Figure S1. Mechanism of ON induced MM cell death.
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1 Figure S1. Mechanism of ON induced MM cell death. To elucidate whether ON induced decrease of viability of MM cells was associated with induction of apoptosis, the number of apoptotic cells was assessed by Annexin V/PI double staining. MM.1S and NCI-H929 cells were treated with control or ON for 24 hours followed by subsequent flow cytometry. ON treatment significantly induced apoptosis in both the cell lines.
2 Figure S2. PD less significantly induces apoptosis in MM1.S cell and does not induce apoptosis in NCI-H929 cells. MM.1S and NCI-H929 cells were treated with control or PD for 24 hours. Induction of apoptosis was assessed by Annexin V/PI double staining and subsequent flow cytometry. PD treatment does not significantly induce apoptosis in MM1.S as compared to ON treatment. Howover there was no apoptotic cell death observed in NCI-H929 cells.
3 Figure S3. PD induces cell cycle arrest in MM1.S cells and not in NCI-H929 cells. PD treated MM1.S and NCI-H929 cells were analyzed by EdU cell proliferation assay and flow cytometry. PD treatment at 60nM and 250nM for 24h induced an elevation of the number of cells in the G1 phase of the cell cycle in MM1.S cells while the number of cells in the G1 phase remain unchanged in the NCI-H929 cells compared with that in the control. This observation suggested that PD did not modulate the cell cycle distribution in NCI-H929 cells even at higher concentration.
4 Figure S4. ON induces cell cycle arrest in MM cells. Cell proliferation is well correlated to the regulation of cell cycle progression. We chose to investigate the possible effect of ON on cell cycle progression. ON treated cells were analyzed by EdU cell proliferation assay and flow cytometry. There was significant dosedependent alteration of cell numbers in different cell cycle phases at 24h compared with that in the control. This data indicate that ON treatment leads to the induction of G1/S arrest in MM cells.
5 Figure S5. Molecular effects of treatment with PD in MM cell lines. MM1.S and NCI-H929 cells were treated for 3 different time points (4hr, 6hr and 12hr) at two diferent doses of PD (250nM and 1000nM). Both MM cell lines showed a significant repression of phosphorb levels at higher dose of PD while there was no change in the levels of phosphos6k in MM1.S and a slight decrease in NCI-H929 cells at 6hr and 12hr time points. This data suggested that PD does not have significant effect on phosphos6k levels as compared to the significant effect of ON in both MM cell lines.
6 Figure S6. Effects of ARK5 depletion on MM cell viability by sirna transfected MM1.S and NCI-H929 cells. Cells were transfected with ARK5-specific sirna or with scrambled sirna and after incubation for 48 hours cell viability was evaluated by fluorometric resazurin reduction assay (CellTiter-Blue) with the percentage of viable cells as compared with control. Values are mean +/- SD and p values between groups are indicated.
7 Figure S7. Anti-MM activity of SRT1720. MM1.S and NCI-H929 cell lines were treated with or without SRT1720 at the indicated concentrations for 24hr, followed by assessment for cell viability. Data presented are means ± standard error (n = 2).
8 Figure S8. Effect of ARK5 silencing by sirna induces apoptosis in MM. Silencing of ARK5 results in apoptosis of MM1.S and NCI-H929 cells. MM cells were transfected with ARK5 and scrambled sirna and examined for apoptosis by flow cytometry. Percentage of apoptotic cells (Annexin-V or Annexin-V/propidium iodide positive) is depicted on the axes. The above result confirm that silencing of ARK5 induced apoptosis in MM cells.
9 Figure S9. Effect of ARK5 silencing by sirna enhances G1/S cell cycle arrest in MM. Flow cytometry analysis showed the G1/S cell cycle arrest was noticed in ARK5 sirna transfected cells compared to negative control in both MM1.S and NCI-H929 cells as confirmed by EdU cell proliferation assay.
10 Figure S10. ON overcomes bone marrow stromal cell (BMSC) induced growth of MM Cells. To assess effects of stromal cells on drug activity, a co-culture model of MM cells with human BMSCs (HS-5) was employed. MM.1S and NCI-H929 cells were cultured alone or with BMSCs in the presence or absence ofon cofirmed by Annexin V/PI staining. Data suggest that MM-bone marrow microenvironmental factors are ineffective in protecting MM cells from the CDK4/6 and ARK5 inhibitor ON
11 Figure S11. In vivo MM xenograft ON inhibition. ON treatment does not affect animal weight. Mice from MM1.S and NCI-H929 xenograft experiments treated with control or 100 mg kg 1 On were weighed at the time of calliper measurements.
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