CDK9 Inhibition and Estrogen Independent Growth of Different Cell Lines (CAN 508; 30µM) 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 MCF7:WS8 MCF7:5C MCF7:2A MCF7/LCC1 MCF7/LCC2 MCF7/LCC9 Fraction Growth versus Vehicle 4 - Day Assay Figure S4 P-CDK9 MCF7:WS8 MCF7:5C MCF7:2A LCC1 LCC2LCC9 β-actin A B
pRb ser 795 Total Rb pRb ser 780 beta-actin Veh 1 CAN 508 (100μM) Treatment (in Hrs) 2 4 12 24 Figure S5
MCF10A 0.0 0.2 0.4 0.6 0.8 1.0 1.2 20µM30µM40µM Fraction Growth versus Vehicle 10058-F4 (cMYC Inhibitor) Fraction Growth versus Vehicle 0.0 0.2 0.4 0.6 0.8 1.0 1.2 5µM10µM30µM CAN 508 (CDK9 Inhibitor) Effect of cMYC inhibition OR CDK9 inhibition on the growth of MCF10A Cells (Immortal Mammary Epithelial Cells) Figure S6 A B
Supplementary Figure Legends Figure S1. Estrogen independent growth and levels of cMYC mRNA and protein in MCF7 and MCF7:5C cells. (A) Estrogen independent growth of MCF7:5C and MCF7 cells over 6 day period. Un-treated cells were grown and total DNA was measured on day 2, 4 and 6 after seeding. The data is represented as fold change in growth. (B) “S” phase cells were assessed by cell cycle analysis of MCF7:5C and MCF7 cells growing in absence of estrogen. Inset: Western blot of cMYC protein in MCF7 and MCF7:5C cells. Beta actin was used as a control for loading. Figure S2. cMYC depletion inhibits estrogen independent growth and reduces phosphorylation of retinoblastoma (Rb) protein in MCF7:5C cells. (A) Growth of MCF7:5C cells were measured after depletion of cMYC using two separate siRNA and compared with control non-targeting siRNA. Total DNA was assessed after 2 and 4 days of growth and the fold change was calculated after dividing by the DNA content on the day of start of the experiment (Day ‘0’). (B) Status of phosphorylated Rb protein (serine 795 and serine 780) after depletion of cMYC in MCF7:5C cells. The numbers below each band represents the fold change in band intensity normalized by total Rb protein. Figure S3. cMYC mRNA stability in MCF7 and MCF7:5C cells. Pulse and chase experiment was performed using 5-ethynyl uridine (uridine analog) to pulse the cells for 24 hours and chased for 60 and 120 minutes with fresh media. The data shows the fraction of 5-ethynyl uridine containing cMYC RNA remaining in MCF7 and MCF7:5C cells relative to the start of chase time. Figure S4. Total CDK9 levels and effect of its inhibition on estrogen-independent growth. (A) Protein levels of CDK9 was assessed using western blotting in MCF7, MCF7:5C, MCF7:2A, MCF7/LCC1, MCF7/LCC2 and MCF7/LCC9 cells. (B) Total DNA was measured to assess the growth of MCF7, MCF7:5C, MCF7:2A, MCF7/LCC1, MCF7/LCC2 and MCF7/LCC9 cells after 4 days of treatment with CDK9 inhibitor, CAN-508 (30µM). Data is presented as the fraction of growth of the respective vehicle treatments. All data points are average of four replicates and the error bars represent the standard deviation. Figure S5. CDK9 inhibition decreases the phosphorylation of Rb protein in MCF7:5C cells. Western blot of phosphorylated Rb protein (serine 795 and serine 780) after inhibition of CDK9 by CAN 508 at indicated time. Figure S6. Effect of cMYC or CDK9 inhibition on the growth immortalized mammary epithelial cells (MCF10A). (A) MCF10A cells were treated with indicated concentration of cMYC inhibitor 10058-F4 for six days and the growth assay was performed by measuring the total DNA. (B) MCF10A cells were treated with indicated concentration of CDK9 inhibitor, CAN 508, for six days and the growth assay was performed by measuring the total DNA. Data is presented as the fraction of growth of the respective vehicle treatments.