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. 2019 Dec 4;8:e49930. doi: 10.7554/eLife.49930

Figure 4. Human miR-1 regulates autophagy by controlling TBC1D15 expression.

(A) WB and (B) quantification of LC3-II normalised to α-tubulin from HeLa cells expressing Scr or miR-1 mimics +/- bafilomycin. Data are mean fluorescence intensities of bands ± SEM (n = 3–5). **p<0.01, ***p<0.001 (one-way ANOVA with Dunnett’s correction). (C) WB and (D) quantification of LC3-II normalised to α-tubulin from HeLa cells expressing empty vector (control) or TBC1D15 overexpression vector +/- bafilomycin. Data are mean fluorescence intensities of bands ± SEM normalised to α-tubulin (n = 5). n.s. not significant to the control, ***p<0.001 (two-way ANOVA with Bonferroni correction). (E) IF images of HeLa cells stably expressing mRFP-GFP-LC3 and transfected with empty vector (control) or TBC1D15 overexpression vector. Scale bar, 10 μm. (F) Quantification of green and red vesicles and (G) red/green vesicle ratio from (E) ± SEM (n = 3, 12–14 cells per replicate). **p<0.01, ***p<0.001 (Student’s t-test). (H) WB and (I) quantification of HeLa cells co-transfected with Scr or miR-1 mimic together with empty vector or TBC1D15 overexpression vector +/- bafilomycin. Data are mean fluorescence intensities of LC3-II bands normalized to α-tubulin ± SEM (n = 7). **p<0.01, ***p<0.001 (two-way ANOVA with Bonferroni correction).

Figure 4.

Figure 4—figure supplement 1. miR-1 and TBC1D15 control autophagy.

Figure 4—figure supplement 1.

(A) Quantification of the mean number of LC3-positive vesicles per HeLa cell ± SEM expressing scrambled miRNA (Scr) or independent miR-1 mimics immunostained with antibodies against LC3 (n = 3). *p<0.05, **p<0.01 (one-way ANOVA with Dunnett’s correction). (B) WB of HeLa cells transfected with Scr siRNA or siRNA against TBC1D15 in the presence or absence of bafilomycin (400 nM for 4 hr). (C) Mean fluorescence intensities of LC3-II WB bands ± SEM normalized to α-tubulin (n = 4). *p<0.05, **p<0.01 (Student’s t-test).
Figure 4—figure supplement 2. miR-1 overexpression induces autophagy flux.

Figure 4—figure supplement 2.

(A) IF images of HeLa cells stably expressing mRFP-GFP-LC3 and transfected with scrambled miRNA (Scr) or miR-1 mimic. Scale bar, 10 μm. (B) Quantification of mean number of green and red vesicles per cell and (D) the red/green vesicle ratio ± SEM (n = 4). **p<0.01, ***p<0.001 (Student’s t-test).