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IL6R-STAT3-ADAR1(P150) interplay in 1q21(amp) MM
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Figure 1. IL6R and ADAR1-P150 are closely associated with 1q21 copy number and IL6 stimulation. (A) Gene expression levels of IL6R and ADAR1 in the Multiple Myeloma Research Consortium (MMRC) patients’ dataset (left) and human multiple myeloma cell lines (HMCL) (right) according to 1q21 status. 0- no copy number gain (wildtype, WT), 1- one copy gain, ≥2- two or more copy gains. IL6R and ADAR1 copy number and expression values were determined as described in the Online Supplementary Information. (B) Basal protein (left) and mRNA (right) expression of IL6R and ADAR1 in HMCL as detected by western blot and real time quantitative poly- merase chain reaction (RT-qPCR). (C) Correlation of ADAR1 with IL6R in different patients’ datasets computed using the Pearson correlation test. ‘r’ is the correlation coef- ficient and P-values indicate whether the correlation coefficient is different from 0. (D, E) Western blot analysis of IL6/STAT3 pathway components (D) and ADAR1 expres- sion (E) in cells with 1q21(amp) (U266 and H929) and 1q21(WT) (OCIMY5 and KMS12BM) upon IL6 stimulation (10 ng/mL) at different time points. The top band in total ADAR1 refers to the P150 isoform, while the bottom band is the P110 isoform. The single P150 band in the second blot was probed with a specific antibody that can only detect P150 but not P110 (Ab126745). (F) Western blot and RT-qPCR analysis of protein (left) and mRNA (right) expression of ADAR1 and STAT3 pathway markers in the XG6 cell line which was cultured in conditions with gradual IL6 deprivation (3 ng/mL, 2 ng/mL, 1 ng/mL) for 24 h. (G) U266 was stimulated with IL6 (10 ng/mL) for 8 h before the IL6-containing medium was washed off, and the cells were resuspended in IL6-free medium again for 24 h. ADAR1 and STAT3 pathway components were checked for expression via western blot (left) and qRT-PCR (right). *P<0.05, **P<0.01, ***P<0.0001. MMRC: Multiple Myeloma Research Consortium; HMCL: human multiple myeloma cell lines; WT: wildtype; IL6: interleukin-6.
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