Fig 2: GJA8 promotes autophagy in HLE cells. (A and B) A shows immunoblot for LC3, and GJA8 from HLE cells in normal complete (NC) media and nutrient starvation media for 4 h (Sta4h). B shows the quantitative analysis of the relative intensity of GJA8 and LC3-II normalized to the TUBG control (n = 3 independent experiments). (C and D) Immunoblot for LC3 from HLE cells in NC media transfected with the different doses of GJA8-Flag. (D) Shows the quantitative analysis of the relative intensity of LC3-II compared to the TUBG control (n = 3 independent experiments). (E-H) E shows representative images for endogenous ATG16L1 and GJA8 in the NC group. (F) shows representative images for endogenous LC3 and GJA8 in NC and Sta4h groups. (G) is the quantitative analysis of the average number of endogenous LC3 puncta per cell. H is the quantitative analysis of the average number of colocalized GJA8 and LC3 puncta per cell (n = 3 wells, 3 independent experiments, > 50 cells per experiment). (I and J) I is representative images of LC3 and GJA8-GFP or GFP in HLE cells transfected with GJA8-GFP or GFP-vector. (J) The quantitative analysis of the average number of LC3 puncta per cell (n = 3 wells, 3 independent experiments, > 50 cells per experiment). Scale bars: 10 µm. Mean ± SEM, N.S., p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001

Fig 3: Autophagy is downregulated in lens fiber cells in gja8b mutant zebrafish. (A and B) Electron micrographs of lens fiber cells (LFC) and lens epithelial cells (LEC) in 36 hpf WT and gja8b mutants. Yellow arrowheads: autophagic vacuoles (AV). Scale bar: 100 nm. B is the number of AVs per area (40 µm2) in the lens of WT and gja8b mutants (n = 60 cells from 6 lenses). (C and D) Immunoblot for endogenous Map1lc3b from WT and gja8b mutant zebrafish. (D) The quantitative analysis of the relative intensity of Map1lc3b-II compared to the Tubg1 (tubulin, gamma 1) control (n = 3 independent experiments). Mean ± SEM, N.S., p > 0.05, *p < 0.05, ****p < 0.0001

Fig 4: 3-MA treatment blocks the rescue effects of rapamycin in gja8b mutant zebrafish lenses. (A) Shows representative images of the distribution of Kdel and Mip, and (B) shows representative images of nuclei and F-actin in the lens of WT and WT zebrafish treated with 4 mM 3-MA at 72 hpf. (C) Average number of lens fiber cell nuclei per lens (n > 30 zebrafish for each group). (D) Statistical analysis of the severity of lens defects in 72 hpf WT, gja8b mutants, gja8b mutants treated with rapamycin, and gja8b mutants treated with 25 μM rapamycin and 4 mM 3-MA (n > 120 zebrafish for each group). (E) Shows representative images of the distribution of Kdel and Mip, and (F) shows representative images of nuclei and F-actin in the lens of gja8b mutants treated with 25 μM rapamycin and gja8b mutants treated with 25 μM rapamycin plus 4 mM 3-MA at 72 hpf. (G) Average number of lens fiber cell nuclei per lens (n > 30 zebrafish for each group). (H) Shows the concentration of endogenous Map1lc3b-I and Map1lc3b-II in WT, WT treated with 4 mM 3-MA, gja8b mutants treated with 4 mM 3-MA, WT treated with 25 μM rapamycin and 4 mM 3-MA, and gja8b mutants treated with 25 μM rapamycin and 4 mM 3-MA. Short exp and long exp: short exposure and long exposure. (I) Shows the quantitative analysis of relative intensity of Map1lc3b-II normalized to the Tubg1 control (n = 3 independent experiments). Scale bars: 50 μm. Mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001