Fig. 3

H19 lncRNA is required for the activation of autophagy. a Autophagic vacuoles stimulated by 10 μM tamoxifen for 12 h in MCF7/TAMR cells that stably expressed shH19 or shControl (shCtrl) were analyzed using a Cyto-ID autophagy detection assay. Stv: starvation. The bar graphs show the quantification of the relative fluorescence data. The data are presented as the mean ± SD of three independent experiments. Student’s t test was used for statistical analysis. b In MCF7/TAMR cells that stably expressed shH19 or shCtrl, LC3 expression was analyzed by Western blotting. Cells were treated without or with CQ to perform flux analysis. The value next to each blot is the quantification of the relative expression of the indicated band normalized to GAPDH expression. c. Autophagic vacuoles stimulated by 10 μM tamoxifen for 12 h in MCF7/TAMR cells that were stably infected with the H19 lentiviral vector or the control lentiviral vector were analyzed using a Cyto-ID autophagy detection assay. Stv: starvation. The bar graphs show the quantification of the relative fluorescence data. The data are presented as the mean ± SD of three independent experiments. Student’s t test was used for statistical analysis. d LC3 expression in MCF7/TAMR cells that were stably infected with the H19 lentiviral vector or the control lentiviral vector was analyzed by Western blotting. Cells were treated without or with CQ to perform flux analysis. The value next to each blot is the quantification of the relative expression of the indicated band normalized to GAPDH expression. e The distribution of autophagic vacuoles that contained mCherry-EGFP-LC3 in MCF7/TAMR cells overexpressing H19 was analyzed by confocal microscopy. The bar graphs show the quantification of the fluorescent puncta data. The data are presented as the mean ± SD of three experiments. The scale bars indicate 20 μm. Two-way ANOVA was used for statistical analysis. f, g MCF7/TAMR cells with knockdown (f) or overexpression (g) of H19 were analyzed by annexin V/PI double staining. The bar graphs show the percentage of apoptotic cells. The data are presented as the mean ± SD of three independent experiments. Student’s t test was used for statistical analysis. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group. n.s. indicates no significant difference