Fig 2: MED13L promotes malignant phenotypes of NSCLC cells. (A) Significantly elevated MED13L expression was observed in NSCLC tissues compared with normal specimens (Shandong cohort) (n = 46). (B) Concurrent chemoradiotherapy or radiotherapy treated NSCLC patients with low MED13L expression exhibited significantly prolonged PFS and OS (Jiangsu cohort) (n = 108). (C) Silencing of MED13L expression (si13L-1 and si13L-2) evidently inhibited PC9, A549 and H1299 cell growth. (D) Both si13L-1 and si13L-2 suppressed clonogenicity of NSCLC cells. (E) Mice with PC9 xenografts were treated with 4 × 3 Gy X-ray radiation. (F) Growth and weights of xenografts with stably ectopic miR-4497 or stably depleted MED13L treated with radiation or not (n = 4/group). No differences of mice weight between different groups were observed. (G) HE, Ki67 and MED13L staining of tumors from different groups. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by paired Student's t test (A) or by unpaired Student's t test (C, F).

Fig 3: Silencing MED13L globally diminishes H3K27ac signals. (A) Upper panel: Line plots showing the distribution of H3K27ac ChIP-Seq signals (TSS ± 3 kb). Lower panel: Heatmap showing ChIP-Seq signals at H3K27ac peak regions (TSS ± 3 kb), rank ordered by intensity of H3K27ac peaks based on reads per million mapped reads (RPM). Lines, peaks; color scale of peak intensity is shown at the bottom. (B) Integrative analyses of miR-4497 and MED13L (si13L-1 and si13L-2) co-regulated downstream genes with significant changes of H3K27ac modification (n = 247). (C) The KEGG pathway analyses of 247 genes with evident H3K27ac changes upon MED13L silencing via miR-4497 or its siRNAs. (D) ChIP-Seq profiles for H3K27ac at PRKCA, SOS2, AKT3, PAK4, or ARAF gene. (E) RT-qPCR analyses of PRKCA, SOS2, AKT3, PAK4, and ARAF gene expression in PC9 and A549 cells after forced expression of miR-4497 or silencing of MED13L. (F) Ectopic miR-4497 or silencing of MED13L can down-regulate PRKCA expression. ACTIN levels were measured as loading controls in Western blot. (G) Chromatin regions 1, 2, 3, 4, 5 and 6 around the TSS of PRKCA are detected using P300 ChIP-qPCR. (H) ChIP-qPCR results showed P300 can directly bind to chromatin regions 2, 3 and 5, but not regions 1, 4 and 6 in NSCLC cells. Silencing of MED13L by miR-4497 or siRNAs could markedly reduce P300 binding to regions 2, 3 and 5. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Student's t test, ns: not significant.

Fig 4: MED13L bridges the CDK8 kinase module with the core Mediator. (A, B) Immunoprecipitation (IP) experiments using antibodies to MED13L, MED6, MED8, MED1, MED4, MED23, MED12, CCNC and CDK8 in PC9 and A549 cells. (C) A carton model indicating the role of MED13L as a vital linker between the core Mediator and the kinase module. (D) IP experiments between P300, MED13L and MED12 in NSCLC cells.