Fig 2: G9a depletion inhibit proliferation of MM cells in vitro. a RNA-seq performed on KMS12BM transfected with lentiviruses expressing shNT or shG9a (n = 2). Gene set enrichment analysis plots of negative enrichment (FDR = 0.41) for NFκB pathway when comparing shG9a#1 and #2 versus shNT. NER = -1.0079; FDR q-value = 0.41. NES, normalized enrichment score. b Immunoblots show G9a, p-RelB, RelB and proapoptotic factor Bim (β-actin as loading control) in indicated MM cell lines infected with lentiviruses expressing shNT or shG9a. c Immunoblots show RelB expression levels in representative KMS12BM xenografts with transfected shNT or shG9a lentivirus. d mRNA expression of RelB and proapoptotic factors BMF, BCL2L11 (Bim) in JJN3 cells treated by DMSO control or UNC0642 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$IC_{50}$$\end{document}IC50 for 72 h. (n = 3; mean ± SD; Two-tailed Student’s t test compared with DMSO treated control; *P < 0.05 and ***p < 0.001). e Whole cell lysates from JJN3 cells treated by DMSO control or UNC0642 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$IC_{50}$$\end{document}IC50 for 72 h were analyzed for the indicated proteins by immunoblotting

Fig 3: NF-κB signals were regulated by LL-2-CM and CMT-64-CM. BMDMs cultured in LL-2-CM and CMT-64-CM were harvested at different time points for western blot analysis. Dynamic alterations of canonical NF-κB (p-p65 and p65), non-canonical NF-κB (p-RelB, RelB, and p100/p52), and overall NF-κB (p-IKK, IKK \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{\alpha }$$\end{document}α, and IKK \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document}β) in LL-2-CM treated BMDMs A-I and CMT-64-CM treated BMDMs J-R were shown. Full-length blots are presented in Supplementary Fig. 3 and Supplementary Fig. 4

Fig 4: Rescue RelB in G9a depleted MM cells eliminates inhibition of proliferation. a Immunoblots show expression levels of G9a and RelB in KMS12BM transfected with lentiviruses expressing shNT or shG9a and RelB recovered G9a depleted KMS12BM cells after long term culture. b Proliferation assay on RelB recovered G9a-depleted JJN3 cells with control JJN3 cells infected by lentiviruses expressing shNT. Luminescent cell viability assay was conducted with CellTiter-Glo® at indicated time points. The viability at day0 was measured as 1 and following cell viability was normalized accordingly. Two-tailed Student’s t test, *p < 0.05, for comparisons of shNT, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RelB^{ + }$$\end{document}RelB+ shG9a#1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RelB^{ + }$$\end{document}RelB+ shG9a#2 versus shG9a. c Tumor volumes over time of KMS12BM xenografts generated with shNT transfected or shG9a transfected/RelB-recovered KMS12BM cells. (n = 3, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 for comparisons of shNT, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RelB^{ + }$$\end{document}RelB+ shG9a#1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RelB^{ + }$$\end{document}RelB+ shG9a#2 versus shG9a, Bonferroni corrected one-way ANOVA). d Final individual and mean tumor volumes of KMS12BM xenografts generated with shNT transfected or shG9a transfected/RelB-recovered KMS12BM cells at day 31. (Bonferroni corrected one-way ANOVA comparing all groups, **p < 0.01, ****p < 0.0001)

Fig 5: Cytosolic calcium participated in conditioned medium-mediated NF-κB signal and LPS-mediated canonical NF-κB signal. BMDMs treated by LL-2-CM and CMT-64-CM with or without BAPTA-AM for 2 h were collected for western blot. Canonical NF-κB (p-p65 and p65), non-canonical NF-κB (p-RelB, RelB, and p100/p52), and overall NF-κB (p-IKK, IKK \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{\alpha }$$\end{document}α, and IKK \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document}β) were shown and calculated A-I. LPS elicited canonical NF-κB in BMDMs, which was suppressed by BAPTA-AM J-L. Full-length blots are presented in Supplementary Fig. 5 and Supplementary Fig. 6. P values were calculated by one-way ANOVA and Tukey test