Figure 4
From: EGR-1/ASPP1 inter-regulatory loop promotes apoptosis by inhibiting cyto-protective autophagy
ASPP1 promotes EGR-1 levels in a feedback loop (a and c) The expression levels of ASPP1 and EGR-1 were analyzed by WB (left) and real-time RT-PCR (right) after being transfected with pcDNA3.1-ASPP1-V5 or empty vector control in HCT116 p53+/+ (a) or HCT116 p53−/− cells (c). (b and d) The expression levels of ASPP1 and EGR-1 were analyzed by WB (left) and real-time RT-PCR (right) after Crispr/Cas9-mediated ASPP1 silence in HCT116 p53+/+ (b) or HCT116 p53−/− cells (d). (e) Cytoplasmic and nuclear fractions were subjected to the WB assay with antibodies specifically targeting ASPP1 or EGR-1 in HCT116 cells in the presence or absence of Quercetin. β-Actin and Histone3.1 were used as cytoplasm and nuclear markers, respectively. (f) The expression of ASPP1 or EGR-1 were detected by WB after SiRNA-mediated ASPP1 knockdown, with or without MG132 treatment. β-Actin was used as loading control. EGR-1 expression level was quantified by ImageJ software. **P<0.01, in comparison with DMSO-treated si-control cells; #P<0.05, in comparison with DMSO-treated si-ASPP1 cells. (g) The cytoplasmic and nuclear fractions were separated in HCT116 cells transfected with the empty vector control or pcDNA3.1-ASPP1-v5, in the presence or absence of Quercetin. EGR-1 and ASPP1 were detected by WB assay. β-Actin and Histone3.1 were used as cytoplasm and nuclear markers, respectively. ImageJ was used to quantify EGR-1 levels. The bar graph showed ASPP1-induced EGR-1 fold changes. *P<0.05, nucleus versus cytoplasm; #P<0.05, Quercetin versus DMSO
