Skip to main content
. 2020 Mar 20;31(5):962–982. doi: 10.1681/ASN.2019070712

Figure 4.

Figure 4.

Basal autophagy is dysfuncational in CTNS iPSCs. (A) Representative Western blot against autophagosome marker LC3B-II and β-actin from wild-type (WT) and CTNS iPSCs and graph displaying quantification of three independent experiments. (B and C) Percentage of cells with yellow-only puncta (autophagosomes) and red-only puncta (autolysosomes) treated with DMSO or 400 nM BafA1 for 4 hours (representative of n=30 cells, from ten random fields per condition containing approximately one to three cells in three independent experiments). One-way AVOVA performed, the data are mean±SEM. ****P<0.0001 relative to WT. (D) Cells transfected with tandem mCherry-LC3B-GFP plasmid showing red and yellow puncta. Nuclei counter stain was 4′,6-diamidino-2-phenylindole. Scale bar, 10 µm. (E and F) Percentage of yellow-only and red-only puncta after exogenous expression of CTNS in CTNS−/− iPSCs. Two-tailed unpaired t test performed, the data are mean±SEM. *P<0.05. (G) Sucrose (50 mM) treatment for 24 hours on WT iPSCs to induce a cystinotic phenotype. Percentage of yellow-only puncta shown. Two-tailed unpaired t test performed, the data are mean±SEM. ****P<0.0001. (H and I) Effects of drug treatments on percentages of yellow and red puncta; CTNS iPSCs treated with 1 mM cysteamine alone, 100 nM everolimus alone, or a combination of both for 24 hours. One-way ANOVA performed, data plotted as mean±SEM (n=30 cells from ten random fields per condition containing approximately one to three cells in three independent experiments). ****P<0.0001, WT versus CTNS−/− and CTNSKO; $$$$P<0.0001, CTNS−/− versus CTNS−/− 100 nM everolimus and CTNS−/− combination; ####P<0.0001, CTNSKO versus CTNSKO 100 nM everolimus and CTNSKO combination. (J and K) Average number of Magic Red vesicles per cell over 10 µm2 in WT iPSCs and CTNS−/− or CTNSKO iPSCs treated with 3 mM 3-ΜΑ and 100 nM everolimus for 24 hours. One-way AVOVA performed, values are mean±SEM (n=300 cells from five to eight random fields per condition, 20 cells per field, three independent experiments). *P<0.05, **P<0.01, ***P<0.001.