American Society of Hematology

Figure 3

$Figure 3. ATRA abrogates sorafenib-mediated upregulation of Bcl6 via reduced STAT3 signaling. (A) Molm14 cells were treated with sorafenib (Sor, 20 nM) and/or ATRA (100 nM) for 6 to 48 hours, and mRNA expression of Bcl6 was measured in triplicate by qPCR relative to TATA-binding protein (TBP). Error bars indicate average fold change vs DMSO ± SD (**P < .01). Data are representative of 3 independent experiments. (B) Expression of phospho-FLT3 (pFLT3), total FLT3, Bcl6, and β-tubulin in Molm14 cells following 24-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM), representative of 4 independent experiments. (C) Bcl6 expression in Molm14 cells by western blotting following treatment with sorafenib (20 nM), AC220 (10 nM), or TTT-3002 (5 nM) alone or in combination with ATRA (100 nM) for 24 hours as in panel B, represented as average Bcl6 expression relative to β-tubulin vs DMSO for at least 3 independent experiments ± SD. (D) Molm14 cells were treated with sorafenib (20 nM) and/or ATRA (100 nM) for 12 or 24 hours, and mRNA expression of Mcl-1 was measured in triplicate by qPCR relative to TBP. Error bars indicate average fold change vs DMSO ± SD. Data are representative of 3 independent experiments. (E) Expression of Mcl-1 and β-tubulin in Molm14 cells following 24-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM), with fraction of Mcl-1/β-tubulin relative to DMSO control indicated below each blot. (F) Expression of phospho-STAT5 (pSTAT5), total STAT5, phospho-STAT3 (pSTAT3), and total STAT3 in Molm14 cells following 48-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM), with fraction of phospho-protein/total protein relative to DMSO indicated below each blot. Data are representative of 3 independent experiments. (G) Surface expression of CD126 (interleukin-6 receptor) in leukemia cell lines treated with sorafenib (20 nM) and/or ATRA (100 nM) for 48 hours, represented as relative CD126 mean fluorescence intensity (MFI) vs DMSO control. Error bars represent average of 3 independent experiments ± SD. (H) Expression of Bcl6 in Molm14 and THP-1 cells expressing NS shRNA or Bcl6-targeted (Bcl6_1, Bcl6_2, or Bcl6_3) shRNAs. Ratio of Bcl6 to β-tubulin relative to NS shRNA control is indicated below the blot. Data are representative of 3 independent experiments. (I) Annexin V binding in Molm14 and THP-1 cells with NS shRNA or Bcl6-targeted (Bcl6_1, Bcl6_2, Bcl6_3) shRNAs following 48-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM). Error bars represent average of 3 independent experiments ± SD (**P < .01).$

ATRA abrogates sorafenib-mediated upregulation of Bcl6 via reduced STAT3 signaling. (A) Molm14 cells were treated with sorafenib (Sor, 20 nM) and/or ATRA (100 nM) for 6 to 48 hours, and mRNA expression of Bcl6 was measured in triplicate by qPCR relative to TATA-binding protein (TBP). Error bars indicate average fold change vs DMSO ± SD (**P < .01). Data are representative of 3 independent experiments. (B) Expression of phospho-FLT3 (pFLT3), total FLT3, Bcl6, and β-tubulin in Molm14 cells following 24-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM), representative of 4 independent experiments. (C) Bcl6 expression in Molm14 cells by western blotting following treatment with sorafenib (20 nM), AC220 (10 nM), or TTT-3002 (5 nM) alone or in combination with ATRA (100 nM) for 24 hours as in panel B, represented as average Bcl6 expression relative to β-tubulin vs DMSO for at least 3 independent experiments ± SD. (D) Molm14 cells were treated with sorafenib (20 nM) and/or ATRA (100 nM) for 12 or 24 hours, and mRNA expression of Mcl-1 was measured in triplicate by qPCR relative to TBP. Error bars indicate average fold change vs DMSO ± SD. Data are representative of 3 independent experiments. (E) Expression of Mcl-1 and β-tubulin in Molm14 cells following 24-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM), with fraction of Mcl-1/β-tubulin relative to DMSO control indicated below each blot. (F) Expression of phospho-STAT5 (pSTAT5), total STAT5, phospho-STAT3 (pSTAT3), and total STAT3 in Molm14 cells following 48-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM), with fraction of phospho-protein/total protein relative to DMSO indicated below each blot. Data are representative of 3 independent experiments. (G) Surface expression of CD126 (interleukin-6 receptor) in leukemia cell lines treated with sorafenib (20 nM) and/or ATRA (100 nM) for 48 hours, represented as relative CD126 mean fluorescence intensity (MFI) vs DMSO control. Error bars represent average of 3 independent experiments ± SD. (H) Expression of Bcl6 in Molm14 and THP-1 cells expressing NS shRNA or Bcl6-targeted (Bcl6_1, Bcl6_2, or Bcl6_3) shRNAs. Ratio of Bcl6 to β-tubulin relative to NS shRNA control is indicated below the blot. Data are representative of 3 independent experiments. (I) Annexin V binding in Molm14 and THP-1 cells with NS shRNA or Bcl6-targeted (Bcl6_1, Bcl6_2, Bcl6_3) shRNAs following 48-hour treatment with sorafenib (20 nM) and/or ATRA (100 nM). Error bars represent average of 3 independent experiments ± SD (**P < .01).

This Feature Is Available To Subscribers Only