Fig 2: Inducible gene expression was suppressed in MED13-LKO mice under different carbohydrate-containing feedings. FASTED for 16 h fasted with water, WATER for normal chow supplemented with water, SUCROSE for normal chow supplemented with 30% sucrose containing water, and FRUCTOSE for normal chow supplemented with 30% fructose containing water. A, B, and C) Real-time quantitative RT-PCR (qRT-PCR) of lipogenic genes (Fasn, Chrebp-β, and Srebf1c), gluconeogenic genes (G6pc and Pck1), and constitutively active and unaffected by nutrient signal genes (Mlx and Chrebp-α) performed in livers of the Med13fl/fl (WT) or MED13-LKO (KO) mice (n = 9 per group, 3 separate experiments were conducted). P values were determined by Student's t-test (p < 0.05). Values are the mean ± SEM.

Fig 3: The RNA Pol II transcription Mediator complex is important for survival and PARPi sensitivity in BRCA2-depleted cells. (A) Western blots performed to determine the efficacy of sgRNAs targeting CCNC, CDK8, MED24 and MED25. HEK293A BRCA2 mAID-EGFP cells were infected with lentiviruses expressing the indicated sgRNAs and selected with puromycin. After treatment with Dox and IAA for 24 hrs, cells were collected and lysates were blotted with the indicated antibodies. (B) Results of long-term clonogenic assays performed using cells transduced with the indicated sgRNA constructs and given the indicated treatments. 5×103 cells were seeded in each well and incubated with the indicated treatments for 7 or 14 days. Colonies were fixed and stained with 0.5% crystal violet. Ola, olaparib. Hereafter BRCA2+/+ cells represent HEK293A BRCA2 mAID-EGFP cells without IAA and Dox treated cells; BRCA2-/- cells represent HEK293A BRCA2 mAID-EGFP cells with IAA and Dox treated cells. (C) Quantification of relative cell survival in the clonogenic assays in (B). All treated groups were normalized to the indicated untreated group. And the relative cell growth was compared with LentiV2 (V2) which was set as 1. The data are presented as means (±SD). **P < 0.01; ***P < 0.001; ns, not significant (Student t-test). (D) Western blots performed to determine the efficacy of sgRNAs targeting MED12 and MED13. (E) Results of long-term clonogenic assays performed to determine the viability of the cells in D. 5 × 103 cells were seeded in each well and incubated with the indicated agents for 14 days. Colonies were fixed and stained with 0.5% crystal violet. (F) Quantification of relative cell survival in the clonogenic assays in (E). The data are presented as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test).

Fig 4: Fructose supplementation reduced insulin signaling and suppressed the downregulation of the Mediator kinase module. A) Western blotting and relative densitometric analysis of hepatic nuclear MED13, MED12, CDK8, CCNC, nuclear ChREBP, and nucleolin loading control. B) Western blotting and relative densitometric of total liver including nuclear and cytosolic fractions (n = 6 per group, representative duplicate from 3 separate experiments shown on gels). P values were determined by Student's t-test (p < 0.05). Values are the mean ± SEM. Identical letters indicate values that are not statistically different from each other (p > 0.05). Multiple sample comparisons were determined by one-way ANOVA followed by the Turkey-Kramer multiple comparisons test.

Fig 5: Fructose induction of endogenous gene expression in primary hepatocytes isolated from the control and MED13-LKO mice. Primary mouse hepatocytes were isolated from the WT (Med13fl/fl) or KO (Med13-LKO) mice. The cells were cultured on collagen-coated plates in DMEM without glucose (−) or with 45 mM of fructose (+) for 5 h. Cellular RNA was isolated and subjected to qRT-PCR for A)Med13, B)G6pc, C)Fasn, D)Chrebp-β, E)Mlx, F)Foxo1, and G)Usf1 mRNA. P values were determined by Student's t-test (p < 0.05). Values are the mean ± SD.