Fig. 8: Loss of DENND6A impairs nutrient dependent lysosomal positioning and autophagic flux.

a Unstarved or Earle’s Balanced Salt Solution (EBSS) starved HeLa cells were fixed, stained with LAMP1 antibody and imaged using confocal microscopy (Leica SP8). The cell periphery is outlined by a white dotted line. Scale bar = 10 µm. Yellow arrows indicate the presence of peripheral lysosomes. b Quantification of cumulative distribution of LAMP1 intensity in experiments performed in (a) (under starvation condition); mean ± SEM; two-tailed extra sum of F-squares test following nonlinear regression and curve fitting; n = 28, 29, 30 cells, corresponding to WT, DENND6A KO1 and DENND6A KO2, from 3 replicates. c Immunoblot showing LC3B-II protein levels under various conditions (unstarved; EBSS starved; and EBSS starved + Bafilomycin A1 (BafA1)) Immunoblot probed with anti-LC3B-II, anti-p62 and anti-HSC70 antibodies. d Quantification of experiment in (c); means ± SEM; two-way ANOVA (**P ≤ 0.0025; ***P ≤ 0.0005; ****P ≤ 0.0001; n = 3 from three replicates). e HeLa WT and DENND6A KOs cells from c were fixed and stained with LC3B-II antibody and DAPI. The cell periphery is outlined by a white dotted line. Scale bar = 25 µm. f Quantification of experiment in (e); means ± SEM; Kruskal-Wallis test ****P ≤ 0.0001; n = (32, 43, 37); (36, 34, 42); (34, 41, 34) cells, corresponding to WT, DENND6A KO1 and DENND6A KO2 in unstarved; EBSS; EBSS + BafA1, from 3 replicates).