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. 2017 Apr 10;7(7):1890–1900. doi: 10.7150/thno.19135

Figure 2.

Figure 2

TAM activated SQSTM1 transcription in a Nrf2-dependent manner. A, SQSTM1 mRNA levels in RL95-2 (left), AN3CA (middle) or MCF7 (right) cells incubated with different concentrations of 4-OH-TAM for 24 hours were measured by qRT-PCR. B, ChIP-PCR experiment was performed to detect the binding of Nrf2 to SQSTM1 promoter in AN3CA cells before and after 4 hours of 4-OH-TAM (20μM) treatment. IgG was used as the negative control. The experiments were performed in triplicate and repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. C, p62-pro/pGL2 or pGL2 vector were co-transfected with Nrf2 plasmid or vector in AN3CA cells. Forty-eight hours later, SQSTM1 promoter activity was measured by dual luciferase reporter assay. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. D, Forty-eight hours after transfection of Nrf2 siRNAs, 4-OH-TAM (15 μM) was added for another 24 hours, and the effect of 4-OH-TAM on SQSTM1 and Nrf2 protein expression in AN3CA cells transfected with or without Nrf2 siRNAs were analyzed by Western blotting. E, Effects of 4-OH-TAM (as in D) on SQSTM1 mRNA expression in AN3CA cells transfected with or without Nrf2 siRNAs were analyzed by qRT-PCR. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. F, Effects of 4-OH-TAM on growth of AN3CA cells transfected with or without Nrf2 knockdown were analyzed by MTS assay. Forty-eight hours after transfection of Nrf2 siRNAs, various concentrations of 4-OH-TAM were added for another 72 hours. Two way ANOVA test was used to compare the statistical difference (NC vs siNrf1 #1, p<0.01; NC vs siNrf1 #1, p<0.01). The experiments were repeated three time. Representative results from one triplicated experiment were shown as Mean±SD. G, Nrf2 inhibitor brusatol (Bru) (40 or 80 nM) was added together with 4-OH-TAM (15 μM) for 24 hours in AN3CA cells. SQSTM1 expression was determined by western blotting. H, SQSTM1 mRNA expression in AN3CA cells treated with brusatol (Bru) and 4-OH-TAM incubation (as in G) were analyzed by qRT-PCR. The results were shown as in E. I, The growth of AN3CA cells treated with various concentrations of brusatol (Bru) and 4-OH-TAM (15 μM) incubation were explored by MTS assay. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD.