Figure 5

Autophagy markers in iPSC-derived rod photoreceptor cells. Immunostaining of pNrl-EGFP-positive cells (green: A, D, G and J) and immunocytochemistry using anti-LC3 antibody (magenta: B, E, H and K), and their merged images (C, F, I, L). Arrowheads indicated pNrl-EGFP- and LC3-double positive cells. Scale bar: 20 μm. The autophagy marker molecules LC3 (M, P), ATG5 (N, Q), and ATG7 (O, R) were analyzed using real-time PCR in the pNrl-EGFP-positive rod photoreceptor cells derived from each iPSC line in the absence of treatment (M-O) and in #5 iPSC-derived cells following therapeutic treatment (P-R), both at 5 weeks of culture. Autophagy was suppressed in the #5Rw and B7 iPSC-derived cells and in the #5 iPSC-derived cells with each treatment. N = 3 for all. Rapa., rapamycin; Salub., salubrinal. *p < 0.05, ***p < 0.001. Mean ± SD (with each p-values of marked by *) for #5, #5Rw, B7, B7#Rm in (M) 1 ± 0.010, 0.830 ± 0.005 (p < 0.0001), 0.881 ± 0.008, 0.943 ± 0.003 (p = 0.002); (N) 1 ± 0.007, 0.747 ± 0.005 (p < 0.0001), 0.836 ± 0.005, 1.06 ± 0.006 (p < 0.0001); (O) 1 ± 0.004, 0.849 ± 0.003 (p < 0.0001), 0.842 ± 0.003, 1.04 ± 0.003 (p < 0.0001). Mean ± SD relative to cont. (with each p-values of marked by *), for Cont.. Rapa., PP242, AICAR, NQDI-1, Salbr. for (P) 1 ± 0.010, 0.601 ± 0.001, 0.873 ± 0.011, 0.784 ± 0.004, 0.885 ± 0.004, 0.757 ± 0.002 (all, p < 0.0001); (Q) 1 ± 0.007, 0.599 ± 0.001, 0.867 ± 0.004, 0.772 ± 0.003, 0.872 ± 0.0003, 0.683 ± 0.001 (all, p < 0.0001); (R) 1 ± 0.004, 0.675 ± 0.001, 0.924 ± 0.003, 0.818 ± 0.002, 0.879 ± 0.001, 0.818 ± 0.004 (all, p < 0.0001). These data were obtained by technical triplicate and not by biological triplicate. Statistical analyses in M-O and P-R were carried out by Student’s T test and by One-way ANOVA Dunnett’s test, respectively.