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. 2018 Aug 17;9:1129. doi: 10.3389/fphys.2018.01129

FIGURE 3.

FIGURE 3

Collagen VI ablation leads to defects in autolysosome formation and TFEB translocation. (A) Immunofluorescence for LAMP-2, showing normal lysosomes in WT fibroblasts and enlarged lysosomes in Col6a1-/- fibroblasts. The insets show a magnification of the respective boxed areas. Scale bar, 25 μm. (B) Quantification of cells containing at least two enlarged lysosomes. Data represent the mean percentages ± SEM of cells with enlarged lysosomes, ad determined from thirty images per condition. (C) Western blot analysis of LAMP-2 in WT and Col6a1-/- fibroblasts, in complete medium (S+) or following serum withdrawal for 3 h (S–). (D) Densitometric quantification of the relative LAMP-2/β-actin ratio of three independent western blot experiments, as in (C). The value for WT fibroblasts in complete medium was arbitrarily set to 1. (E) qRT-PCR analysis of Lamp2 mRNA levels. (F) Co-immunostaining for LAMP-2 and LC3 in WT and Col6a1-/- fibroblasts, following 3 h serum withdrawal. The right panels show a higher magnification of the boxed area of the respective merged image. Col6a1-/- fibroblasts display impaired autophagosome (green puncta) fusion with lysosomes (red) (arrowheads). Scale bar, 25 μm. (G) Co-localization rate of LC3 and LAMP-2 staining in complete medium (S+) or following serum withdrawal for 3 h (S–). Mean percentages ± SEM were calculated for at least fifteen images per condition. (H) Representative fluorescence microscopy images of WT and Col6a1-/- fibroblasts in complete medium and after 3 h serum withdrawal, following transfection with a GFP-TFEB expression construct (green). Nuclei were stained with Hoechst (blue). Scale bar, 25 μm. (I) Quantification of transfected WT and Col6a1-/- fibroblasts showing either cytosolic or nuclear GFP-TFEB, as shown in (G). Data represents the mean of at least three independent experiments, and were analyzed by ANOVA test with Bonferroni correction. ∗P < 0.05; ∗∗P < 0.001; ∗∗∗P < 0.0001; #P < 0.001 for the comparison between WT complete medium and WT no serum; § P < 0.001 for the comparison between Col6a1-/- complete medium and Col6a1-/- no serum. S+, complete medium; S–, 3 h serum withdrawal.