Fig. 4.
Autophagy enhances stemness in MB. a qPCR analysis of autophagy-related genes mRNA in different MB cell lines. GADPH and B2M were used as internal control. DAOY cells were arbitrarily defined as 1.00. Data are expressed as the mean value ± SEM (n = 6). Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test. (*p < 0.05; ***p < 0.001). b Both D283-Med and DAOY cells were treated with CQ (20 μM) for 1 h. Levels of LC3, P62, and ACTIN were analysed by WB. Densitometric analysis of LC3 II levels over ACTIN is also shown. Data are expressed as the mean ± SEM (n = 3) and analysed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test (**p < 0.01). c Both ATG7 and BECLIN 1 expression was downregulated in D283-Med cells using specific RNAi oligonucleotides (siATG7 or siBECLIN 1, respectively) or unrelated oligos as negative control (siCTRL). Levels of CD133, BECLIN 1 and ACTIN were analysed by WB. Densitometric analysis of CD133 levels over ACTIN is also shown. Data are expressed as the mean ± SEM (n = 3) and analysed by unpaired Student's t-test (**p < 0.01). The downregulation of ATG7 was analysed by qPCR. d D283-Med cells were treated as in (c). The percentage of plasma membrane (PM) CD133 positive cells was assessed by flow-cytometry. Representative histograms of CD133 mean of fluorescence intensity (MFI) are shown. Data are expressed as the mean value ± SEM (n = 3). Data were analysed by unpaired Student's t-test (**p < 0.01). e D283-Med cells were treated with two different concentration of CQ (5 and 10 μM, respectively) for 48 h. Levels of CD133, NESTIN, LC3 and ACTIN were analysed by WB. f D283-Med cells were treated as in (e) for different time points. NESTIN mRNA levels were analysed by qPCR. GADPH and B2M were used as internal control. Data are expressed as the mean value ± SEM (n = 3). Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. (*p < 0.05, **p < 0.01). g GSAA enrichment plot showing that loss of ATG7 in D283-Med cells (3 independent experiments) results in downregulation of the pluripotency of stem cell pathway. Data are generated from RNA-seq analyses. h DAOY cells were cultured as medullospheres for 3 and 7 days. Levels of p-ULK1 758, p-ATG14, LC3, P62 and ACTIN were analysed by WB. i AMBRA1-silenced D283-Med cells (shAMBRA1) were transfected with empty, AMBRA1WT and AMBRA1AA mutant constructs, respectively. Levels of AMBRA1, CD133 and ACTIN were analysed by WB. Densitometric analysis of CD133 levels over ACTIN is also shown (right panel). Data are expressed as the mean ± SEM (n = 3) and analysed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test. (*p < 0.05)