Figure 4
DOX washout of shRNA knockdown of IKZF1 or IKZF3 but not c-MYC or IRF4 leads to a partial rescue of their respective anti-proliferative effects in MM cells. (a) Western blot analysis showing the relative expression of Ikaros, Aiolos, c-Myc and IRF4 in MM1.S shIKZF1 cells cultured with or without DOX (Dox, 0.01 μg/ml) for 4 consecutive (D4) days in five distinct cultures marked with superscripts 1 through 5 (see Results section for details). The five parallel cultures consisted of culture 1 (−DOX), culture 2 (+DOX), culture 3 (DOX was washed out after day 1 (+DOX/WashD1)), culture 4 (DOX was washed out after day 2 (+DOX/WashD2)), or culture 5 (DOX was washed out after day 3 (+DOX/WashD3)), followed by continuous culture up to 4 days while the samples were collected each day (D11–5 through D41−5). Maximum protein reductions are visualized by D4 (red arrow), or partial knockdown (green arrow) following washout after D1. (b) Measurement of proliferation by 3H-thymidine incorporation in MM1.S shIKZF1 cells in parallel samples to the experiment (see Results) described in part (a). Similar DOX-washout experiments as described in a followed by measurement of 3H-thymidine incorporation similar to part b were performed with shIKZF3 (c), shMYC (d) or shIRF4 (e) MM1.S cells.
