and fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS for three times. Then, cells were blocked with 2% BSA in PBST (0.1% Triton X-100 in PBS) for 1 h and incubated with primary antibody for 2 h [GJA8(sc-50432) 1:200 from Santa Cruz, LC3(2775) 1:200 from Cell Signaling Technology], and washed with PBST for triple; second antibody for 1 h (Alexa Fluor488-conjugated antibodies, Alexa Fluor555-conjugated antibodies, and DAPI from Life Technologies), and washed with PBST for triple. Images were captured using an Olympus FluoView 1000 confocal microscope at 40 ×.
The GFP plasmid vector was used to test the possible alterations of the autophagic proteins in the IF, and no differences between in the GFP plasmid vector and the normal control were observed (Supplementary Figure S1). Thus, in our paper, we use the normal group (the NC group) as the control to explain the alteration of autophagic proteins with the overexpression of GJA8, and to prove that the autophagy process occurred in human lens epithelial (HLE) cells.