Fig. 5.

Upregulation of autophagy in the heterozygous retinas. a Western blot analysis showed that LC3B-II was upregulated and SQSTM1 was downregulated in the heterozygous retinas in comparison with the wild-type retinas. GAPDH was used as an internal control. b, c Quantification of LC3B-II and SQSTM1 expression levels in retinas in (a). The results were representative of 3 independent experiments and represented as means ± SD. The data were analyzed using Student’s t-test. *p < 0.05; **p < 0.01. d Real-time quantitative PCR of Sqstm1 in these retinas. β-actin was used as an internal control. e Immunofluorescence analysis of GCP4 in wild-type and heterozygous retinas using anti-GCP4 antibody. GCP4 was mainly expressed in OS, IS, OPL, IPL, and GCL of retina in both wild-type and heterozygous mice. The nuclei were stained with Hoechst reagent. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 50 μm. f–i HCQ treatment resulted in accumulation of both LC3-II and SQSTM1 proteins in both wild-type and heterozygous retinas. The retina lysates were analyzed by immunoblotting with antibodies as indicated. GAPDH was used as an internal control. g, h Quantification of LC3B-II and SQSTM1 protein levels in retinas in (f). The results were representative of 3 independent experiments and represented as means ± SD. The data were analyzed using one-way ANOVA followed by Bonferroni posttest. *p < 0.05; **p < 0.01; ***p < 0.001. i Real-time quantitative PCR of Sqstm1 in retinas in (f). β-actin was used as an internal control. The data were analyzed using one-way ANOVA followed by Bonferroni posttest. j Immunofluorescence analysis of the LC3B puncta in the heterozygous and wild-type retinas of 2, 4, and 8 months using anti-LC3B antibody. LC3B puncta were mainly located in the cytoplasm of photoreceptor cells in both heterozygous and wild-type retinas (red arrows). Some LC3B puncta were also observed in the nuclei of ONL in heterozygous retinas (red arrowheads). The nuclei were stained with Hoechst reagent. The enlarged images were originated from the white squares. k Statistic analysis of LC3 puncta per cell using Student’s t-test. Three retinas from three mice were sectioned at indicated age and three sections were counted per retina. Data were represented as means ± SD. *p < 0.05; **p < 0.01. l Transmission electron microscopy of autophagosomes in photoreceptor cells. Representative images of autophagosomes in the cytoplasm of photoreceptor cells. The images in the white squares in panels l1, l3, l5, and l7 were enlarged and showed in panels l2, l4, l6, and l8, respectively. Avi, initial autophagic vacuole; Avd, degradative autophagic vacuole. Scale bars are indicated in each image