Fig 2: Loss of MED12 restores Homologous Recombination in BRCA-deficient cells. (A, B) DR-GFP assay showing that MED12 co-depletion with two different siRNAs restores HR efficiency in BRCA1-depleted and BRCA2-depleted 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). (C) DR-GFP assay showing that TGFß pathway inhibition with LY2109761 (TGFßRi) suppresses the restoration of HR induced by MED12 depletion in BRCA2-knockdown 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). (D) RAD51 immunofluorescence experiment showing that MED12 depletion partly restores RAD51 foci formation upon CPT treatment in HeLa-BRCA2KO cells. At least 75 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unequal variance). (E, F) Western blots showing that MED12 depletion in HeLa-BRCA2KO cells does not affect the levels of RAD51 or ATM upon CPT (E) or HU (F) treatment.

Fig 3: 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 4: MED12 depletion promotes chemoresistance through activation of the TGFß pathway. (A) Western blots showing that treatment of HeLa-BRCA2KO cells with TGFßRi for 24h abolishes the SMAD2 phosphorylation induced by MED12 depletion. (B, C) Clonogenic survival experiments showing that treatment with 20 µM LY2109761 (TGFßRi) restores olaparib (B) and cisplatin (C) sensitivity in MED12-depleted HeLa-BRCA2KO cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (two-way ANOVA). (D, E) Cellular viability experiments showing that treatment with 20 µM LY2109761 (TGFßRi) restores olaparib (D) and cisplatin (E) sensitivity in MED12-depleted HeLa-BRCA2KO cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (2-way ANOVA).

Fig 5: Bioenergetic dynamics are regulated by menin and MAPs in T47D and MCF-7 cells. (A) and (B) Glycolytic and OXPHOS ATP productions in T47D (A) or MCF-7 (B) cells infected with vehicle, shMEN1, shKMT2A, shMED12, shWAPL, or shGATA3 lentivirus. Statistics represented the difference of glycolytic or OXPHOS ATP production between shRNA knockdown and vehicle controls. (C) and (D) Bar charts representing mitochondrial functions in the single knockdown of MEN1, KMT2A, MED12, WAPL, or GATA3 and their vehicle control in T47D (C) or MCF-7 (D) cells. (E) Schematic summary of mitochondrial dynamics affected by the knockdown of MEN1 or MAPs. (F,G) Bar charts represented the glycolytic functions in T47D (F) or MCF-7 (G) cells subject to gene knockdown by shMEN1, shKMT2A, shMED12, shWAPL, or shGATA3 lentivirus. (H) Schematic summary of glycolytic functions affected by the knockdown of MEN1 or MAPs. Data are presented as mean ± S.D. (n = 15–20 technical-replicate wells). Statistical significance was performed by an unpaired two-tailed Student’s t-test between treated groups and corresponding controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.