Quercetin enhances the anti-tumor effects of BET inhibitors by suppressing hnRNPA1 - Supplementary Data

Figure S1: Both Quercetin and hnRNPA1 knockdown enhance the anti-tumor effects of BET inhibitors. A. Cancer cells were treated with DMSO, OTX-015 (0.5 μmol/L), Quercetin (20 μmol/L), or a combination of OTX-015 (0.5 μmol/L) and Quercetin (20 μmol/L) for 24 hours. The cells were analyzed for cPARP-1 and GAPDH by western blot analysis. B. Cancer cells embedded in 3D collagen were treated with DMSO, OTX-015 (0.5 μmol/L), Quercetin (20 μmol/L), or a combination of OTX-015 (0.5 μmol/L) and Quercetin (20 μmol/L) for 72 hours. The effect on proliferation was determined by WST-1 assay. Two-way ANOVA analysis was performed. *, p<0.05 ****, p<0.0001. Error bars represent SD from three technical replicates. Results are representative of three independent experiments. C. Thyroid cancer cells K1 and 8505c were transfected with siCtrl or sihnRNPA1 for 48 hours and then treated with OTX-015 (0.5 μmol/L) for 24 hours. Expression of hnRNPA1, cPARP-1 and GAPDH were evaluated by western blot analysis. D. Thyroid cancer cells K1 and 8505c were transfected with control siRNA (siCtrl) or hnRNPA1-targeting siRNA (sihnRNPA1) for 48 hours, embedded in 3D collagen and treated with OTX-015 (0.5 μmol/L) for additional 48 hours. The effect on cell proliferation was determined using the WST-1 assay. Two-way ANOVA analysis was performed. ***, p<0.001 ****, p<0.0001. Error bars represent SD from three technical replicates. Results are representative of three independent experiments.

Figure S2: Quercetin decreases hnRNPA1 protein. A. Thyroid and pancreatic cancer cells were treated with Quercetin (20 μmol/L) for 24, 48, or 72 hours, and the lysates were analyzed for hnRNPA1 and GAPDH by western blot analysis. The results are representative of three biological replicates. B. Thyroid and pancreatic cancer cells, treated with increasing concentrations of Quercetin for 24 hours, were analyzed for hnRNPA1 by qPCR. Error bars represent SEM from three independent experiments.

Figure S3: Co-treatment of Quercetin and JQ1 decreases Survivin while having no effects on other apoptosis regulating proteins. CD18 and K1 cancer cells were treated with DMSO, JQ1 (1 μmol/L), Quercetin (20 μmol/L), or a combination of JQ1 (1 μmol/L) and Quercetin (20 μmol/L) for 24 hours. Cell lysates were collected and analyzed for apoptosis-related proteins using the Proteome Profiler Human Apoptosis Array ARY009, and the pixel density for Bad, Bax, Bcl-2, and Survivin were quantified by ImageJ. Error bars represent SD from two technical replicates. The results are representative of two independent experiments.

Figure S4: : The combination treatment of Quercetin and BET inhibitor JQ1 was well-tolerated. Mice were weighed daily, and the effect of inhibitors on mouse weight at the end of treatment was compared with weight at the start of treatment.

Figure S5: Representative IHC images for cleaved caspase-3 and Ki67 for CD18 tumors. CD18 xenograft tumors were stained for Ki67 and cleaved caspase-3 as described in Materials and Methods. Images were taken at 10X. 

Table S1: Calculations for coefficients of drug interaction.

Table S2: Effect of hnRNPA1 knockdown on JQ1-mediated changes in proteins in the ARY009 apoptosis array.

Table S3: Effect of Quercetin on JQ1-mediated changes in proteins in the ARY009 apoptosis array.