Fig 5. SFN treatment impairs STAT5A-1*6-mediated survival and growth of Ba/F3 cells.
(A) Schematic representation of the growth and survival behavior of non-induced and doxycycline (Dox)-induced Ba/F3-tet-on-1*6 in the presence or absence of IL-3. Non-induced Ba/F3-tet-on-1*6 cells are IL-3-dependent (-Dox, +IL-3), like parental Ba/F3 cells. IL-3-growing cells express active endogenous STAT5 (WTON). Upon IL-3 withdrawal, endogenous STAT5 activity is turned off (WTOFF) and non-induced Ba/F3-tet-on-1*6 cells rapidly undergo apoptosis (-Dox, -IL-3). Treatment of Ba/F3-tet-on-1*6 cells with doxycycline results in the expression of the constitutively active STAT5A-1*6 mutant (1*6ON) [76], allowing cells to survive and eventually grow in the absence of IL-3 (+Dox, -IL-3). In these conditions, endogenous wild-type STAT5 is inactive (WTOFF). (B, C) Non-induced and Dox-induced Ba/F3-tet-on-1*6 cells were grown in the absence or presence of IL-3, and were treated with 2 μM SFN or left untreated, as indicated, for up to 11 days. Living and dead cells were counted every day by trypan blue exclusion (B) and were harvested for Western blot analysis (C). The ‘no Dox, no IL-3’ control cells shown in lane 2 of the Western blot were harvested after 6 hours of IL-3 withdrawal. The FLAG-specific antibody recognizes flag-tagged STAT5A-1*6 in Dox-induced cells; pSTAT5 detects phosphorylated STAT5 (endogenous WT in IL-3-growing non-induced cells and STAT5A-1*6 in Dox-induced cells); STAT5A-specific antibodies (STAT5A and STAT5A+B) recognize both endogenous WT STAT5A and induced STAT5A-1*6; STAT5B-specific antibodies (STAT5A+B) recognize endogenous WT STAT5B proteins; α-tubulin was used as a loading control. Western blot analysis showed that STAT5A-1*6 was overexpressed compared to endogenous STAT5A and STAT5B upon treatment with doxycycline. Moreover, expression of STAT5A-1*6 remained constant over time regardless of the presence of SFN. By contrast, STAT5A-1*6 phosphorylation decreased after 5 days of treatment with 2 μM SFN.