Fig 2: 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 3: Med23 deficiency does not affect histological classification of tumors.a Lung sections from KrasG12D/+;Med23+/+ mice and KrasG12D/+;Med23f/f mice at 16 weeks post Adeno Cre infection were stained for SFTPC, CCSP, or NKX2.1. Lymph node sections from both groups were stained with H&E. b Kaplan‒Meier survival curves for KrasG12D/+;Med23+/+ mice and KrasG12D/+;Med23f/f mice (n = 15 per group). c Total RNA was extracted from lung tumors from both groups at 20 weeks post-infection and then analyzed by qRT‒PCR to detect the mRNA levels of Med23, Med1, Med16, Med24 and Med28 (n = 7 per group). d Total protein was extracted from lung tumors from both groups at 25 weeks post-infection and then analyzed by Western blotting to detect the protein levels of MED23, MED16 and MED24. MED16 and MED24 were used as loading control for MED23. n = 3 mice per group, and two separate tumors were taken from each mice for Western blot analysis. Data are presented as the means ± SEMs. ***P < 0.001.

Fig 4: Inflammation-related genes were downregulated in Med23-deleted mice.a Heatmap analysis of differentially expressed genes between KrasG12D/+;Med23+/+ mice and KrasG12D/+;Med23f/f mice at 20 weeks post-infection. GO analysis of downregulated (b) and upregulated (c) genes (fold change > 2) in KrasG12D/+;Med23f/f mice. The black line indicates the gene counts and the blue bars indicate the statistics. d GSEA plot showing enrichment of antigen processing and presentation of a peptide antigen via MHC class I in KrasG12D/+;Med23+/+ mice. e The mRNA levels of B2m were confirmed by qRT‒PCR (n = 7 per group). f GO analysis of downregulated genes (fold change > 2) in MED23-knockdown A549 cells. The black line indicates the gene counts, and the blue bars indicate the statistics. g qRT‒PCR analysis of inflammation-related genes in lung tumors for both groups at 20 weeks post-Adeno Cre infection (n = 7 per group). h qRT‒PCR analysis of inflammation-related genes in A549 cells (n = 3 per group). i The mRNA level of B2M in A549 cells was analyzed by qRT‒PCR (n = 3 per group). The effect of Med23 knockdown (j) and Med23 overexpression (k) identified with a B2m promoter-luciferase reporter assay is shown (n = 3 per group). l The effect of overexpression of Med1 and Med24 on the B2m promoter-luciferase reporter assay is shown (n = 3 per group). Data are presented as the means ± SEMs. * P < 0.05, ** P < 0.01, *** P < 0.001.