Figure 5 | Cell Death & Disease

Figure 5

From: Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity

Figure 5

ATG gene depletion and pharmacological inhibition of autophagy significantly enhances apoptosis in ATRA-treated SKBR3 cells. (a) SKBR3 and MDA-MB453 cells were treated with increasing concentrations of ATRA (AT) for 2, 4 and 6 days as indicated. Western blot analysis for cleaved (cl.) caspase-3 and GAPDH was performed. ImageJ software was used to quantify bands. Raw values were normalized to GAPDH. Caspase-3 levels of SKBR3 cells treated with 1 μM ATRA for 4 days were arbitrarily set to 100% as expression levels at day 0 or 2 were undetectable or weak, respectively. (b) ATG7 and cleaved caspase-3 western blot analysis of control or shATG7-transduced SKBR3 and MDA-MB453 cells treated with 1 μM ATRA for 2 and 4 days. Cleaved (cl.) caspase-3 expression levels were quantified as in (a). (c) Western blot analysis and quantification of cleaved (cl.) caspase-3 in control and shATG7-transduced SKBR3 cells treated with 1 μM ATRA for 4 days. Raw values for cleaved capsase-3 were normalized to GAPDH using the ImageJ software. Vehicle-treated control cells were arbitrarily set to 100%. Bars and s.d. are representative of five independent experiments. (d) Knockdown efficiency of ATG7 and statistics thereof. Normalization was performed as described in (c). (e) Caspase-3/7 activity was measured using Caspase-Glo-3/7 assay from Promega. RLU values were calculated according to manufacture's recommendations and normalized to the cell number. Values are shown for SKBR3 and MDA-MB453 control or shATG7-transduced cells treated with 1 μM ATRA for 2 and 4 days, respectively. Standard deviations are shown from three independent experiments. (f) Annexin V/PI fluorescence-activated cell sorting (FACS) analysis of control or ATG7-depleted SKBR3 cells at day 2, 4 and 6 of treatment with 1 μM ATRA. Bars represent five independent experiments. (g) Control, ATG5- and VPS34 SKBR3- knockdown cells were treated and analyzed as in (e). (h) Caspase-3/7 activity of SKBR3 cells treated with ATRA, increasing concentrations of chloroquine or a combination thereof as indicated. Analysis as in (e). Mann–Whitney U-test: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001

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