Fig 2: The expression of MED16 is associated with breast cancer tumorigenesis. (A,B) Gene expression data of MED16 in breast cancer and normal tissues from the TCGA and GEO databases showed that MED16 mRNA level was overexpressed in human breast cancer (C) RT-PCR was adopted to detect the mRNA expression level of MED16 in tumour and adjacent tissues (n = 25). (The values are compared to normal group and are represented as the means ± S.E.M (ns: not significant, ** p < 0.01). (D) MED16 expression in breast cancer subtypes from the TCGA cohort. (E,F) Western blot and RT-qPCR were used to detect the MED16 expression in different subtype BC cell lines. (G) IHC staining of MED16 expression in normal and ER+ BC tissues (the bar on the figure represents 50 µm), mostly located in the cytoplasm.

Fig 3: Knockdown of MED16-induced autophagy and inhibited PI3K/AKT/mTOR pathway in ER+ BC cells. (A) Autophagy marker P62, LC3A/B protein expression in T47D-WT and TamR-T47D. (B) Autophagy-related marker P62, LC3A/B protein expression in overexpression of MED16 and knockdown of MED16 in MCF7 and T47D. (C) The effect on control (Parental), MED16 knockout (KO2) and autophagy inhibitor CQ treated (KO2 + CQ) MCF7 cells with different doses of 4OH-TAM at 72 h. (ns: not significant, ** p < 0.01; *** p < 0.001) (D) Autophagy marker LC3A/B protein expression in 4OH-TAM and CQ treated in control (Parental), MED16 knockout (KO2) MCF7 cells. (E) mTOR, phospho-mTOR, PI3K, phospho-PI3K, AKT, phospho-AKT protein expression in overexpression of MED16 and knockdown of MED16 compared with control MCF7 cells. (F) Effect on control (Parental), MED16 knockout (KO2) and mTOR activator-treated (KO2 + MHY1485) MCF7 cells with different doses of 4OH-TAM at 72 h.

Fig 4: Knockdown of MED16 hinders proliferation of BC cells. (A,B) RT−qPCR and western blot revealed the expression of MED16 was overexpressed and inhibited remarkably in MCF7 and T47D. (C) Growth curve of MCF7 and T47D cells infection of MED16−negative control and MED16−knockdown was displayed. Relative proliferation ratio against day 0 were observed for 5 days (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (D) Colony formation of MED16−overexpression cells and MED16-knockdown cells compared with MED16-negative control cells. (E) Representative fluorescence images of Edu staining of MCF7 and T47D cells. Proliferating MCF7 and T47D positively stained with Edu showed red colour. Cell nuclei stained with DAPI showed blue colour. The length of bar represents 750 μm, chart of proliferating cells (red) to total cells (blue). (F) Knockdown of MED16 induced G1−phase cell cycle arrested. Flow cytometric assay was employed to analyse the cell cycle distribution. Percentage of cells in the G1 phase was evidently increased, compared with the control group. (G) c-MYC, cyclinD1 protein expression in MCF7 and T47D cells with control (Vector) and MED16-overexpressed cells, control (shNC) and MED16−knockdown cells (sh1, sh2).

Fig 5: Downregulation of MED16 reduces tamoxifen sensitivity in ER+ breast cancer cells. (A) Gene expression data of MED16 in T47D wild-type and tamoxifen-resistant T47D from GEO database (GSE125738). (B) 4OH-Tamoxifen sensitivity of tamoxifen-resistant T47D. (ns: not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) (C) Protein level of MED16 in WT and TAMR of T47D. (D) The MED16 protein expression levels of different time gradients at 1 μM 4OHT treatment. (E) Effect on control (vector) and MED16-OE, control (Parental) and MED16 knockout (KO1, KO2) MCF7 cells with different doses of 4OH-TAM at 72 h respectively. (F) Colony formation assay of untreated and treated with 4OHT (1 μM)in control (Parental) and MED16 knockout (KO2) MCF7 cells. (G) A micrograph showing mammosphere formation in control (Parental) and MED16 knockout (KO2) MCF7 cells. The length of bar represents 750 μm.