Fig. 2: Rapamycin induces autophagy in PC3 cells.

a PC3 cells were treated for 48 h with 10, 50 or 100 nM rapamycin. MTT viability assay was performed. Data are mean ± SD of six independent biological samples (n = 6). Statistical analysis was performed using Student’s t test (*p < 0.05 vs. control). Each experiment was repeated three times. b WB analysis of LC3 was performed with lysate of cells treated for 48 h with different doses of rapamycin (10, 50, 100 nM). Fifteen micrograms of protein extract was loaded in SDS-gel electrophoresis. Detection of autophagy was analysed by quantification of LC3-II/LC3-I ratio. Relative optical density was determined by ImageJ software. Experiments were performed independently three times and a representative blot is shown. c TFEB localization was carried out by IF after treatment with 100 nM rapamycin for 48 h. Nuclei were stained with DAPI. Scale bar, 20 μm. d LC3 mRNA expression levels were analysed by RT-qPCR after treatment with 100 nM rapamycin for 48 or 72 h. Data were normalized to the amount of RplP0 mRNA. Data are mean ± SD of four independent biological samples (n = 4). Statistical analysis was performed using Student’s t test (*p < 0.05 vs. control 72 h). e WB analysis of LC3 was performed with lysate of cells treated for 24, 48 or 72 h with 100 nM rapamycin. Detection of autophagy was analysed by quantification of LC3-II/LC3-I ratio. Relative optical density was determined by ImageJ software. Experiments were performed independently three times and a representative blot is shown. f Cells were treated with 100 nM rapamycin for 48 h and CQ (10 μM) or NH₄Cl (2.5 mM or 5 mM) for the last 24 h before their collection. LC3 levels were analysed by WB and relative optical density of LC3II/I was determined by ImageJ software. Experiments were performed independently three times and a representative blot is shown. g LC3 puncta were analysed by IF utilizing anti-LC3 antibody followed by FITC-conjugated secondary antibody. Cells were treated with 100 nM rapamycin alone for 48 h or in combination with 10 μM CQ for the last 24 h. Nuclei were stained with DAPI. Scale bar, 20 μm. h p62 mRNA expression levels were detected by RT-qPCR after treatment with 100 nM rapamycin for 48 or 72 h. Data were normalized to the amount of RplP0 mRNA. Data are mean ± SD of four independent biological samples (n = 4). Statistical analysis was performed using Student’s t test (*p < 0.01 vs. control 48 h, ##p < 0.01 vs. control 48 h). i Cells were treated with 100 nM rapamycin for 48 or 72 h. Twenty micrograms of protein extract was analysed by WB. The quantification results were calculated from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-test (*p < 0.05 vs. control 48 h, **p < 0.05 vs. control 72 h). j p62 was analysed by IF using anti-p62 antibody followed by FITC-conjugated secondary antibody in cells treated with 100 nM rapamycin for 48 h. Nuclei were stained with DAPI. Scale bar, 20 μm. k Cells were treated with 100 nM rapamycin for 48 h and 10 μM CQ or 2.5 mM NH₄Cl for the last 24 h before their collection. p62 levels were analysed by WB. Bands relative optical density was determined by ImageJ software. The quantification results were calculated from three independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-test (*p < 0.05 vs. control)