Fig. 2: ERK hyperactivation drives intrinsic CDK4i/6i resistance via cyclin D1.

A A panel of ALM cell lines were treated with palbociclib (500 nM, 24–72 h) before characterization by Western blotting (top panel). Densitometric analysis is shown in the bottom panel (n = 5 in each group). B Cells were treated with palbociclib (500 nM) and/or trametinib (10 nM for WM4324/WM4235, 1 nM for YUSEEP) for 72 h before characterization by Western blotting. C WM4324 spheroids were formed before implantation in collagen and treatment with palbociclib and/or trametinib for 72 h. Spheroids were subsequently stained with a viability stain and imaged by fluorescent microscopy (green indicates living cells, red indicates dead cells). D Cells were treated for up to 4 weeks with palbociclib (30 nM) and/or trametinib (0.3 nM) before colonies were fixed and stained with crystal violet. Quantification is shown in the right panel. E WM4235 cells were treated with palbociclib (500 nM) and/or trametinib (10 nM) for 24 h before subsequent staining with EdU. Shown in the panel to the right is quantification. F YUHIMO cells were stably transfected with either Empty Vector (EV) or DUSP4 (DUSP4 OE) in the presence or absence of palbociclib (500 nM) for 72 h before characterization by Western blotting. G YUHIMO cells were transfected with either EV or DUSP4 for 72 h before treatment with palbociclib (500 nM) for 72 h before proliferative capacity following EdU staining. H Cells were transfected with either non-specific siRNA (siNS) or siCCND1 in the presence or absence of palbociclib (500 nM) for 72 h before characterization by Western blotting. I WM4324 cells were transfected with either non-specific siNS or siCCND1 for 48 h before treatment with palbociclib (500 nM) for 6 h before proliferative capacity following EdU staining (n = 6 in each group). Paired t-test was used to calculate the significance of difference between treatment and control group in figure A. Ordinary One-Way ANOVA and Tukey test were used to perform multiple pairwise comparisons in figure C, E, and G. Two sample t-test was used to compare group means in figure D and I. *p < 0.05 and n = 3 unless otherwise stated throughout panels.