Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: TET3 affects DNA methylation and histone modifications of the MED12, TGFBR2, and TSP1 promoters. a UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative TET3 enrichment over input. n = 3. Red numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red-stripped bars. b Sequences of critical transcription regulatory regions (CTRR) of MED12 , TGFBR2 , and TSP1 . The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. c UtLM cells were transfected with siCon or siTET3 for 48 h, followed by QMSP analysis. n = 3. d UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative enrichment over input. n = 3. All data are representative of at least two independent experiments and are presented as mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: DNA Methylation Assay, Transfection, ChIP-qPCR, Methylation

Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: H19 and TET3 co-express with fibroid-promoting genes in vivo. a , c RT-qPCR analyses were performed on RNAs extracted from human fibroids and matched myometrium tissues. Spearman’s correlation showed positive correlations between expression of H19 and TET3 ( a , left panel), as well as TET3 and its target genes MED12 , TGFBR2 , and TSP1 ( c ) in a statistically significant manner. No correlation between expression of H19 and HMGA2 at the RNA level was detected ( a , right panel). Spearman’s correlation coefficient, p -values, and sample numbers are presented. b Results of western blotting analysis of HMGA2 in human fibroids and matched myometrium. n = 3. Data are representative of two independent experiments and are presented as mean ± SEM

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Mediator Subunit MED16 Collaborates with UBP1-TFCP2 to Control Transcriptional Activation or Repression via Promoter Positional Specificity

doi: 10.1101/2025.08.12.669905

Figure Lengend Snippet: (A) Co-IP experiment with antibodies against endogenous CDK8, MED1, and MED12 in 293T whole cell lysis. The resultant immunoprecipitates were washed by washing buffers containing 0%, 5%, and 10% of 1,6-hexanediol respectively. Input: 0.5% of the total lysate was loaded. (B) Co-IP experiment with antibodies against endogenous CDK8, MED1, and MED12 in HeLa nuclear extract. The resultant immunoprecipitates were washed by washing buffers containing 10% of 1,6-hexanediol or 10% 2,5-hexanediol. Input: 0.2% of the total HeLa nuclear extract was loaded. (C) Co-IP experiment with antibodies against endogenous MED1 in 293T whole cell lysis. The resultant immunoprecipitated was washed by washing buffers containing 150 mM, 300 mM, and 500 mM of NaCl respectively. Input: 0.2% of the total HeLa nuclear extract was loaded. (D) Gel filtration chromatography of HeLa nuclear extract. 500µl HeLa nuclear extract was applied to Superose 6 column then was run in Buffer D. Column fractions of 500µl were collected. The Void (void volumn) of Superose 6 is based on the volume of effluent required for the elution of blue dextran (molecular mass of ∼2000 kDa). The molecular weight of corresponding fractions was detected by protein standards. (E) Immunoblots of anti-MED16 immunoprecipitation in HeLa nuclear extract and gel filtration factions No.29 to No.31. The immunoprecipitated proteins were detected with indicated antibodies by western blotting. HeLa NE input: 0.2% of the total HeLa nuclear extract was loaded. (F) Immunoblots of anti-TFCP2 immunoprecipitation in HeLa nuclear extract and gel filtration factions No.29 to No.31. The immunoprecipitated proteins were detected with indicated antibodies by western blotting. HeLa NE input: 0.2% of the total HeLa nuclear extract was loaded.

Article Snippet: Antibody for Western blot include the following MED23 (Abcam, ab200351), MED1 (Bethyl Lab, A300-793A), MED24 (Bethyl, A301-472A), MED6 (Santa Cruz, sc-9434), MED12 (Bethyl Lab, A300-774A), MED16 (Bethyl Lab, A303-668A), CDK8 (Abcam, ab115155), UBP1 (Protientech, 67318-1-Ig), TFCP2 (Proteintech, 15203-1-AP), YY1 (Proteintech, 66281-1-Ig), P300 (Abcam, ab14984), HDAC1 (Santa Cruz, sc-7872), PolII (Abcam, ab816), PolII-pSer5 (Millipore, 04-1572), TFIIB (Santa Cruz, sc-274D), TFIIH (Santa Cruz, sc-293), H3 (Abcam, ab1791), H3ac (Abcam, ab4729), β-Actin (Proteintech, 66009-1-Ig), GAPDH (Proteintech, 60004-1-Ig), Flag-tag (Sigma-Aldrich, F1804), His-tag (Proteintech, 66005-1-Ig), GST-tag (Invitrogen, 10004D).

Techniques: Co-Immunoprecipitation Assay, Lysis, Immunoprecipitation, Filtration, Chromatography, Molecular Weight, Western Blot

Journal: bioRxiv

Article Title: Mediator Subunit MED16 Collaborates with UBP1-TFCP2 to Control Transcriptional Activation or Repression via Promoter Positional Specificity

doi: 10.1101/2025.08.12.669905

Figure Lengend Snippet: Venn diagram of MED1, MED12, CDK8 and MED16 interacting proteins identified by IP-MS. MED16 specific interacting proteins were highlight on the right.

Article Snippet: Antibody for Western blot include the following MED23 (Abcam, ab200351), MED1 (Bethyl Lab, A300-793A), MED24 (Bethyl, A301-472A), MED6 (Santa Cruz, sc-9434), MED12 (Bethyl Lab, A300-774A), MED16 (Bethyl Lab, A303-668A), CDK8 (Abcam, ab115155), UBP1 (Protientech, 67318-1-Ig), TFCP2 (Proteintech, 15203-1-AP), YY1 (Proteintech, 66281-1-Ig), P300 (Abcam, ab14984), HDAC1 (Santa Cruz, sc-7872), PolII (Abcam, ab816), PolII-pSer5 (Millipore, 04-1572), TFIIB (Santa Cruz, sc-274D), TFIIH (Santa Cruz, sc-293), H3 (Abcam, ab1791), H3ac (Abcam, ab4729), β-Actin (Proteintech, 66009-1-Ig), GAPDH (Proteintech, 60004-1-Ig), Flag-tag (Sigma-Aldrich, F1804), His-tag (Proteintech, 66005-1-Ig), GST-tag (Invitrogen, 10004D).

Techniques: Protein-Protein interactions

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: MED12 is over-expressed in glioblastoma patients and cell lines. GlioVis (a web application for data visualization and analysis to explore brain tumors expression datasets) was used for expression analysis of kinase subunits in TCGA-GBM (GBM (n) = 528, non-tumor (n) = 10) glioblastoma patient dataset. a Graph showing expression pattern of kinase subunits (MED12, MED13, CDK8, CCNC, MED13L, MED12L, CDK19) in TCGA_GBM patients (HG-U133A) versus non-tumor control. MED12L levels are shown from Rembrandt dataset. b Analysis of MED12 over-expression in glioblastoma in (i) Rembrandt patient dataset (GBM n = 219 non-tumor n = 28) (ii) Gravendeel patient dataset (GBM n = 159 non-tumor n = 8) (iii) Murat patient dataset (GBM n = 80 non-tumor n = 4). c Graph showing fold change in expression of MED12 in a cohort of 25 Indian glioblastoma patients versus non-tumor control (n = 3). d Graph showing fold change in expression of MED12 across three glioblastoma cell lines (A172, U87MG and T98G) versus normal brain RNA (Agilent MVP total brain mRNA). GAPDH was used for normalization. e Representative images of IHC of two glioma patients (patient id: 1572, gender: female, age: 55, glioma: high grade, antibody used: HPA003184; patient id: 1578, gender: male, age: 56, glioma: high grade, antibody used: HPA003184) showing over-expression in MED12 protein. Image was retrieved from the Human Protein Atlas. The graphical data points represent mean ± S.D of at least three independent experiments (* represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Expressing, Control, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: Clinical association of MED12 expression across different grades of glioma and its correlation with patient prognosis: a expression of MED12 across different subtypes of gliomas. Graph from GlioVis showing higher expression of MED12 in astrocytoma and oligodendroglioma as compared to glioblastoma in (i) TCGA_GBMLGG dataset (ii) Rembrandt dataset. (iii) Gravendeel dataset. b Expression of MED12 across low grade gliomas. Graph from GlioVis showing higher expression of MED12 in grade II and grade III tumors as compared to glioblastoma in (i) TCGA_GBMLGG dataset. (ii) Rembrandt dataset. (iii) Gravendeel dataset. c Prognosis of glioblastoma patients in relation to MED12 over-expression (i) Kaplan–Meier curve from GlioVis (CGGA dataset) showing significant correlation of MED12 high expression with poor patient prognosis in glioblastoma. (ii) Kaplan–Meier curve from Prognoscan database displaying significant correlation of MED12 high expression with poor patient prognosis in glioblastoma. (iii) Kaplan–Meier curve from GlioVis (Murat dataset) database displaying significant correlation of MED12 high expression with poor patient prognosis in glioblastoma. (iv, v, vi) Kaplan–Meier curve from GlioVis (TCGA_GBM, Rembrandt and Gravendeel dataset) database displaying no significant correlation of MED12 expression with patient prognosis in glioblastoma. d Prognosis of glioma patients in relation to MED12 over-expression. (i) Kaplan–Meier curve from Prognoscan analysing GSE4412-GPL96 dataset showing high expression of MED12 promotes patient prognosis in glioma. (ii) Kaplan–Meier curve from GlioVis analysing TCGA_GBMLGG dataset showing high expression of MED12 promotes patient prognosis in astrocytoma. (iii) Kaplan–Meier curve from CGGA showing high expression of MED12 promotes patient prognosis in grade II glioma

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Expressing, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: MED12 promotes cellular proliferation in glioblastoma. a, b Graph showing relative proliferation using MTT assay in cells transfected with MED12 siRNA or universal negative control siRNA in a A172 cells b T98G cells. c, d Graph showing relative proliferation using MTT assay in cells transfected with MED12 over-expressing plasmid or PC DNA 3.1(+) in c A172 cells d T98G cells. e Graph showing relative proliferation using cyquant cell proliferation assay in A172 cells having modulation in MED12 levels using MED12 siRNA and over-expressing plasmids as well as their respective controls. f Graph showing relative proliferation using cyquant cell proliferation assay in T98G cells having modulation in MED12 levels using MED12 siRNA and over-expressing plasmids as well as their respective controls. g, h Colony formation assay results in T98G and A172 cells upon g MED12 knockdown or h MED12 over-expression [(i) images of colonies (ii) graphical representation of fold change in number of colonies]. The graphical data points represent mean ± SD of at least three independent experiments (* represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: MTT Assay, Transfection, Negative Control, Expressing, Plasmid Preparation, CyQUANT Assay, Proliferation Assay, Colony Assay, Knockdown, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: MED12 promotes cellular migration in glioblastoma: a images of T98G and A172 cells showing wound healing assay post MED12 knockdown b graphical representation of the data showing relative migration. c Microscopic images of T98G and A172 cells showing wound healing assay post MED12 over-expression. d Graphical representation of the data showing relative migration. e Microscopic Images showing transwell migration assay post MED12 knockdown in T98G and A172 cells. f Graphical representation of the data. g Microscopic Images showing transwell migration assay post MED12 over-expression in T98G and A172 cells. h Graphical representation of the quantification of transwell migration assay using ImageJ. The graphical data points represent mean ± SD of at least three independent experiments (*represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Migration, Wound Healing Assay, Knockdown, Over Expression, Transwell Migration Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: MED12 inhibits apoptosis in glioblastoma: substrate based caspase 3/7 glo assay was performed to check for apoptosis. a, b Graph showing fold change in relative caspase 3/7 activity in cells having MED12 over-expression and knockdown in a A172 cells b T98G cells. c FACS based detection of annexin-V externalisation was checked in A172 cells post MED12 knockdown. d, e Western blotting was performed to detect cleaved PARP protein levels in A172 and T98G cells having d MED12 knockdown and e having MED12 over-expression. The actin blot was run on a different gel. The western blotting experiment was performed in duplicates. The graphical data points represent mean ± SD of at least three independent experiments (*represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Glo Assay, Activity Assay, Over Expression, Knockdown, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: MED12 physically interacts with Vitamin D Receptor and regulates the Vitamin D receptor pathway in glioblastoma: a schematic of work-flow for analysis of pathways affected by MED12 in glioblastoma. b Bar graph showing five topmost pathways affected by MED12 in glioblastoma cells. The data from whole transcriptome analysis post MED12 knockdown was analysed and significantly de-regulated genes (fold change ≥ ± 1.5, p value < 0.05) were used for pathway analysis using wiki-pathways. c, d Graph showing fold change in expression of genes of VDR pathway analysed by qRT-PCR post MED12 knockdown in c A172 and d T98G cells. e Surface representation of MED12 complex (blue colour) and VDR (yellow colour). f Interaction analysis of MED12 with VDR, LigPlot + image representing the hydrogen bond interactions between the MED12 (labelled in green colour) and VDR (labelled in pink colour). g, h Co-Immunoprecipitation results establishing physical interaction between MED12 and VDR. A172 (g), T98G (h) cells over-expressing MED12 and a flag tagged VDR were subjected to immunoprecipitation using MED12 antibody. Post precipitation western blotting with anti-flag antibody was performed to check for physical interaction between MED12 and VDR. Western Blotting with anti-MED12 was performed to confirm successful precipitation. The graphical data points represent mean ± SD of at least three independent experiments (*represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: MED12 enhances VDR mediated transcription induction of BCL6 and inhibits p53 expression in glioblastoma: a graph and gel image showing Chromatin Immunoprecipitation results indicating that enrichment of BCL6 promoter by VDR decreases significantly post MED12 knockdown. The experiment was performed in duplicates. b Graph showing fold change in p53 expression analysed by qRT-PCR post MED12 knockdown and over-expression in A172 cells. c Graph showing fold change in p53 expression analysed by qRT-PCR post MED12 knockdown and over-expression in T98G cells. d, e Western blotting results showing effects of MED12 modulation on BCL6 and p53 protein levels in A172 and T98G cells in d MED12 over-expressing cells e cells having MED12 knockdown. The western blotting experiment was performed in duplicates. The actin blot was run on a different gel. The graphical data points represent mean ± SD of at least three independent experiments (*represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Expressing, Chromatin Immunoprecipitation, Knockdown, Quantitative RT-PCR, Over Expression, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 is overexpressed in glioblastoma patients and serves as an oncogene by targeting the VDR/BCL6/p53 axis

doi: 10.1007/s00018-021-04056-6

Figure Lengend Snippet: Inhibition of apoptosis and p53 levels by MED12 is BCL6 mediated: simultaneous over-expression of BCL6 and MED12 knockdown was performed in A172 and T98G cells. a, b Graph showing fold change in p53 expression analysed by qRT-PCR upon simultaneous over-expression of BCL6 and MED12 knockdown in a A172 cells, b T98G cells. c, d Western blotting results showing effects of simultaneous knockdown of MED12 and BCL6 over-expression in c A172 cells, d T98G cells. The western blotting experiment was performed in duplicates. The actin blot was run on a different gel. e, f Graph showing fold change in relative caspase 3/7 activity in cells having BCL6 over-expression and MED12 knockdown in e A172 cells f T98G cells. g Cartoon summarizing the role of MED12 in glioblastoma. The illustration was created with BioRender.com. The graphical data points represent mean ± SD of at least three independent experiments (*represents p value < 0.05 and ** represents p value < 0.001). Error bars denote ± SD

Article Snippet: The membrane was incubated with primary antibody overnight (1:1000 dilution of MED12 antibody cell signaling technology #4529, 1:1000 dilution of cleaved parp antibody cell signaling technology #9541S, 1:200 dilution of p53 antibody Santacruz biotechnology # sc-126, 1:250 dilution of BCL6 antibody Thermo scientific #MA5-11493, 1:1000 dilution of β-actin antibody cell signaling technology #4970) at 4 °C followed by incubation with secondary antibody for 2 h (Santa Cruz Biotechnology anti-mouse # SC-2005, anti-rabbit# SC-2357, Dilution 1:7000).

Techniques: Inhibition, Over Expression, Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of CDK8 gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Genome Wide, CRISPR, shRNA, In Vitro, Cell Counting, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) The time course of the experiment. CH12F3-2A cells were transfected with either siControl or siMed12 and CIT stimulated CIT (+) as indicated and analyzed by flow cytometry [fluorescence-activated cell sorting (FACS)]. ( B ) Representation of siMed12 resistance wild-type Med12 R (WT R ) used for the CSR rescue experiment. Asterisk (*) mark shows the site of the si resistance. The FACS analysis showing the effect of different concentrations of WT R on IgA complementation efficiencies. ( C ) The reverse transcription quantitative PCR (RT-qPCR) graph showing the effect of siControl and siMed12 (20 pmol) on germline transcripts after normalization with endogenous β2 microglobulin (β2m). ( D ) LM-PCR showing the amplified Sμ region in respective samples followed by Southern blot analysis with 5′-Sμ–specific probe. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of respected samples served as an internal control. The triangles indicate 3× dilution in the DNA amount. Right: The ImageJ analysis showing the normalized band intensities in the respective samples. ( E ) The chromatin immunoprecipitation (ChIP)–qPCR estimating the γH2AX occupancy in Control/siMed12-treated cells. Values were normalized to the DNA input signals followed by the maximum value in each dataset. ( F ) Schematic view of the long-range interactions (LRI) that occurred at the IgH locus after CIT stimulation, which brings Sμ-Sα into close proximity. The representative gel picture of 3C-PCR, detecting the LRI between different IgH regions. GAPDH served as a loading control. Right: The ImageJ analysis showing the normalized band intensities in the respective samples. ( G ) PCR amplification scheme to detect IgH/c-Myc chromosomal translocations. Southern blot analysis of PCR-amplified fragments with a Myc-specific probe in indicated samples. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05); n.s. indicates insignificant difference.

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Transfection, Flow Cytometry, Fluorescence, FACS, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Southern Blot, Chromatin Immunoprecipitation, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) Schematic representation of various Med12 deletion mutants used in the study. ( B ) FACS analysis showing the IgA rescue efficiency by WT R and Med12 R deletion mutants in Med12 KD CH12F3-2A cells. Mock contains empty backbone vector. ( C ) LM-PCR assay detecting the DNA break in WT R and corresponding domain deleted constructs, followed by Southern blot with the 5′-Sμ–specific probe. GAPDH was used as internal control. The triangles indicate a 3× dilution in the DNA amount. Right: The ImageJ analysis showing the quantitative band intensities in the respective samples. ( D ) 3C-PCR analysis in WT R and indicated Med12 deletion constructs in Med12 KD cells. GAPDH PCR of the cross-linked DNA sample served as a loading control. Right: The ImageJ analysis showing the quantification of the band intensities in respective samples. ( E ) The schematic showing the overview of experiment. The Med12 LS and PQL domains tagged with V5 and/or Flag were cotransfected in human embryonic kidney (HEK) 293T cells as depicted (right) and probed as indicated antibodies. ( F ) Top: Schematic representation of the WT R Med12 protein and its associated various disease linked point mutations scattered over LS and PQL domains. The different color code corresponds to linked disease. Bottom: The IgA rescue efficiency in disease linked Med12 mutations in Med12 KD CH12F3-2A cells. ( G ) LM-PCR assay for estimation AID-induced DNA break rescue in WT R and corresponding disease-linked CSR-defective mutants in Med12 KD cells. Right: The ImageJ analysis showing the quantification of the band intensities in respective samples. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05). XLID, X-linked intellectual disability.

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Plasmid Preparation, Construct, Southern Blot, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) Schematic representation depicting the Med12 kinase module and core Mediator function involved in transcription regulation. Med12 kinase module consists of four proteins (Med12, Med13, CcyC, and cdk8), and Med13 act as an anchor and connects the kinase module to the core Mediator complex. CA inhibits the Med12 kinase activity by binding to Cdk8 and inhibits the RNAPII phosphorylation and gene transcription. ( B ) The bar plot showing the effect of CA on IgA switching. ( C ) Egr1 transcript abundance was estimated by RT-qPCR and served as positive control. ( D ) The bar plot showing the effect of Cdk8 knockdown (KD) on IgA switching and ( E ) corresponding RT-qPCR analysis. The values have been normalized to endogenous GAPDH. ( F ) Top: Schematic representation of UL-linked point mutations found on Med12. Bottom: The HEK293T cells were transfected with indicated Med12 constructs, and later, flag IP was performed. Mock shows untransfected cells. ( G ) The bar plot showing the effect of IgA complementation by UL-linked Med12 point mutations in siMed12 CH12F3-2A cells. ( H and I ) The effect of Med13 and Med13L KD on IgA switching. The CH12F3-2A cells were transfected with either siMed13 or siMed13L and CIT (+) for 24 hours and analyzed by FACS. The samples have been collected and processed for RT-qPCR analysis and plotted after the GAPDH normalization. ( J ) The schematic showing the stepwise dissection of the kinase domain and their components requirement for CSR. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05); n.s. indicates insignificant difference.

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Activity Assay, Binding Assay, Quantitative RT-PCR, Positive Control, Transfection, Construct, Dissection, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) Schematic showing the different regions present at the IgH locus; the black bar showing the position of the primers used for ChIP-qPCR amplification. ( B to D ) The ChIP-qPCR showing the enrichment in control and Med12 and/or p300 KD cells using the indicated antibodies. The values were normalized to the DNA input signals, followed by the maximum value in each dataset. ( E ) The bar plot showing p300 KD by siRNA and inhibition of p300 histone acetyltransferase (HAT) (p300 In ) activity by C646 (5 nM) and their effect on CSR. The samples were transfected and analyzed as mentioned before. ( F to H ) The ChIP-qPCR showing the relative enrichment in control and KD cells as indicated. The antibody used for ChIP is indicated on each panel. ( I to K ) The RT-qPCR data showing the effect of Med12 and p300 KD or p300 In on indicated transcripts relative to control. The data were normalized with endogenous β2m abundance. ( L ) Schematic showing the role of Med12-dependent differential epigenomic regulation at S and 3′RR regions. We propose that Med12 activates the 3′RR enhancer through p300, which marks H3 histone acetylation (black boll). The enhancer RNA (eRNA) produced from the activated enhancers regulates the AID-induced DNA break formation by regulating the histone marks γH2AX (red boll) and H3K4me3 (purple boll) at S regions. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05).

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Amplification, Inhibition, Activity Assay, Transfection, Quantitative RT-PCR, Produced, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) The constructs showing the dead Cas9 (dCas9) fused with p300core HAT domain (dCas9-p300 C ) and expressed from cytomegalovirus (CMV) promoter. The small guide RNA (sgRNA) designed for hs1.2 and hs4 enhancers were expressed from the U6 promoter. The cotransfection of dCas9-p300 C and either sgRNAs (hs1.2/hs4) in CH12-F3-2A cells showing site-specific enhancer activation at 3′RR. ( B ) The ChIP-qPCR showing the enrichment of H3K27ac histone in dual-transfected dCas9-p300 C with either sgRNAs in Med12 KD cells. ( C ) Corresponding RT-qPCR data showing the enhancer transcripts level. ( D ) The bar plot showing the IgA rescue efficiency by dCas9-p300 C –mediated enhancer activation in Med12 KD CH12F3-2A cells. The black bar and gray bar showing the control samples. The left blue bars showing the rescue efficiency in WT R cells. The right two blue bars showing the dual-transfected dCas9-p300 C with either sgRNAs (hs1.2 and hs4) in Med12 KD cells. ( E ) LM-PCR assay estimating DNA break rescue in indicated samples as described above. Right: The ImageJ analysis showing the band intensities in the respective samples. ( F and G ) The ChIP assay was performed using the indicated antibodies, followed by qPCR showing the enrichment at S regions in dual-transfected dCas9-p300 C with sgRNAs (hs1.2 and hs4) in Med12 KD cells. ( H ) The 3C-PCR assay showing long-range interaction (LRI) in dual-transfected dCas9-p300 C with Med12 KD cells. Bottom: The ImageJ analysis showing the band intensities in the respective samples. ( I ) The ChIP-qPCR showing the AID enrichment in dual-transfected dCas9-p300 C with sgRNAs in Med12 KD cells. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05).

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Construct, Cotransfection, Activation Assay, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A and B ) The ChIP-qPCR showing the occupancy of Jmjd6 at 3′RR in control and either Jmjd6- or Med12-depleted cells. ( C ) Top: The FACS data showing the effect of Jmjd6 and Carm1 KD by respective siRNAs. Bottom left: The RT-qPCR data showing the KD efficiency by respective samples, and the data were normalized with β2m abundance. ( D ) The bar plot showing the effect of Jmjd6 and Carm1 KD on 3′RR transcripts. ( E and F ) The ChIP assay was performed by indicated the antibodies, followed by qPCR in control and Jmjd6 KD cells. ( G ) The bar plot showing the IgA rescue efficiency in dual-transfected dCas9-p300 C with hs4 sgRNAs in Jmjd6 and/or Carm1 KD cells. ( H ) The FACS analysis showing the IgA rescue efficiency in methylation-defective Med12 mutants in Med12 KD cells. The position of the mutations at PQL domain is shown at the right. ( I ) The RT-qPCR analysis showing the rescue of hs1.2 and hs4 transcripts in WT R and ΔPQL Med12 mutant in Med12 KD cells. ( J ) The schematic representation showing the sequential steps of Med12 workflow in CSR. Med12 is methylated by Jmjd6/Carm1 complex (magenta) at different positions (black boll). Methylation recruits p300 protein to 3′RR, which marks histone H3K27 acetylation and activate the enhancers. Activated enhancers were transcribed into eRNA, which regulates H3K4me3 at S region and recruits DNA break and repair complex for CSR. The result summarizes the means ± SD of three independents experiments and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05). NHEJ, nonhomologous end joining.

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Quantitative RT-PCR, Transfection, Methylation, Mutagenesis, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) FACS data showing the effect of antisense oligos (ASOs) on IgA switching. ( B ) RT-qPCR data showing the effect of ASOs on GLTs and hs4 transcripts relative to control. ( C ) Left: LM-PCR assay showing the effect of AID-induced DNA breaks in ASOs treated cells. Bottom panel shows the semi-qPCR analysis of GAPDH of respected samples as an internal control. Middle: The ImageJ analysis showing the normalized band intensities in the respective samples. Right: The Western blotting (WB) showing the effect of both ASOs on AID protein expression. ( D ) The ChIP assay was performed using the indicated antibodies, followed by qPCR showing the enrichment in control and ASO1 KD cells. ( E ) 3C assay showing the LRI in control and ASO1 KD cells. GAPDH PCR of the cross-linked DNA sample served as a loading control. ( F ) Schematic representation of Med12 protein showing the position of mutations found in nonspecific XLID disease. The IgA rescue efficiency has been determined in XLID-associated triple mutant (HQT) in Med12 KD cells. ( G ) The RT-qPCR showing the effect of Med12 HQT mutant on 3′RR transcription. ( H ) The proposed model showing the role of Med12 in enhancer activation. Med12 recruits p300 at 3′RR enhancers, which, in turn, catalyzes the H3K27ac histone acetylation. Med12-p300 complex initiates the enhancer activation, which transcribed into the eRNA. The eRNA produced from the 3′RR and recruits Med12, H3K4me3 methyltransferase, and DNA break complex at S regions. In the absence of Med12, p300 does not recruit to 3′RR, resulting in inactivation of enhancers and perturbed DNA break and S-S synapse formation. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05).

Article Snippet: The V5 tag was PCR amplified from Med12-pLX307 (Addgene, no. 98350) and fused to C-terminals of each domain using In-Fusion HD Cloning Kit (Takara).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Mutagenesis, Activation Assay, Produced, Two Tailed Test

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: List of recurrent mutations found in 27 WGS and 400 capture sequencing cases

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Sequencing, Mutagenesis

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: Ranking of recurrently mutated genes with bases on mutation rate

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Mutagenesis

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: MED12 mutations with three different pathogenicity scores

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Mutagenesis

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: Schematic diagram of distribution of 14 MED12 mutations identified in 12 colorectal cancer cases.

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques:

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: 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.

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Sequencing, Methylation, Mutagenesis

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: (A) Expression of MED12 in colorectal cancer (CRC) cell lines. Proteins were isolated from eight CRC cell lines and immunoblotted with antibodies against MED12 and GAPDH. (B) Representative images of fluorescence immunostaining for MED12 in four selected CRC cell lines and confirm results from western blot analysis. (C and D) CRC cells were treated with increasing doses of 5-fluorouracil (FU) for 48 hours and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (C) and apoptosis by flow cytometry after staining the cells with annexin V/propidium iodide (D). 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.

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Expressing, Isolation, Fluorescence, Immunostaining, Western Blot, Flow Cytometry, Staining, Control

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: 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 (6×10 2 ) 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)-β-R2, p-ERK1/2, ERK1/2, E-cadherin, N-cadherin, vimentin, Twist and β-actin for equal loading. (E) Representative images of fluorescence immunostaining for MED12, TGF-β-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.

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Transfection, Small Interfering RNA, Clonogenic Assay, Staining, Flow Cytometry, Isolation, Fluorescence, Immunostaining, Control

Journal: Gut

Article Title: MED12 is recurrently mutated in Middle Eastern colorectal cancer

doi: 10.1136/gutjnl-2016-313334

Figure Lengend Snippet: 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 (6×10 2 ) 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)-β-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-β-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.

Article Snippet: Plasmid DNA encoding human MED12 was purchased from OriGene.

Techniques: Expressing, Transfection, Plasmid Preparation, Clonogenic Assay, Staining, Flow Cytometry, Isolation, Fluorescence, Immunostaining, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

doi: 10.1007/s00018-025-05791-w

Figure Lengend Snippet: MED12 mutation induces resistance to RTK inhibitors in NSCLC via the suppression of its expression and predicts poor survival to RTK inhibitors in NSCLC patients A Cell viability of the ceritinib-resistant H3122 cell line (H3122CR) was assessed using the MTS assay. B Targeted sequencing analysis of H3122CR identified the presence of the L1283P (3848T > C) mutation in the MED12 gene, represented as a proportion (%) among various gene mutations. C Western blotting analysis confirmed decreased MED12 expression in both ceritinib-resistant and mutant MED12 overexpression cell line compared to the control group. D Western blot analysis validated the efficient knockout of MED12 in the generated knockout (KO) cell lines, exhibiting a notable reduction in MED12 protein expression compared to the control group. E , F MTS assay was performed to evaluate the sensitivity of MED12 KO cell lines to RTK inhibitors (ceritinib, alectinib, lorlatinib, and osimertinib). Cell viability of MED12 KO cell lines was compared to the respective control cell lines. Re-expression of wild-type MED12 in KO cells restored RTK inhibitor sensitivity to levels comparable to the parental cells. G Kaplan-Meier survival analysis of progression-free survival (PFS) for EGFR-TKI or ALKi treated NSCLC patients with MED12 mutations compared to those without. The hazard ratio (HR) was 1.979 (p-value = 0.0246), indicating an unfavorable prognosis for patients with MED12 mutations following RTKi treatment

Article Snippet: MED12 ( NM_005120 ) Human Tagged ORF Clone (CAT#: RC217206) was purchased from OriGene Technologies.

Techniques: Mutagenesis, Expressing, MTS Assay, Sequencing, Western Blot, Over Expression, Control, Knock-Out, Generated

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

doi: 10.1007/s00018-025-05791-w

Figure Lengend Snippet: MED12 mutation, L1283P, induces its proteasomal degradation, which lead to break of MED12 complex. A Western blot analysis showing the protein expression levels of MED12 complex components (MED12, CDK8, MED13, CCNC) in the parental cell line (H3122) and MED12 knockout (KO) cell line with mutant MED12 overexpression (OE) before and after treatment with 2 µM MG132 (proteasome inhibitor) for 24 h. B Co-immunoprecipitation was performed to assess the direct interaction between the mutant MED12 and ubiquitin. Protein lysates from the MED12 KO/mutant MED12 OE cell line were immunoprecipitated with anti-MED12 antibody, followed by immunoblotting with anti-ubiquitin antibody. C Fluorescence imaging of GFP-tagged MED12 in the MED12 KO/mutant MED12 OE cell line before and after treatment with MG132, demonstrating the blockade of ubiquitin-mediated proteasomal degradation

Article Snippet: MED12 ( NM_005120 ) Human Tagged ORF Clone (CAT#: RC217206) was purchased from OriGene Technologies.

Techniques: Mutagenesis, Western Blot, Expressing, Knock-Out, Over Expression, Immunoprecipitation, Ubiquitin Proteomics, Fluorescence, Imaging

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

doi: 10.1007/s00018-025-05791-w

Figure Lengend Snippet: Inflammatory cytokines release by MED12 mutation induce RTK inhibitor resistance through only the MEK/ERK pathway activation, not the PI3K/AKT pathway. A Gene Set Enrichment Analysis (GSEA) of RNA-seq data from NSCLC patients with MED12 mutations, as well as MED12 knock-out H3122 and PC9 cell lines, showing significant enrichment in the cytokine-cytokine receptor interaction gene set of the KEGG_LEGACY subset. B Western blot analysis confirming elevated chromatin-bound MED1 levels in MED12 knock-out H3122 and PC9 cell lines compared to parental cell lines. C Olink proteomics analysis of culture media from MED12 knock-out cell lines, identifying increased levels of inflammatory cytokines. D Cell image of the trans-well insert co-culture system used to expose parental NSCLC cell lines to cytokines released from MED12 knock-out cell lines, resulting in increased RTKi resistance, assessed by clonogenic assay. Schematic of the trans-well co-culture system was created with BioRender.com. E Western blot analysis showing the activation of both AKT and ERK1/2 pathways in parental cell lines exposed to cytokines from MED12 knock-out cell lines. F In H3122/MED12 KO cell lines, inhibition of the AKT pathway and activation of the ERK1/2 pathway were observed by Western blotting analysis. G Western blot analysis demonstrating the reactivation of the AKT pathway and suppression of the ERK1/2 pathway in H3122/MED12 KO cell lines after overexpression of wild type MED12

Article Snippet: MED12 ( NM_005120 ) Human Tagged ORF Clone (CAT#: RC217206) was purchased from OriGene Technologies.

Techniques: Mutagenesis, Activation Assay, RNA Sequencing, Knock-Out, Western Blot, Co-Culture Assay, Clonogenic Assay, Inhibition, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

doi: 10.1007/s00018-025-05791-w

Figure Lengend Snippet: MEK inhibitor, trametinib, alone overcomes RTK-induced resistance by repression of MED12 expression by its mutation. A MTS assay was performed to assess the cell viability of MED12 KO cell lines and control cell lines treated with ceritinib/osimertinib and trametinib. B Caspase 3/7 assay was performed to evaluate apoptosis in MED12 KO cell lines and control cell lines treated with ceritinib, osimertinib, or trametinib, with or without co-treatment of the pan-caspase inhibitor Z-VAD-FMK. C Western blot analysis showing the expression of the apoptosis marker cleaved PARP in MED12 KO cell lines treated with trametinib compared to control groups. Ceritinib and trametinib were each administered at a concentration of 200 nM for 72 h. D Tumor volume measurements of xenograft mouse models implanted with parental cell line (H3122, n = 3) and MED12 knockout (KO) NSCLC cells (H3122/MED12 KO, n = 3) and treated with daily administration of vehicle, ceritinib, or trametinib for three weeks. E Representative images of tumors obtained from the xenograft mouse model showing the differences in tumor growth among the treatment groups. F Immunohistochemistry (IHC) analysis performed on tumor tissues derived from the xenograft mouse model to evaluate the expression levels of Ki-67, a proliferation marker, and MED12

Article Snippet: MED12 ( NM_005120 ) Human Tagged ORF Clone (CAT#: RC217206) was purchased from OriGene Technologies.

Techniques: Expressing, Mutagenesis, MTS Assay, Control, Western Blot, Marker, Concentration Assay, Knock-Out, Immunohistochemistry, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

doi: 10.1007/s00018-025-05791-w

Figure Lengend Snippet: Inhibiting physical interaction between MED12 and YAP leads to increased PTEN expression and subsequent inhibition of the AKT pathway. A Co-immunoprecipitation was performed to confirm the direct interaction between MED12 and YAP. Protein lysates from the cell lines were immunoprecipitated with anti-MED12 antibody, followed by immunoblotting with anti-YAP antibody. B Western blot analysis showing increased levels of phospho-YAP (ser127) and PTEN in the MED12 KO cell line compared to the control, indicating the inhibition of the PI3K/AKT pathway. C Restoration of wild type MED12 expression in the MED12 KO cell line resulted in decreased levels of phospho-YAP (ser127) and PTEN, indicating the reactivation of the PI3K/AKT pathway. D The expression levels of miR-29, a mediator of PTEN suppression by YAP, were downregulated in the MED12 KO cell line and restored after wild-type MED12 recovery, as determined by qPCR analysis. E Western blot analysis showing the effects of YAP overexpression (YAP, YAP-5SA, YAP-S94A) in both the parental (H3122) and MED12 KO (H3122/MED12 KO) cell lines. Changes in PTEN and phospho-AKT (p-AKT) levels were evaluated. F The expression levels of miR-29 in the different YAP overexpressed cell lines were evaluated by qRT-PCR. G Resistance to ceritinib and trametinib in the various YAP overexpressed cell lines was assessed using MTS assay

Article Snippet: MED12 ( NM_005120 ) Human Tagged ORF Clone (CAT#: RC217206) was purchased from OriGene Technologies.

Techniques: Expressing, Inhibition, Immunoprecipitation, Western Blot, Control, Over Expression, Quantitative RT-PCR, MTS Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

doi: 10.1007/s00018-025-05791-w

Figure Lengend Snippet: MEK inhibitor could be the most suitable treatment option for MED12 mutation-induced RTK inhibitor-resistant NSCLC. Created with BioRender.com Schemas illustrating the identified mechanisms of MED12 mutation-mediated resistance to RTK inhibitions. A Targeting EGFR or ALK in EGFR-mutated or EML4-ALK/wild-type MED12 cells. B Although MED12 mutation-induced inflammatory cytokine induces both PI3K/AKT activation and MEK/ERK activation, MED12 mutation blocks PI3K/AKT activation by PTEN induction via YAP regulation. Therefore, MEK inhibitor alone provides sufficient benefit for MED12-mutated RTKi-resistant NSCLC. Solid lines indicate the effects

Article Snippet: MED12 ( NM_005120 ) Human Tagged ORF Clone (CAT#: RC217206) was purchased from OriGene Technologies.

Techniques: Mutagenesis, Activation Assay