Fig 2: Sanger sequencing electropherogram of inactive/active allele of MED12 gene on chromosome X in eight female cases harbouring MED12 mutations. Top panel: sequencing electropherogram of enriched methylated DNA indicating no mutation was identified on the inactive allele caused by methylation. Lower panel: sequencing electropherogram of enriched unmethylated and unbounded methylated DNA. WT, wild type.

Fig 3: Forced expression of MED12 decreases the chemoresistance in colorectal cancer (CRC) cell lines. (A and B) CRC cells were transfected with either empty vector or MED12 pcDNA for 48 hours and clonogenic assay were performed. Cells (6×102) after post-transfection were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet (A). Colony numbers in the entire dish were counted (B). (C) CRC cells were transfected with either empty vector or MED12 pcDNA and subsequently treated with 50 and 100 µM 5-fluorouracil (FU) for 48 hours. Following treatment, cells were analysed for apoptosis by flow cytometry. (D) COLO-320 and HT29 cells were transfected with either empty vector or MED12 pcDNA for 48 hours. Proteins were isolated and immunoblotted with antibodies against MED12, transforming growth factor (TGF)-ß-R2, p-ERK1/2, ERK1/2, E-cadherin, N-cadherin, vimentin, Twist and GAPDH for equal loading. (E) Representative images of fluorescence immunostaining for MED12, TGF-ß-R2 and E-cadherin in COLO-320 and HT29 cells after post-transfection with MED12 pcDNA. Data presented in the bar graphs are the mean±SD of three independent experiments. *Indicates a statistically significant difference compared with control with p<0.05.

Fig 4: Silencing of MED12 increases the chemoresistance in colorectal cancer (CRC) cell lines. (A and B) CRC cells were transfected with either scrambled siRNA or MED12-specific small interfering RNA (siRNA) for 48 hours and clonogenic assay were performed. Cells (6×102) after post-transfection were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet (A). Colony numbers in the entire dish were counted (B). (C) CRC cells were transfected with either scrambled siRNA or MED12 siRNA and subsequently treated with 50 and 100 µM 5-fluorouracil (FU) for 48 hours. Following treatment, cells were analysed for apoptosis by flow cytometry. (D) LOVO and DLD1 cells were transfected with either scrambled siRNA or MED12-specific siRNA for 48 hours. Proteins were isolated and immunoblotted with antibodies against MED12, transforming growth factor (TGF)-ß-R2, p-ERK1/2, ERK1/2, E-cadherin, N-cadherin, vimentin, Twist and ß-actin for equal loading. (E) Representative images of fluorescence immunostaining for MED12, TGF-ß-R2 and E-cadherin in LOVO and DLD1 cells after post-transfection with MED12 siRNA. Data presented in the bar graphs are the mean±SD of three independent experiments. *Indicates a statistically significant difference compared with control with p<0.05.

Fig 5: Schematic diagram of distribution of 14 MED12 mutations identified in 12 colorectal cancer cases.