Fig 2: Dexamethasone downregulates GM-CSF-induced IRF4 expression in human monocytes and mouse macrophages(A and B) Human monocytes were treated with either GM-CSF (20 ng/mL) and Dex (100 nM) alone or together for 16 h. A) IRF4, IRF5, and IRF8 mRNA expression (n = 8) and B) protein expression in whole cell lysates (western blotting) with anti-IRF4, anti-IRF8, and anti-β-actin antibodies (n = 4).(C and D) Human monocytes were pre-treated with mifepristone (1μM) for 30 min before treated with GM-CSF alone or together with Dex for 16 h. C) IRF4 mRNA expression (n = 4) and D) IRF4 and β-actin protein expression in whole cell lysates (n = 4).(E and F) BMM were treated with GM-CSF (20 ng/mL) and Dex (100 nM) alone or together for 16 h. E) Irf4 mRNA expression (n = 5) and F) IRF4 and β-actin protein expression in whole cell lysates (n = 4). The data are graphed as scatterplots with bars indicating mean ± SEM. p values were obtained using one-way ANOVA with Tukey post-test, where ∗p < 0.05, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. Original Western blots are presented in Figure S1.

Fig 3: Biological functions of activator protein‐1 (AP‐1) family proteins in multiple myeloma (MM). (A) We determined the correlation of expression levels between IKZF1‐target genes (y‐axis) and the indicated genes (x‐axis) using the GenomicScape tool (http://www.genomicscape.com). 32 The data were extracted from DNA microarray analyses of gene expression in newly‐diagnosed MM patients. 53 (B) Cell lysates were prepared from the indicated MM cell lines and analyzed by immunoblotting using antibodies against c‐FOS, FOSB, c‐JUN and GAPDH (loading control). (C) We examined the expression of FOS, FOSB and JUN transcripts in normal plasma cells (n = 22), CD138‐positive cells derived from patients with monoclonal gammopathy of undetermined significance (MGUS; n = 44) and MM cells from newly‐diagnosed patients (n = 12). The data were extracted from DNA microarray analyses deposited in the GenomicScape database by the Arkansas group. 53 (D) Left panel: We transduced the indicated MM cell lines with shRNA against FOS or scrambled sequences (sh‐control) and evaluated the expression of candidate IKZF1‐target genes (IRF4, SLAMF7, BSG and CD48) along with GAPDH (Control) and FOS (for the evaluation of knockdown efficiency) using quantitative real‐time reverse transcription‐PCR after 24 h. The data were quantified by the 2–∆∆Ct method using GAPDH as a reference and are shown as the fold changes against the values of sh‐control‐transfected cells. *p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. Right panel: Correlation between the expression levels of FOS mRNA and those of IRF4 or CD48 mRNA in MM cell lines.

Fig 4: A selective activator protein‐1 (AP‐1) inhibitor augments the effects of lenalidomide in murine multiple myeloma (MM) models. (A) Luciferase‐expressing MM.1S cells were inoculated subcutaneously into the right thigh of NOD/SCID mice (1 × 106 cells/mouse). When the inoculated tumour became measurable (defined as day 1), we started treatment with vehicle alone (0.9% NaCl, twice a week), 20 mg/kg T‐5224 (twice a week), 10 mg/kg lenalidomide (twice a week), or the combination of T‐5224 and lenalidomide (20 mg/kg T‐5224 and 10 mg/kg lenalidomide, twice a week) intraperitoneally for two weeks. Left panel: Representative photographs of mice on day 1 and day 25 (original magnification: 2×). Right panel: Quantitative data of ex vivo bioluminescence imaging on day 1 and day 25 expressed in photons/s (photons/s). The mean ± S.D. (bars) is shown. p‐Values were determined by one‐way ANOVA with Student–Newman–Keuls multiple comparison test. (B) Left panel: Representative photographs of tumours resected on day 25 (original magnification: ×2). Right panel: The mean ± S.D. (bars) of tumour weight. *p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (C) Upper panel: Tumor sections were prepared from mice and stained with hematoxylin and eosin (HE). Lower panel: Serial tumour sections were immunostained for IRF4 (green) and CD138 (red), followed by counterstaining with DAPI (blue). Merged images are shown at the bottom. The data are representative of several independent experiments. (D) We transplanted luciferase‐expressing KMS26 cells (1 × 106 cells/mouse) into BALB/c mice via tail‐vein injection. After confirming the engraftment of transplanted cells on day 14 via ex vivo luciferase assays, we assigned recipients to four groups (n = 5), which were standardized on the basis of total luciferase activity, and started the treatments with vehicle alone (0.9% NaCl, three times a week), 20 mg/kg T‐5224 (three times a week), 10 mg/kg lenalidomide (three times a week), or the combination of T‐5224 and lenalidomide (20 mg/kg T‐5224 and 10 mg/kg lenalidomide, three times a week) intraperitoneally for 3 weeks. p‐Values and hazard ratios (95% confidential interval) were calculated by log‐rank test between the combined treatment group (red) and the lenalidomide‐alone (green) or T‐5224‐alone treatment group (blue). No significant differences were observed between the vehicle‐treated group (Control) and the lenalidomide‐alone or T‐5224 alone‐treatment group.

Fig 5: IRF4 depletion alleviated kidney damage of MRL/lpr mice. A Western blotting for IRF4 protein expression in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus; ELISA or biochemical kit for levels of plasma creatinine (B), urine ALB (C), plasma ANA (D) and plasma urea nitrogen (E) in MRL/lpr mice injected with control and AAV9-IRF4-sh virus. F Immunofluorescence staining for kidney IgG and C3 deposition in kidneys MRL/lpr mice injected with control and AAV9-IRF4-sh virus, Scale bar, 50 μm; G HE, PAS and MASSON staining for pathological condition in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus, Scale bar, 50 μm; H Flow cytometry for proportion of kidney plasma cells in MRL/lpr mice injected with control and AAV9-IRF4-sh virus. *P<0.05, **P<0.01, ***P<0.001