Journal: PLOS One

Article Title: Impact of MED12 mutation and CDK8 activity on uterine leiomyoma growth and response to gonadotropin-releasing hormone agonist treatment

doi: 10.1371/journal.pone.0338485

Figure Lengend Snippet: (A) Representative western blot image; CTD-pS5 phosphorylated by the CDK8-MED12 complex and non-phosphorylated CTD protein (p–) are indicated in the upper part of the panel. Protein extracts were immunoprecipitated with anti-MED12 antibodies and immunoblotted with anti-CDK8 and anti-MED12 antibodies as indicated in the middle of the panel. Protein extracts were immunoblotted with anti-CDK8, anti-MED12, and anti-β-actin antibodies as indicated in the lower part of the image. (B–F) Graph of each protein band intensity according to MED12 status (WT or MUT) in GnRH agonist-treated (GnRH agonist+) and -untreated (GnRH agonist–) groups. * p < 0.05; ** p < 0.01. CTD, C-terminal domain (of the largest subunit of RNA polymerase II); CTD-pS5, C-terminal domain with phosphorylated 5th serine residue; GnRH, gonadotropin-releasing hormone; IP, immunoprecipitated; MUT, mutant; WT, wild type.

Article Snippet: Rabbit polyclonal anti-MED12 antibody (1 μg) (Bethyl Laboratories, Inc., Montgomery, TX) was cross-linked to 12.5 μL Dynabeads protein G (Thermo Fisher Scientific) and mixed with 380 μL (tissues) or 1 mL (cells) of protein extract at 4°C overnight.

Techniques: Western Blot, Immunoprecipitation, Residue, Mutagenesis

Journal: PLOS One

Article Title: Impact of MED12 mutation and CDK8 activity on uterine leiomyoma growth and response to gonadotropin-releasing hormone agonist treatment

doi: 10.1371/journal.pone.0338485

Figure Lengend Snippet: Primary leiomyoma cells were treated with Snx ± EP for 4 days followed by CDK8 phosphorylation assay and western blotting analysis. Results for the leiomyoma cells derived from two patients (LM1 and LM2) are shown. Data are normalized to vehicles and presented as the means of three independent experiments. (A) Representative western blot image: the CTD-pS5 protein is indicated in the upper part of the panel. Cell extracts were immunoprecipitated with an anti-MED12 antibody and immunoblotted with anti-CDK8 and anti-MED12 antibodies, as indicated in the middle of the panel. Protein extracts were immunoblotted with anti-CDK8, anti-MED12, and anti-ERα antibodies, as indicated in the lower part of the panel. Anti-β-actin was used as a loading control. (B) Quantification of phosphorylated CTD protein and immunoprecipitated protein. (C) Quantification of protein extracts. Protein levels were quantified, and expression data are presented relative to β-actin. The data are normalized to the control and presented as the mean of three independent experiments. Two-way ANOVA showed an interaction between Snx and EP at CTD-pS5 in LM2, and post-hoc test showed a significant difference between EP (–) Snx (–) and other groups. (*, p < 0.05; **, p < 0.01). CTD, C-terminal domain (of the largest subunit of RNA polymerase II); CTD-pS5, C-terminal domain with phosphorylated 5th serine residue; EP, 17-β estradiol (E2) + progesterone (P4); ERα, estrogen receptor alpha; IP, immunoprecipitated; Snx, Senexin B.

Article Snippet: Rabbit polyclonal anti-MED12 antibody (1 μg) (Bethyl Laboratories, Inc., Montgomery, TX) was cross-linked to 12.5 μL Dynabeads protein G (Thermo Fisher Scientific) and mixed with 380 μL (tissues) or 1 mL (cells) of protein extract at 4°C overnight.

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Immunoprecipitation, Control, Expressing, Residue

Journal: Molecular Medicine

Article Title: Functional characterization of the MED12 p.Arg1138Trp variant in females: implications for neural development and disease mechanism

doi: 10.1186/s10020-025-01365-5

Figure Lengend Snippet: MED12 patient-derived iPSCs and protein expression. A Schematic indicating the MED12 variant location and known protein domains. B MED12 3D modelling utilizing AlphaMissense indicating position and change of amino acid. C and D Targeted amplicon sequencing of gDNA and cDNA showing read counts and the percentage of reads aligning to MED12 WT or MED12 VUS. E Immunofluorescent staining of the variant and WT iPSCs indicating cellular localization of MED12 protein. White bar, 60 μm

Article Snippet: Membranes were blocked overnight at 4 °C with Intercept ® (TBS) Blocking Buffer (LI-COR Biosciences), then incubated with rabbit anti-human MED12 monoclonal antibody (1:1000; clone BLR084G; Bethyl Laboratories, USA) and/or β-actin antibody (1:2000; MA5-15729; Life Technologies, Australia).

Techniques: Derivative Assay, Expressing, Variant Assay, Amplification, Sequencing, Staining

Journal: Molecular Medicine

Article Title: Functional characterization of the MED12 p.Arg1138Trp variant in females: implications for neural development and disease mechanism

doi: 10.1186/s10020-025-01365-5

Figure Lengend Snippet: Neural disease modelling for MED12_WT and MED12_VUS. iPSCs were induced to differentiate into neural progenitor cells and examined for changes in morphology, marker expression, and MED12 protein levels at indicated timepoints. A Light-microscopy images of NPCs at days 18 and day 24 showing bipolar cells with long dendrites. B Flow cytometry gating strategy for iPSCs and NPCs during neural cell differentiation. C and D Bar graphs show the percentage of live cells expressing stem or neural markers, respectively. Timepoints as indicated. MED12_WT (blue), and MED12_VUS, (red). Mixed-model two-way ANOVA with Bonferroni’s multiple comparison test. ( n = 3 group). * p < 0.05; ** p < 0.01. E MED12 western blot in NPCs at day 24 of neural cell differentiation, and F Bar graph indicates MED12 protein expression, normalized to β- Actin expression, in MED12_WT and MED12_VUS NPCs

Article Snippet: Membranes were blocked overnight at 4 °C with Intercept ® (TBS) Blocking Buffer (LI-COR Biosciences), then incubated with rabbit anti-human MED12 monoclonal antibody (1:1000; clone BLR084G; Bethyl Laboratories, USA) and/or β-actin antibody (1:2000; MA5-15729; Life Technologies, Australia).

Techniques: Marker, Expressing, Light Microscopy, Flow Cytometry, Cell Differentiation, Comparison, Western Blot

Journal: Molecular Medicine

Article Title: Functional characterization of the MED12 p.Arg1138Trp variant in females: implications for neural development and disease mechanism

doi: 10.1186/s10020-025-01365-5

Figure Lengend Snippet: Changes in MED12 and components of the MKM during neural cell differentiation. MED12_WT and MED12_VUS iPSCs were stimulated for neural cell differentiation for transcriptomics analysis. A Upset plots showing DEGs common to WT and VUS differentiation at days 18 and 24. B Comparison of NPCs to public NPCs sourced from the ARCHS4 dataset. C GSEA GO-BP geneset enrichment indicates upregulation of neural pathways at day 18 and day 24 in NPCs compared to respective iPSCs. D and E Box plots indicate down-regulation of stem cell markers at and up-regulation of neural cell markers at day 24. F Box plots indicate changes in transcript expression for components of the MKM. G Bar graph shows the change in the MED12/MED12L ratio during differentiation. Boxplots adjusted p-value < 0.05; Bar graph, One tailed, unpaired t-test, p < 0.05

Article Snippet: Membranes were blocked overnight at 4 °C with Intercept ® (TBS) Blocking Buffer (LI-COR Biosciences), then incubated with rabbit anti-human MED12 monoclonal antibody (1:1000; clone BLR084G; Bethyl Laboratories, USA) and/or β-actin antibody (1:2000; MA5-15729; Life Technologies, Australia).

Techniques: Cell Differentiation, Comparison, Expressing, One-tailed Test

Journal: Molecular Medicine

Article Title: Functional characterization of the MED12 p.Arg1138Trp variant in females: implications for neural development and disease mechanism

doi: 10.1186/s10020-025-01365-5

Figure Lengend Snippet: MED12 variant alters gene expression and neural development. MED12_WT and MED12_VUS iPSCs were differentiated into NPCs and transcriptomic profiles of MED12_VUS and MED12_WT NPCs were compared. A Heatmap of top differentially expressed genes. B Treeplot of GO-BP terms enriched when comparing NPCs. Colour indicates normalised enrichment score, representing overall direction of enrichment. Red (positive) represents overall up-regulation, blue (negative) represents overall down-regulation

Article Snippet: Membranes were blocked overnight at 4 °C with Intercept ® (TBS) Blocking Buffer (LI-COR Biosciences), then incubated with rabbit anti-human MED12 monoclonal antibody (1:1000; clone BLR084G; Bethyl Laboratories, USA) and/or β-actin antibody (1:2000; MA5-15729; Life Technologies, Australia).

Techniques: Variant Assay, Gene Expression

Journal: Molecular Medicine

Article Title: Functional characterization of the MED12 p.Arg1138Trp variant in females: implications for neural development and disease mechanism

doi: 10.1186/s10020-025-01365-5

Figure Lengend Snippet: Neural cells carrying the MED12 variant show altered cell growth, specification, and ribosomal complex formation. The MED12_WT and MED12_VUS iPSCs were stimulated for neural cell differentiation, and transcriptomics performed using GSEA and the GO-CC data set. Treeplot demonstrates enriched GO-CC terms when comparing MED12_VUS NPCs to MED12_WT NPCs at days 18 and 24. Terms are clustered based on similarity in gene set. Colour indicates direction of enrichment, with red representing overall upregulation of gene set, blue representing overall downregulation

Article Snippet: Membranes were blocked overnight at 4 °C with Intercept ® (TBS) Blocking Buffer (LI-COR Biosciences), then incubated with rabbit anti-human MED12 monoclonal antibody (1:1000; clone BLR084G; Bethyl Laboratories, USA) and/or β-actin antibody (1:2000; MA5-15729; Life Technologies, Australia).

Techniques: Variant Assay, Cell Differentiation

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: The primary antibody was purchased from Cell Signaling Technology, and the following antibodies were used: MED12 (#14360), β-actin (#8457), phospho-AKT (#4060), phospho-ERK1/2 (#9101), phospho-ALK (#3341), cleaved PARP (#5625), CDK8 (#17395), MED13(#91684), CCNC (#68179), YAP (#14074), phospho-YAP (#13008), PTEN (#9559), and ubiquitin (#3933).

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: The primary antibody was purchased from Cell Signaling Technology, and the following antibodies were used: MED12 (#14360), β-actin (#8457), phospho-AKT (#4060), phospho-ERK1/2 (#9101), phospho-ALK (#3341), cleaved PARP (#5625), CDK8 (#17395), MED13(#91684), CCNC (#68179), YAP (#14074), phospho-YAP (#13008), PTEN (#9559), and ubiquitin (#3933).

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: The primary antibody was purchased from Cell Signaling Technology, and the following antibodies were used: MED12 (#14360), β-actin (#8457), phospho-AKT (#4060), phospho-ERK1/2 (#9101), phospho-ALK (#3341), cleaved PARP (#5625), CDK8 (#17395), MED13(#91684), CCNC (#68179), YAP (#14074), phospho-YAP (#13008), PTEN (#9559), and ubiquitin (#3933).

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: The primary antibody was purchased from Cell Signaling Technology, and the following antibodies were used: MED12 (#14360), β-actin (#8457), phospho-AKT (#4060), phospho-ERK1/2 (#9101), phospho-ALK (#3341), cleaved PARP (#5625), CDK8 (#17395), MED13(#91684), CCNC (#68179), YAP (#14074), phospho-YAP (#13008), PTEN (#9559), and ubiquitin (#3933).

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: The primary antibody was purchased from Cell Signaling Technology, and the following antibodies were used: MED12 (#14360), β-actin (#8457), phospho-AKT (#4060), phospho-ERK1/2 (#9101), phospho-ALK (#3341), cleaved PARP (#5625), CDK8 (#17395), MED13(#91684), CCNC (#68179), YAP (#14074), phospho-YAP (#13008), PTEN (#9559), and ubiquitin (#3933).

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: The primary antibody was purchased from Cell Signaling Technology, and the following antibodies were used: MED12 (#14360), β-actin (#8457), phospho-AKT (#4060), phospho-ERK1/2 (#9101), phospho-ALK (#3341), cleaved PARP (#5625), CDK8 (#17395), MED13(#91684), CCNC (#68179), YAP (#14074), phospho-YAP (#13008), PTEN (#9559), and ubiquitin (#3933).

Techniques: Mutagenesis, Activation Assay

Journal: Scientific reports

Article Title: The MicroRNA miR-454 and the mediator complex component MED12 are regulators of the androgen receptor pathway in prostate cancer.

doi: 10.1038/s41598-025-95250-0

Figure Lengend Snippet: Fig. 2. Effects of siMED12 and miR-454 on protein expression in LNCaP Abl EnzR cells. Western hybridization for protein detection of MED12, AR, and c-Myc in LNCaP Abl EnzR cells transfected with siMED12, miR-454-3p or combined siMED12/miR-454-3p. (A) Mean protein expression of three independent experiments. All the experiments represent three independent biological replicates. (B) Representative Western hybridization results.

Article Snippet: The primary antibodies used were monoclonal rabbit MED12 (clone D9K5J; Cell Signaling, Frankfurt a.M., Germany; 1:1000), monoclonal rabbit AR (clone D6F11; Cell Signaling, 1:1000), monoclonal rabbit AR-V7 (clone RM7; RevMAb Biosciences, Hamburg, Germany; 1:1000), monoclonal rabbit c-Myc (clone Y69, Abcam, Cambridge, UK; 1:1000) and monoclonal rabbit GADPH (clone 14C10, Cell Signaling; 1:1000).

Techniques: Expressing, Western Blot, Hybridization, Transfection

Journal: Scientific reports

Article Title: The MicroRNA miR-454 and the mediator complex component MED12 are regulators of the androgen receptor pathway in prostate cancer.

doi: 10.1038/s41598-025-95250-0

Figure Lengend Snippet: Fig. 3. Effects of siMED12 and miR-454 on protein expression in DuCaP EnzR cells. Western hybridization for protein detection of MED12, AR, AR-V7 and c-Myc in DuCaP EnzR cells transfected with siMED12, miR-454- 3p or combined siMED12/miR-454-3p. (A) Mean protein expression of three independent experiments. All the experiments represent three independent biological replicates. (B) Representative Western hybridization results.

Article Snippet: The primary antibodies used were monoclonal rabbit MED12 (clone D9K5J; Cell Signaling, Frankfurt a.M., Germany; 1:1000), monoclonal rabbit AR (clone D6F11; Cell Signaling, 1:1000), monoclonal rabbit AR-V7 (clone RM7; RevMAb Biosciences, Hamburg, Germany; 1:1000), monoclonal rabbit c-Myc (clone Y69, Abcam, Cambridge, UK; 1:1000) and monoclonal rabbit GADPH (clone 14C10, Cell Signaling; 1:1000).

Techniques: Expressing, Western Blot, Hybridization, Transfection