Page 235 - Haematologica May 2020

IL6R-STAT3-ADAR1(P150) interplay in 1q21(amp) MM
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Figure 2. STAT3 transcriptionally regulates ADAR1-P150 expression. (A) Wildtype (STAT3+/+) and STAT3 knockout (STAT3-/-) mouse liver cell lines were treated with IL6 (10 ng/mL) for different periods and the protein expression profile was checked with western blotting. (B) Western blot analysis of H929 cells treated with STAT3 inhibitors, STA-21 (10 mM for 24 h and 48 h) and LLL12 (for 24 h at 1.25 mM and 2.5 mM). (C) Top panel: real-time quantitative polymerase chain reaction (RT-qPCR) analysis of STAT3 mRNA expression after siRNA-mediated-STAT3 knockdown in H929 cells (24 h). Bottom panel: MCL1 and ADAR1 mRNA expression 24 h after STAT3 knockdown. (D) H929 transfected with pCDNA plasmid transcribing for constitutively-activated STAT3 (STAT3-CA) and its dominant negative mutant (STAT3-DN) (24 h). STAT3-CA is artificially phosphorylated at Y705, while the STAT3-DN is transcriptionally defective and has a gain-of-function of inhibiting endogenous STAT3 expression. Top panel: the transfection efficiency was checked with qRT-PCR. Bottom panel: ADAR1 mRNA expression after STAT3 overexpression. CA: constitutively activated, DN: dominant negative. (E) Top panel: depiction of primers (purple stars) designed to encompass different regions of ADAR1 promoters. Promoters 1B and 1C transcribe for the P110 isoform and promoter 1A transcribes for the P150 isoform. Bottom panel: H929 cells were stimulated with IL6 (10 ng/mL for 6 h) and STAT3 enrichment on the different regions of ADAR1 promoter was investigated with chromatin immunoprecipitation and qPCR with primers depicted in the top panel. (F, G) H929 cells were transfected with a luciferase-reporter vector encoding for either P110 or P150 promoter regions. At 24 h after transfection, the cells were treated with (F) either phosphate-buffered saline (PBS) or IL6 (10 ng/mL) for 8 h or (G) further transfected with either STAT3-CA or STAT3-DN and the cells were left to grow for another 24 h. The luciferase activity was analyzed with a Tecan plate reader. EV: empty vector.
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