Fig 2: CLC-7 silencing results in weak lysosomal acidification. Cardiomyocytes were transfected with CLC-7 siRNA or pretreated with 100 nM CQ for 2 h, and then treated with 200 nM rapamycin for 6 h. (A) Lysosomal acidification was assessed using lysosomal probes. Scale bar, 20 µm. (B) Relative green fluorescence was quantified. ANOVA. *P<0.05, **P<0.01 vs. untreated control. n=6. (C) Cathepsin D protein expression levels were detected using western blotting and (D) semi-quantified. ANOVA. **P<0.01 vs. untreated control. n=6. CLC-7, H(+)/Cl(-) exchange transporter 7; CQ, chloroquine; siRNA, small interfering RNA; NCsi, negative control siRNA; ns, not significant.

Fig 3: CLC-7 silencing causes autophagosome accumulation. Cardiomyocytes were transfected with 200 nM CLC-7 siRNAs or pretreated with 100 nM CQ for 2 h, then treated with 200 nM rapamycin for 6 h. (A) Cardiomyocytes were incubated with antibodies against LC3 (green) and LAMP1 (red) and viewed via confocal microscopy. Scale bar, 5 µm. (B) Quantitative results are shown in the bar chart. Autolysosomes (yellow dots) and autophagosomes (green dots) were counted per cell in three microscopic fields per group. The mean number was calculated. ANOVA. *P<0.05, **P<0.01, autolysosome; ##P<0.01, autophagosome. n=5. (C) Electron micrograph showing the morphology of autophagosomes and autolysosomes in cardiomyocytes; triangles represent autophagosomes, arrows represent autolysosomes. Scale bar, 500 nm. Independent experiments were repeated three times. CLC-7, H(+)/Cl(-) exchange transporter 7; siRNA, small interfering RNA; NCsi, negative control siRNA; LC3, microtubule associated protein 1 light chain 3; LAMP1, lysosomal-associated membrane protein 1; CQ, chloroquine; ns, not significant.

Fig 4: Autophagy substrates accumulate following CLC-7 silencing. (A, B, D and F) Expression levels of autophagy-related proteins and CLC-7 were measured using western blotting, (E) RT-qPCR and (G and H) immunofluorescence. (C) Cell viability was assessed using CCK-8 and LDH assays following transfection with CLC-7 siRNAs with or without CQ pretreatment. (A) Western blot analysis of (B) expression levels of autophagy-related proteins following transfection with CLC-7 siRNAs or NCsi. *P<0.05, **P<0.01 vs. NCsi group. n=3. (C) Cardiomyocyte viability analysis using CCK-8 and LDH assays. *P<0.05, **P<0.01, ***P<0.001. n=5. (D) Western blot analysis of (F) expression levels of autophagy-related proteins following transfection with CLC-7 siRNAs with or without CQ (100 nM for 2 h) pretreatment. *P<0.05, **P<0.01 vs. untreated control. n=3. (E) LC3 and p62 mRNA expression levels were assessed using RT-qPCR. ANOVA, n=6. (G) Quantification of (H) immunofluorescence analysis of the localization and protein expression levels of CLC-7 following transfection with CLC-7 siRNAs. Scale bar, 5 or 10 µm. ANOVA. *P<0.05 vs. untreated control. n=5. CLC-7, H(+)/Cl(-) exchange transporter 7; siRNA, small interfering RNA; CQ, chloroquine; CCK-8, Cell Counting Kit-8; LDH, lactate dehydrogenase; NCsi, negative control siRNA; LC3, microtubule associated protein 1 light chain 3; RT-qPCR, reverse transcription-quantitative PCR; ns, not significant; OD, optical density.

Fig 5: Schematic depiction of CLC-7-associated lysosomal acidification and autophagosome degradation. CLC-7, H(+)/Cl(-) exchange transporter 7; v-ATPase, vacuolar-type H+-ATPase.