Fig. 3

TAK-981 upregulates GR expression in primary multiple myeloma and cell lines through downregulation of miR-130b. A GR expression is associated with Dex sensitivity in primary MM cells. GR (NR3C1) mRNA expression was assessed by q-PCR and presented as relative level normalized to GAPDH. IC₅₀ values were calculated by GraphPad Prism 8. Pearson correlation analysis showed a significant negative correlation negative correlation between GR RNA (NR3C1) and IC₅₀ for Dex in 15 primary MM. B NR3C1 mRNA level was decreased and (C) miR-130b level was increased in dose-dependent manner in primary MM cells treated with TAK- 981. Primary MM cells from 5 relapsing patients were treated with TAK-981 at indicated concentrations for 48 h. Total RNA extracted and NR3C1 mRNA and miR-130b level was measured by q-PCR. Data presented as mean ± SD, n = 5 (D) TAK-981 treatment increases GR protein level in primary MM cells. Western blot presents GR protein of 3 primary MM samples treated with or without 0.1 μM TAK-981 for 48 h. Relative protein level was quantified using Image J, normalized to GAPDH, and labeled below each blotting band. E NR3C1 mRNA was increased and miR-130b was decreased in MM1S and H929 cell lines upon TAK-981 treatment. Estimated variation is indicated as SD. P values were derived using a two-tailed Student t test. * p < 0.05, ** p < 0.01, ***p < 0.001 (F) TAK-981 treatment increases GR protein level in MM1S and H929 cell lines. MM1S and H929 cells were treated with or without 20 nM TAK-981 for 48 h. Cell lysates were used for western blot. G GR-repressed gene RRM2 and apoptosis marker cleaved PARP (c-PARP) were measured by western blot in MM1S and H929 cell lines treated with TAK-981, Dex or both for 48 h. GAPDH was used as loading controls. Relative protein level was quantified by Image J and labeled below each blot