Fig. 1: Trehalose induces autophagy in PC3 cells.

a MTT viability assay was performed on PC3 cells treated for 48 h with 20, 50 or 100 mM trehalose. Six independent biological samples for each condition were analysed (n = 6), bar graph represents the mean relative cell viability ± SD. Statistical analysis was performed using Student’s t test. b TFEB localization was carried out by IF after treatment with 100 mM trehalose for 24 h. Nuclei were stained with DAPI. Scale bar, 20 μm. c LC3 mRNA expression was analysed by RT-qPCR after treatment with 100 mM trehalose for 48 or 72 h. Data were normalized to the amount of RplP0 mRNA. Bar graph represents the mean of four independent biological samples (n = 4) ± SD (*p < 0.01 vs. control 48 h, **p < 0.01 vs. control 72 h; Student’s t test). d Autophagy was analysed by quantification of LC3-II/LC3-I ratio by WB analysis of PC3 cells treated for 24, 48 or 72 h with 100 mM trehalose. e LC3 puncta was carried out by IF after treatment with 100 mM trehalose for 48 h. Nuclei were stained with DAPI. Scale bar, 20 μm. f PC3 cells pretreated with or without 1 mM 3-MA for 1 h were exposed to 100 mM trehalose for additional 48 h. WB analysis of LC3 was carried out. Relative optical density of LC3II/I was quantified by ImageJ software. Bar graph represents mean ± SD calculated from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test (*p < 0.05). g PC3 cells were transfected with 50 nM negative control (NC) or ATG5 siRNA and WB of ATG5 was performed. Quantification of LC3 in PC3 cells transfected and treated 48 h with 100 mM trehalose was carried out. h PC3 cells were treated with 100 mM trehalose for 48 h and with 10 μM CQ or 2.5 mM NH₄Cl for the last 24 h before their collection. WB shows LC3 protein levels. Relative optical density of LC3II/I was determined by ImageJ software. Bar graph represents mean ± SD calculated from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test (*p < 0.05). i p62 mRNA expression levels were detected by RT-qPCR after treatment with 100 mM trehalose for 48 or 72 h. Data were normalized to the amount of RplP0 mRNA. Bar graph represents the mean of four biological independent samples (n = 4) ± SD (*p < 0.05 vs. control 48 h, **p < 0.01 vs. control 72 h; #p < 0.05 vs. control 48 h, Student’s t test). j PC3 cells were treated with 100 mM trehalose for 48 or 72 h. Twenty micrograms of protein extract was analysed by WB against p62. The quantification results were calculated over three individual experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-test (*p < 0.05 vs. control 48 h, **p < 0.05 vs. control 72 h; #p < 0.05 vs. control 48 h). k p62 localization was analysed by IF with anti-p62 antibody followed by FITC-conjugated secondary antibody in cells treated with 100 mM trehalose for 48 h. Nuclei were stained with DAPI. Scale bar, 20 μm. l PC3 cells were treated with 100 mM trehalose for 48 h and with 10 μM CQ or 2.5 mM NH₄Cl for the last 24 h before their collection. p62 levels were analysed by WB. Bar graph represents quantification mean ± SD calculated from three independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-test (*p < 0.05)