Fig. 3: Erk1/2i + CDK4/6i treatment triggers cell-cycle arrest and induces apoptosis.

Erk1/2i + CDK4/6i induced cellular effects were evaluated in H929, MM 1S, and U266 cells co-cultured with BMSC-conditioned medium after 48 h treatment with ERK1/2i (0; 0.9; 3 and 6 μM) and CDK4/6i (0; 0.1; 1 and 3uM), alone or in combination. A1 Cell cycle profiles were analyzed using standard DAPI staining. MM-cells in G0/G1, S, and G2/M phases were measured on the BD Fortessa X-20, followed by analysis using ModFit LT software. B1 ERK1/2i + CDK4/6i effects on apoptosis were quantified using annexin V-fluorescein isothiocyanate and 7-AAD staining and flow cytometry; percentages of early apoptotic (early A; Annexin V+/7-AAD−) and late apoptotic (late A, Annexin V+/7-AAD+) events were identified using FlowJo V10 software. A2 and B2 Protein lysates were obtained after treatment of MM-cells with Erk1/2i or CDK4/6i or Erk1/2i + CDK4/6i. Cell lysates were subjected to immunoblotting using the antibodies indicated; GAPDH served as a loading control. A3 Densitometry analysis of protein bands was performed using the Image J software. The color key next to each heatmap shows the protein expression level compared to DMSO. On the heatmaps, protein expression levels are indicated with intensity shades of black color. These analyses showed a dose-dependent overexpression of a cell cycle inhibitor p27 protein (A2), and increased PARP cleavage (B2), the signature of cell death. B3 Caspase (CAS) 3/7 activation in MM cell lines was measured by flow cytometry in response to Erk1/2i, CDK4/6i, and Erk1/2i + CDK4/6i treatment. Relative CAS3/7 activation is summarized as heatmaps; mean fluorescent intensities (MFI) are calculated in comparison with MFI of controls. Each treatment with a specific concentration of Erk1/2i and/or CDK4/6i was done in duplicate, plus/minus the standard error of the mean has been hidden for simplicity. C BH3 profiling to measure early changes in net pro-apoptotic signaling of mitochondria in response to ERK1/2i and CDK4/6i, alone or in combination. C1 Interaction map for BH3 peptides and BH3 mimetics with BCL-2 family proteins. Darker green color indicates Kd<100 nM determined by fluorescence polarization. C2, C3 Heatmap of delta priming response, assessed by dynamic BH3 profiling, in MM cell lines plated and treated for 16 h with ERK1/2i and CDK4/6i, alone or combination. Delta priming = % cytochrome c loss_(drug) − % cytochrome c loss _(DMSO). A, B, C Results are summarized as heatmaps; color-keys are shown next to the each heatmap for data interpretation. Expression levels are indicated with intensity shades of green color.