Figure 3.
dMtmr6, and the mammalian orthologue MTMR8, influence autophagic flux(A) Representative image of fed L3 fat body with control (GFP-negative) and dMtmr6-knockdown cells (white dashed outline, green cells) stained with an antibody to detect ref(2)p and Hoechst to detect nuclei (blue). (B) Quantification of the percentage of cellular area occupied by ref(2)p in control and dMtmr6-knockdown cells of L3 fat body from fed and starved animals (shown in A and C). (C) Representative image of starved L3 fat body with control (GFP-negative) and dMtmr6-knockdown cells (white dashed outline, green cells) stained with an antibody to detect ref(2)p and Hoechst to detect nuclei (blue). (D) Quantification of ref(2)p puncta size in control and dMtmr6-knockdown cells of L3 fat body from fed and starved animals (shown in A and C). (E) Amino acid sequence alignment of the CG3530, MTMR6, MTMR7, and MTMR8 catalytic domain (see Fig. S1 for full sequence alignment). (F) Schematic of the MTMR6 subfamily. Drosophila CG3530 contains an N-terminal FYVE domain. MTMR6, MTMR7, and MTMR8 contain a N-terminal coiled coil domain. (G and H) Representative maximum intensity projection of z-stack image from serum-fed Cos7 cells treated with scrambled siRNA (NC, top) or MTMR8-siRNA (bottom). Cells were stained with Hoechst (blue) to detect nuclei and antibodies to detect p62 (red) and LC3B (green). (I–L) Quantification of the size and number of cellular p62 and LC3B puncta as represented in G and H. (M) Western blot from serum-fed or serum-starved COS7 cells treated without or with Bafilomycin A1, which disrupts autophagosome-lysosome fusion, with quantification of the ratio of p62 to actin (M′) and LC3B-II to LC3B-I (M″). Data are presented as mean ± minimum to maximum, n = 10 each (B and D); and mean ± minimum to maximum (n = 12 images, three experimental replicates; I–L). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001), using paired two-tailed t test (B and D), unpaired two-tailed t test (I–L), and one-way ANOVA (M). Scale bars, 20 µm.

dMtmr6, and the mammalian orthologue MTMR8, influence autophagic flux (A) Representative image of fed L3 fat body with control (GFP-negative) and dMtmr6-knockdown cells (white dashed outline, green cells) stained with an antibody to detect ref(2)p and Hoechst to detect nuclei (blue). (B) Quantification of the percentage of cellular area occupied by ref(2)p in control and dMtmr6-knockdown cells of L3 fat body from fed and starved animals (shown in A and C). (C) Representative image of starved L3 fat body with control (GFP-negative) and dMtmr6-knockdown cells (white dashed outline, green cells) stained with an antibody to detect ref(2)p and Hoechst to detect nuclei (blue). (D) Quantification of ref(2)p puncta size in control and dMtmr6-knockdown cells of L3 fat body from fed and starved animals (shown in A and C). (E) Amino acid sequence alignment of the CG3530, MTMR6, MTMR7, and MTMR8 catalytic domain (see Fig. S1 for full sequence alignment). (F) Schematic of the MTMR6 subfamily. Drosophila CG3530 contains an N-terminal FYVE domain. MTMR6, MTMR7, and MTMR8 contain a N-terminal coiled coil domain. (G and H) Representative maximum intensity projection of z-stack image from serum-fed Cos7 cells treated with scrambled siRNA (NC, top) or MTMR8-siRNA (bottom). Cells were stained with Hoechst (blue) to detect nuclei and antibodies to detect p62 (red) and LC3B (green). (I–L) Quantification of the size and number of cellular p62 and LC3B puncta as represented in G and H. (M) Western blot from serum-fed or serum-starved COS7 cells treated without or with Bafilomycin A1, which disrupts autophagosome-lysosome fusion, with quantification of the ratio of p62 to actin (M′) and LC3B-II to LC3B-I (M″). Data are presented as mean ± minimum to maximum, n = 10 each (B and D); and mean ± minimum to maximum (n = 12 images, three experimental replicates; I–L). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001), using paired two-tailed t test (B and D), unpaired two-tailed t test (I–L), and one-way ANOVA (M). Scale bars, 20 µm.

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