FIGURE 3.
Acriflavine (ACF) targets STAT3/STAT5 signalling in K562 cells. A, Protein extracts from K562 cells cultured for 72 h without (PBS) or with increasing concentrations of ACF were analysed by western blot with the indicated antibodies (n = 3). Actin served as the loading control. B, Cells were cultured with ACF (2 µmol/L) or not (PBS) for the specified times. Protein extracts were then prepared and analysed by immunoblotting with the indicated antibodies (n = 3). Actin served as the loading control. C, qRT‐PCR analysis of STAT5A, STAT5B and STAT3 expression in K562 cells treated or not (PBS) with increasing concentrations of ACF for 72 h. Results are presented as the fold change in STAT3/5A/5B gene expression in treated cells normalized to internal control genes (GAPDH, ACTB, YWHAZ and RPL13a) and relative to the control condition (normalized to 1) (n = 4 done in triplicates, data are mean ± SD, *P < 0.05; one‐sample t test). D, qRT‐PCR analysis of PIM1, cMYC and CISH in K562 cells treated or not with increasing concentrations of ACF for 72 h. Results are presented as the fold change in STAT3/5 target gene expression in treated cells normalized to internal control genes (GAPDH, ACTB and RPL13a) and relative to the control condition (normalized to 1) (n = 4 done in triplicates, data are mean ± SD, *P < 0.05; **P < 0.001; ***P < 0.0001, two‐way ANOVA test). E, K562 cells were cultured in normoxic (21% O2) or hypoxic (1% O2) conditions and treated or not with ACF (2 µmol/L). Protein extracts were then prepared and analysed by western blot with the indicated antibodies (n = 2). Actin served as the loading control