Fig. 4. iBET-BD2 has immunomodulatory activity.

(A) schematic of auxin-inducible degradation strategy for BRD4. Blast^R (Blasticidin resistance) linked in-frame with T2A self-cleaving peptide to FLAG-mAID-BRD4. A separate viral vector was used to express CMV promoter driving expression of OsTIR1-IRES-Puro resistance. (B) FACS analysis of HLA/B/C expression in K562 cells pretreated with DMSO, Auxin (IAA) or I-BET151 for 6hrs followed by stimulation with IFNg (10ng/ml) for 48 hrs and 48 hr incubation with compounds. (C) qRT-PCR analysis of MYC expression in K562 cells following treatment with I-BET151 or IAA (Auxin) for 6hrs. Data shown represent the mean ± SD (n=3) (D) qRT-PCR analysis of TAP1 expression in K562 cells before and after IFNg treatment (6 hours) and/or either IAA (Auxin), IBET151, or DMSO (Vehicle control) for 7 hours (1hr pretreatment), Data shown represent the mean (n=3) +/- SD, ** p>0.01. (E) Compound effects on cellular proliferation and (F) cytokine production in anti-CD3/CD28-stimulated human primary CD4⁺ T cells. Data represent the mean ± SEM (n = 4) (G-H) Efficacy of compounds reducing KLH-induced antibody responses (IgM) in mice. N, naїve (n = 4); V, vehicle; iBET-BD1 (15 mg/kg, s.c., BID), iBET-BD2 (40 mg/kg, s.c., QD), I-BET151 (15 mg/kg, s.c., QD). Data shown as the mean ± SEM (n = 10). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to determine statistical significance compared with respective vehicle controls (***P<0.001, **P<0.005 vs V(QD); ⁺⁺ P<0.005, ⁺ P<0.01 vs V(BID)).