Fig. 6: Effects of Erk1/2i and CDK4/6i on primary MM-cells (A 1, B1, B3).

Dose-response effects of Erk1/2i (Ei), CDK4/6i (Ci), or Erk1/2i + CDK4/6i (Ei + Ci) treatment on MM-cells freshly obtained from MM BM aspirates of patients are summarized as heatmaps. CD138 + PCs selected from MM patient BM using EasySep cell separation, with purity >95% (confirmed using flow cytometry), were treated with Erk1/2i, CDK4/6i, or Erk1/2i + CDK4/6i at the concentrations indicated. On the figures A1, B1 the results are shown as percent (%) of cell death relative to DMSO controls. The % cell death is indicated with intensity shades of green color scale shown at the right edge of each heatmap. Each concentration of Erk1/2i and/or CDK4/6i was tested in triplicate. The results, plus or minus the standard error of the mean, ranged from +0.01 to +0.7, and have been hidden for simplicity. A2 CD138 + PC were collected 48 h after treatment and cell lysates were prepared to measure phosphorylation/total levels of Erk1/2 using InstantOne enzyme-linked immunosorbent assay kits, according to the manufacturer’s protocol. Absorbance was measured at 450 nm. The results are presented as bar graphs. The x-axis shows the samples analyzed, and the y-axis displays the phosphorylation/total Erk1/2 levels as an absorbance. Results obtained from positive and negative control samples are not displayed on the graph. The mean ± S.D. is shown for two independent experiments. A3 Caspase (CAS) 3/7 activation in MM CD138 + PCs from Pt 1 and Pt 2 were measured by flow cytometry in response to ERK1/2i and CDK4/6i treatment, alone or in combination. In this experiment, unfractionated BM cells from MM patients were used, and CAS3/7 activation was measured in MM CD138 gated PCs. The results are summarized as heatmaps; the percentage of mean fluorescent intensity (MFI) was calculated and compared with the MFI of the controls, and presented in shades of red scale shown on the right side of each heatmap. Each treatment with a specific concentration of Erk1/2i and/or CDK4/6i was done in duplicate, plus/minus standard error (+0.01 to +0.75) of the mean MFI have been hidden for simplicity. B2 Functional effects of ERK1/2i and CDK4/6i or ERK1/2i + CDK4/6i treatments were validated at the protein level by immunoblotting. Cell lysates were prepared 2D after treatment from CD138 + PCs selected using a CD138 positive magnetic bead selection method (Stem Cell Technology).