Fig. 4: Effects of ERK1/2i + CDK4/6i on gene and protein expression in MM cell lines.

H929, MM 1S, and U266 cells co-cultured with BMSCs were treated for 48 h with ERK1/2i (Ei) (0; 0.9; 3 and 6 μM) and CDK4/6i (Ci) (0; 0.1, 1, and 3 μM), alone or combination (Ei + Ci). MM-cells were then separated from BMSC using EasySep magnetic bead cell separation method. A Total mRNAs were isolated from MM1S and H929 cells and transcribed into cDNAs. Gene expression profiling was performed using custom TaqMan Gene Expression Array Plates containing primers and probes for detection of RAS and cell cycle signaling pathway genes. Results were analyzed using the relative standard curve method, and final data analyses and heatmaps were generated based on dCt values using Partek Genomic Suite. Expression levels are indicated with intensity shades of green/red colors. GAPDH and TBP genes were used as housekeeping genes. B Protein lysates (20–40 μg of protein/lane) of H929 and MM1S cells were separated on SDS-PAGE, and after blotting onto nitrocellulose membranes were probed with anti- Erk1/2, -p-ERK1/2, -c-myc, -p-S6, p-RSK, -p-RB, and -E2F1 antibodies; GAPDH served as a loading control. C 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.