Fig 2: Loss of MED12 in BRCA-deficient cells promotes chemoresistance. (A, B) Clonogenic survival experiments showing that depletion of MED12, but not of MED13 or CDK8, increases the resistance of HeLa-BRCA2KO cells to olaparib (A) and cisplatin (B). The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance calculated using two-way ANOVA (A) or t-test two-tailed, unequal variance (B). (C) Cellular viability assay showing that depletion of MED12 increases the resistance of DLD1-BRCA2KO cells to cisplatin. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance calculated using two-way ANOVA. (D, E) Cellular viability assay showing that depletion of MED12, but not of MED13, increases the resistance of RPE1-BRCA1KO cells to olaparib (D) and cisplatin (E). The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (two-way ANOVA). (F) Annexin V assays showing a reduction in cisplatin-induced apoptosis upon MED12 depletion in RPE-BRCA1KO cells. The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (t-test two-tailed, unequal variance). (G) Representative clonogenic assay showing that MED12 depletion in PEO1 cells promotes olaparib resistance. (H, I) Analyses of BRCA2-mutant ovarian (H) and bladder (I) TCGA cancer datasets showing that high MED12 levels are associated with increased survival, while low MED12 levels are associated with reduced survival. Mantel-Cox log ranked t test was used for statistical analyses (n = 31, P-value = 0.3234 for the ovarian dataset; n = 38, P-value = 0.4424 for the bladder dataset). The differences observed are not significant, likely because of the small number of BRCA2-mutant samples in the datasets.

Fig 3: Luciferase reporter assay of ChREBP and FOXO1 transfactors in MED13-LKO mice. Isolated primary hepatocytes from adenovirus (Ad)-GFP (Ad-WT) or Ad-Cre (Ad-KO) transduced Med13fl/fl (WT) or Med13fl/flAlb-Cre (KO) mice were used. A) Luciferase reporter assays for the effects of ChREBP-β on ChoRE activity in either Med13fl/fl (WT and Ad-WT) or Med13-LKO (KO and Ad-KO) as indicated. B) Luciferase reporter assays for the effects of FOXO1 on G6pc promoter activity in indicated cells, either from Med13fl/fl (WT and Ad-WT) or Med13-LKO (KO and Ad-KO). P values were determined by Student's t-test (p < 0.05). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and n = 3. The values above each of the error bars indicate the fold stimulation.

Fig 4: 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 5: Glucose metabolic phenotype of the MED13-LKO mice. Twelve-week-old male Med13fl/fl and MED13-LKO mice were fed normal chow (LabDiet #5053) ad libitum as described in Methods (n = 8 per group). A) Representative Western blotting images of liver nuclear proteins (5 separate experiments performed) and relative densitometric analysis of the mediator complex kinase module subunits. B) Glycerol gradient column separation and shift of kinase module subunits to the size that correlates with different complexes shown on gels. Fractions 2–8 represent free subunits, 14–20 represent small complexes, and 24–27 represent large complexes according to the size standards. Five livers of MED13-LKO (left panel) or Med13fl/fl (right panel) mice that were fasted or fed with normal chow and 30% sucrose containing water were pooled and the same amount of liver protein lysate was loaded onto each column as described in Methods. C) Body weight and liver weight were measured in 12-week-old Med13fl/fl and MED13-LKO mice after 4-h normal chow ad libitum feeding. D) Respiratory exchange ratio (RER) over a 24-h period. E) Glucose tolerance test, F) insulin tolerance test, and G) pyruvate tolerance test for Med13fl/fl (red) and MED13-LKO (blue) mice were performed as described in Methods (n = 8 per group). P values were determined by Student's t-test (p < 0.05). Values are the mean ± SEM.