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Cell Signaling Technology Inc anti med12
Anti Med12, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti med12/product/Cell Signaling Technology Inc
Average 92 stars, based on 18 article reviews

anti med12 - by Bioz Stars, 2026-06

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CTCF motif is highly enriched in LP-specific RUNX1-binding regions and LP-specific interaction of RUNX1 with CTCF. (A) ChIP-seq analysis of RUNX1 was performed using Cas9-LPs with or without Notch stimulation. The Venn diagram shows the numbers of reproducible RUNX1 nonpromoter ChIP peaks in LPs (Notch-) and Notch-stimulated LPs for 2 days (Phase 1; Notch+). (B) Top three enriched sequence motifs among the 4,832 LP-specific, the 11,014 Phase 1–specific, and 17,550 shared reproducible RUNX1 peaks between LP and Phase 1 are shown. Data are based on ChIP-seq peaks scored as reproducible in two replicate samples. (C) Myc- and FLAG-tagged RUNX1-ERT2 vectors were retrovirally transduced into Cas9-LPs. Total extracts from Myc-FLAG-RUNX1-ERT2–expressing LPs treated with tamoxifen for 6 h were subjected to two-step affinity purification followed by SDS-PAGE and silver staining. All of the visible bands were analyzed using mass spectrometry analysis. Phase 1 and Phase 2 cells were stimulated with Notch ligand on OP9-DLL4 for 2 and 10 days, respectively. (D) Representative RUNX1-binding molecules in LP, Phase 1, and Phase 2 cells are shown with Mascot scores. The full list of RUNX1-binding molecules is provided in . (E) Total extracts from Mock- or Myc-FLAG-RUNX1-ERT2–transduced and tamoxifen-treated LPs, with or without Notch stimulation, were subjected to IP with anti-FLAG and anti-Myc mAbs followed by immunoblotting with anti-CTCF, anti-Notch1-IC, or anti-Med12 antibodies (left panels). Nuclear lysates (input) were also immunoblotted with anti-CTCF, anti-Notch1-IC, anti-Med12, and anti-Myc (RUNX1) antibodies, whereas cytoplasmic lysates (input) were immunoblotted with anti-tubulin-α mAb (right panels). Data are representative of three independent experiments. IP, immunoprecipitation. Source data are available for this figure: .

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: CTCF motif is highly enriched in LP-specific RUNX1-binding regions and LP-specific interaction of RUNX1 with CTCF. (A) ChIP-seq analysis of RUNX1 was performed using Cas9-LPs with or without Notch stimulation. The Venn diagram shows the numbers of reproducible RUNX1 nonpromoter ChIP peaks in LPs (Notch-) and Notch-stimulated LPs for 2 days (Phase 1; Notch+). (B) Top three enriched sequence motifs among the 4,832 LP-specific, the 11,014 Phase 1–specific, and 17,550 shared reproducible RUNX1 peaks between LP and Phase 1 are shown. Data are based on ChIP-seq peaks scored as reproducible in two replicate samples. (C) Myc- and FLAG-tagged RUNX1-ERT2 vectors were retrovirally transduced into Cas9-LPs. Total extracts from Myc-FLAG-RUNX1-ERT2–expressing LPs treated with tamoxifen for 6 h were subjected to two-step affinity purification followed by SDS-PAGE and silver staining. All of the visible bands were analyzed using mass spectrometry analysis. Phase 1 and Phase 2 cells were stimulated with Notch ligand on OP9-DLL4 for 2 and 10 days, respectively. (D) Representative RUNX1-binding molecules in LP, Phase 1, and Phase 2 cells are shown with Mascot scores. The full list of RUNX1-binding molecules is provided in . (E) Total extracts from Mock- or Myc-FLAG-RUNX1-ERT2–transduced and tamoxifen-treated LPs, with or without Notch stimulation, were subjected to IP with anti-FLAG and anti-Myc mAbs followed by immunoblotting with anti-CTCF, anti-Notch1-IC, or anti-Med12 antibodies (left panels). Nuclear lysates (input) were also immunoblotted with anti-CTCF, anti-Notch1-IC, anti-Med12, and anti-Myc (RUNX1) antibodies, whereas cytoplasmic lysates (input) were immunoblotted with anti-tubulin-α mAb (right panels). Data are representative of three independent experiments. IP, immunoprecipitation. Source data are available for this figure: .

Article Snippet: Five micrograms of antibodies against RUNX1 (Abcam, ab23980, RRID:AB_2184205), CTCF (3418; CST, RRID:AB_2086791), Med12 (A300-774A; Bethyl, RRID:AB_669756), or p300 (57625; CST, RRID:AB_3068009) were prebound to Dynabeads coated with anti-rabbit IgG and incubated overnight at 4°C with the chromatin complexes.

Techniques: Binding Assay, ChIP-sequencing, Sequencing, Expressing, Affinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Western Blot, Immunoprecipitation

Validation of RUNX1, CTCF, and Med12 depletion and identification of RUNX1-regulated genes in LP and Phase 1 cells. (A) sgRNAs against Cbfb , Ctcf , or Med12 were introduced into Cas9-LPs. 3 days after sgRNA transduction, nuclear lysates from retrovirus-infected hNGFR + cells were subjected to immunoblotting for Cbfβ, CTCF, and Med12 antibodies, while cytoplasmic lysates were subjected to immunoblotting with anti-tubulin-α mAb. (B) Volcano plots showing changes of transcriptome profiles between control and Cbfb -deficient LP (left) and Phase 1 cells (right). (C) Heatmap showing changes in the expression of RUNX-dependent and RUNX-repressed genes in LP and Phase 1 following Cbfb deletion. (D) Dot plot showing expression changes of RUNX-regulated DEGs in LP and Phase 1 cells following the disruption of Cbfb . (E) Venn diagrams showing the number of RUNX-dependent genes in LP and RUNX-repressed genes in Phase 1 . Names of the three overlapping genes are shown. (F) Venn diagrams showing the number of RUNX-repressed genes in LP and RUNX-dependent genes in Phase 1 ( , yellow areas). Names of the five overlapping genes are shown. Two independent experiments were performed with similar results (A). Data are presented as the average of three biological replicates (B–F). Source data are available for this figure: .

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: Validation of RUNX1, CTCF, and Med12 depletion and identification of RUNX1-regulated genes in LP and Phase 1 cells. (A) sgRNAs against Cbfb , Ctcf , or Med12 were introduced into Cas9-LPs. 3 days after sgRNA transduction, nuclear lysates from retrovirus-infected hNGFR + cells were subjected to immunoblotting for Cbfβ, CTCF, and Med12 antibodies, while cytoplasmic lysates were subjected to immunoblotting with anti-tubulin-α mAb. (B) Volcano plots showing changes of transcriptome profiles between control and Cbfb -deficient LP (left) and Phase 1 cells (right). (C) Heatmap showing changes in the expression of RUNX-dependent and RUNX-repressed genes in LP and Phase 1 following Cbfb deletion. (D) Dot plot showing expression changes of RUNX-regulated DEGs in LP and Phase 1 cells following the disruption of Cbfb . (E) Venn diagrams showing the number of RUNX-dependent genes in LP and RUNX-repressed genes in Phase 1 . Names of the three overlapping genes are shown. (F) Venn diagrams showing the number of RUNX-repressed genes in LP and RUNX-dependent genes in Phase 1 ( , yellow areas). Names of the five overlapping genes are shown. Two independent experiments were performed with similar results (A). Data are presented as the average of three biological replicates (B–F). Source data are available for this figure: .

Techniques: Biomarker Discovery, Transduction, Infection, Western Blot, Control, Expressing, Disruption

RUNX/CTCF represses CD25 + cell generation in LPs, whereas RUNX/Mediator promotes DN2 development after Notch stimulation. (A) Schematic of the stage-specific deletion of Cbfb , Ctcf , or Med12 in LP and Phase 1 cells using the CRISPR/Cas9 system. (B) Retroviral vectors encoding sgRNAs were introduced into Cas9-LPs. 5 days after sgRNA introduction, hNGFR + CD45 + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. (C and D) Percentage (C) and relative number (D) of CD25 + cells among hNGFR + CD45 + sgRNA-transduced cells from B are shown with SD. (E) 1 day after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. hNGFR + CD45 + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. (F and G) Percentage (F) and relative number (G) of CD25 + cells among hNGFR + CD45 + sgRNA-transduced cells from E are shown with SD. Data in B and E are representative of three independent experiments. Data in C, D, F, and G represent mean values from three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (C, D, F, and G). For LP (Notch-); C, **adjusted P < 0.0001 for sgCbfb; **adjusted P = 0.0064 for sgCTCF. For LP (Notch-); D, **adjusted P < 0.0001 for sgCbfb; **adjusted P = 0.0064 for sgCTCF. For Phase 1 (Notch+); F, **adjusted P = 0.0046 for sgCbfb; **adjusted P = 0.0002 for sgCTCF; **adjusted P = 0.0362 for sgMed12. For Phase 1 (Notch+); G, **adjusted P = 0.0049 for sgCbfb; **adjusted P = 0.0002 for sgCTCF; **adjusted P = 0.0360 for sgMed12. SD, standard deviation.

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: RUNX/CTCF represses CD25 + cell generation in LPs, whereas RUNX/Mediator promotes DN2 development after Notch stimulation. (A) Schematic of the stage-specific deletion of Cbfb , Ctcf , or Med12 in LP and Phase 1 cells using the CRISPR/Cas9 system. (B) Retroviral vectors encoding sgRNAs were introduced into Cas9-LPs. 5 days after sgRNA introduction, hNGFR + CD45 + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. (C and D) Percentage (C) and relative number (D) of CD25 + cells among hNGFR + CD45 + sgRNA-transduced cells from B are shown with SD. (E) 1 day after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. hNGFR + CD45 + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. (F and G) Percentage (F) and relative number (G) of CD25 + cells among hNGFR + CD45 + sgRNA-transduced cells from E are shown with SD. Data in B and E are representative of three independent experiments. Data in C, D, F, and G represent mean values from three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (C, D, F, and G). For LP (Notch-); C, **adjusted P < 0.0001 for sgCbfb; **adjusted P = 0.0064 for sgCTCF. For LP (Notch-); D, **adjusted P < 0.0001 for sgCbfb; **adjusted P = 0.0064 for sgCTCF. For Phase 1 (Notch+); F, **adjusted P = 0.0046 for sgCbfb; **adjusted P = 0.0002 for sgCTCF; **adjusted P = 0.0362 for sgMed12. For Phase 1 (Notch+); G, **adjusted P = 0.0049 for sgCbfb; **adjusted P = 0.0002 for sgCTCF; **adjusted P = 0.0360 for sgMed12. SD, standard deviation.

Techniques: CRISPR, Retroviral, Expressing, Standard Deviation

RUNX1/CTCF represses T-signature genes in LPs, whereas RUNX1/Med12 activates them in Phase 1 cells. (A) ChIP-seq analyses for RUNX1, CTCF, and Med12 were performed using Cas9-LPs with or without Notch stimulation. Venn diagrams show the numbers of reproducible CTCF ChIP peaks overlapping with LP-specific (left) or Phase 1–specific (right) RUNX1 peaks (as shown in ). Genes bound by CTCF at LP-specific RUNX1 peaks (highlighted in yellow) were subjected to GO analysis using the GREAT analysis tool ( https://great.stanford.edu/public/html/ ). The top three GO terms are shown. (B) Venn diagrams show the numbers of reproducible Med12 ChIP peaks overlapping with LP-specific (left) or Phase 1–specific (right) RUNX1 peaks (as shown in ). ChIP peaks co-occupied by LP-specific RUNX1 and Med12, as well as Phase 1–specific RUNX1 and Med12, are highlighted in yellow. The top three GO terms for genes bound by LP-specific RUNX1 and LP-specific Med12-co-occupied peaks (upper), and genes bound by Phase 1-specific RUNX1 and Phase 1-specific Med12 (lower) are shown. Data were obtained from ChIP-seq peaks scored as reproducible in two replicate samples.

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: RUNX1/CTCF represses T-signature genes in LPs, whereas RUNX1/Med12 activates them in Phase 1 cells. (A) ChIP-seq analyses for RUNX1, CTCF, and Med12 were performed using Cas9-LPs with or without Notch stimulation. Venn diagrams show the numbers of reproducible CTCF ChIP peaks overlapping with LP-specific (left) or Phase 1–specific (right) RUNX1 peaks (as shown in ). Genes bound by CTCF at LP-specific RUNX1 peaks (highlighted in yellow) were subjected to GO analysis using the GREAT analysis tool ( https://great.stanford.edu/public/html/ ). The top three GO terms are shown. (B) Venn diagrams show the numbers of reproducible Med12 ChIP peaks overlapping with LP-specific (left) or Phase 1–specific (right) RUNX1 peaks (as shown in ). ChIP peaks co-occupied by LP-specific RUNX1 and Med12, as well as Phase 1–specific RUNX1 and Med12, are highlighted in yellow. The top three GO terms for genes bound by LP-specific RUNX1 and LP-specific Med12-co-occupied peaks (upper), and genes bound by Phase 1-specific RUNX1 and Phase 1-specific Med12 (lower) are shown. Data were obtained from ChIP-seq peaks scored as reproducible in two replicate samples.

Techniques: ChIP-sequencing

ChIP-seq analysis of RUNX1, CTCF, Med12, and Notch1-IC binding in LP and Phase 1 cells. (A) Tag count distributions for RUNX1, CTCF, Med12, and Notch1-IC ChIP peaks around LP-specific RUNX1-binding regions are shown as peak-centered heatmaps. Each lane represents the merged tag directories from two biological replicates. (B) Representative ChIP-seq tracks for RUNX1 and CTCF in LP and Phase 1 around the Lck , Thy1 , and Zap70 loci. CTCF-binding sites co-occupied with LP-specific RUNX1 peaks are labeled with blue rectangles. Data are representative of two independent experiments. (C) Heatmap showing changes in the expression of Thy1 , Lck , and Zap70 in LP following Cbfb deletion. Data are presented as the average of three biological replicates. (D) Top three enriched sequence motifs among the 5,849 LP-specific, the 14,961 Phase 1–specific, and 24,171 shared reproducible Med12 peaks between LP and Phase 1 are shown . Data are based on ChIP-seq peaks scored as reproducible in two replicate samples. (E) Venn diagrams show the number of shared CTCF ChIP peaks overlapping with LP-specific (upper) or Phase 1–specific (lower) Med12 and RUNX1 peaks. (F) Tag count distributions for RUNX1, CTCF, Med12, and Notch1-IC ChIP peaks around Phase 1–specific RUNX1-binding regions are shown as peak-centered heatmaps. Each lane represents the merged tag directories from two biological replicates. (G) ChIP-seq data for Notch1-IC in Phase 1 and Phase 2 were analyzed. Venn diagrams show the number of reproducible Notch1-IC ChIP peaks in Phase 1 and Phase 2 cells. The top three enriched sequence motifs among the 298 reproducible Phase 1 Notch1-IC peaks (upper panel) and 709 reproducible Phase 2 Notch1-IC peaks (lower panel) are shown. (H) Venn diagrams showing the number of reproducible RUNX1 ChIP peaks in LP and Phase 1 cells, along with Notch1-IC ChIP peaks in Notch-stimulated pro-T cells (Phase 1 + Phase 2, n = 845). (I) Tag count distributions for RUNX1, CTCF, Med12, and Notch1-IC ChIP peaks around Notch1-IC–binding regions in Notch-stimulated pro-T cells are shown as a peak-centered heatmap. Each lane represents the merged tag directories from two biological replicates.

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: ChIP-seq analysis of RUNX1, CTCF, Med12, and Notch1-IC binding in LP and Phase 1 cells. (A) Tag count distributions for RUNX1, CTCF, Med12, and Notch1-IC ChIP peaks around LP-specific RUNX1-binding regions are shown as peak-centered heatmaps. Each lane represents the merged tag directories from two biological replicates. (B) Representative ChIP-seq tracks for RUNX1 and CTCF in LP and Phase 1 around the Lck , Thy1 , and Zap70 loci. CTCF-binding sites co-occupied with LP-specific RUNX1 peaks are labeled with blue rectangles. Data are representative of two independent experiments. (C) Heatmap showing changes in the expression of Thy1 , Lck , and Zap70 in LP following Cbfb deletion. Data are presented as the average of three biological replicates. (D) Top three enriched sequence motifs among the 5,849 LP-specific, the 14,961 Phase 1–specific, and 24,171 shared reproducible Med12 peaks between LP and Phase 1 are shown . Data are based on ChIP-seq peaks scored as reproducible in two replicate samples. (E) Venn diagrams show the number of shared CTCF ChIP peaks overlapping with LP-specific (upper) or Phase 1–specific (lower) Med12 and RUNX1 peaks. (F) Tag count distributions for RUNX1, CTCF, Med12, and Notch1-IC ChIP peaks around Phase 1–specific RUNX1-binding regions are shown as peak-centered heatmaps. Each lane represents the merged tag directories from two biological replicates. (G) ChIP-seq data for Notch1-IC in Phase 1 and Phase 2 were analyzed. Venn diagrams show the number of reproducible Notch1-IC ChIP peaks in Phase 1 and Phase 2 cells. The top three enriched sequence motifs among the 298 reproducible Phase 1 Notch1-IC peaks (upper panel) and 709 reproducible Phase 2 Notch1-IC peaks (lower panel) are shown. (H) Venn diagrams showing the number of reproducible RUNX1 ChIP peaks in LP and Phase 1 cells, along with Notch1-IC ChIP peaks in Notch-stimulated pro-T cells (Phase 1 + Phase 2, n = 845). (I) Tag count distributions for RUNX1, CTCF, Med12, and Notch1-IC ChIP peaks around Notch1-IC–binding regions in Notch-stimulated pro-T cells are shown as a peak-centered heatmap. Each lane represents the merged tag directories from two biological replicates.

Techniques: ChIP-sequencing, Binding Assay, Labeling, Expressing, Sequencing

Organization of LP-specific RUNX1/CTCF repressive complex and Phase 1–specific RUNX1/Med12/p300/Notch1-IC activation complex at the Notch3 and Hes1 loci. (A) Experimental scheme for the transcriptome analysis. (B) 5 days after sgRNA introduction, hNGFR + CD45 + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. The heatmap shows the expression changes of representative RUNX1- and CTCF-repressed genes among Notch-activated genes in pro-T cell stages . Data represent the average of three biological replicates. (C and D) Representative ChIP-seq tracks for RUNX1, CTCF, Med12, and p300 in LP and Phase 1 cells, Notch1-IC in LP, Phase 1, and Phase 2 cells, RUNX1 in sgControl- or sgCTCF-transduced LPs, and Med12 in sgControl- or sgCbfb-transduced Phase 1 cells around the Notch3 (C) and Hes1 (D) loci are shown. CTCF-binding sites co-occupied with LP-specific RUNX1 peaks are labeled with blue rectangles, whereas Phase 1–specific RUNX1/Med12/p300/Notch-IC–binding sites are marked with red rectangles. Data are representative of two independent experiments.

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: Organization of LP-specific RUNX1/CTCF repressive complex and Phase 1–specific RUNX1/Med12/p300/Notch1-IC activation complex at the Notch3 and Hes1 loci. (A) Experimental scheme for the transcriptome analysis. (B) 5 days after sgRNA introduction, hNGFR + CD45 + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. The heatmap shows the expression changes of representative RUNX1- and CTCF-repressed genes among Notch-activated genes in pro-T cell stages . Data represent the average of three biological replicates. (C and D) Representative ChIP-seq tracks for RUNX1, CTCF, Med12, and p300 in LP and Phase 1 cells, Notch1-IC in LP, Phase 1, and Phase 2 cells, RUNX1 in sgControl- or sgCTCF-transduced LPs, and Med12 in sgControl- or sgCbfb-transduced Phase 1 cells around the Notch3 (C) and Hes1 (D) loci are shown. CTCF-binding sites co-occupied with LP-specific RUNX1 peaks are labeled with blue rectangles, whereas Phase 1–specific RUNX1/Med12/p300/Notch-IC–binding sites are marked with red rectangles. Data are representative of two independent experiments.

Techniques: Activation Assay, Sequencing, Expressing, ChIP-sequencing, Binding Assay, Labeling

RUNX1, CTCF, Med12, p300, and Notch1-IC–binding at the Tcf7 and Gata3 loci in LP and Phase 1 cells. (A) TPM values for Tcf7 and Gata3 in Cbfb -deficient LPs or Phase 1 cells are shown with SDs. The data represent the mean values of three independent biological replicates. The data were analyzed by a two-sided t test. For Tcf7 mRNA, **P = 0.003. For GATA3 mRNA, **P = 0.0001. (B and C) Representative ChIP-seq tracks for RUNX1, CTCF, Med12, and p300 in LP and Phase 1, Notch1-IC in LP, Phase 1, and Phase 2, Med12 in sgControl- or sgCbfb-transduced Phase 1 cells, and RUNX1 in sgControl- or sgMed12-transduced Phase 1 cells around the Tcf7 (B) and Gata3 (C) loci. Phase 1–specific RUNX1/Med12/p300/Notch-IC–binding sites are labeled with red rectangles, including the Tcf7 enhancer (−31 kb) and T cell–specific Gata3 enhancer (+280 kb) regions. Data are representative of two independent experiments. SD, standard deviation.

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: RUNX1, CTCF, Med12, p300, and Notch1-IC–binding at the Tcf7 and Gata3 loci in LP and Phase 1 cells. (A) TPM values for Tcf7 and Gata3 in Cbfb -deficient LPs or Phase 1 cells are shown with SDs. The data represent the mean values of three independent biological replicates. The data were analyzed by a two-sided t test. For Tcf7 mRNA, **P = 0.003. For GATA3 mRNA, **P = 0.0001. (B and C) Representative ChIP-seq tracks for RUNX1, CTCF, Med12, and p300 in LP and Phase 1, Notch1-IC in LP, Phase 1, and Phase 2, Med12 in sgControl- or sgCbfb-transduced Phase 1 cells, and RUNX1 in sgControl- or sgMed12-transduced Phase 1 cells around the Tcf7 (B) and Gata3 (C) loci. Phase 1–specific RUNX1/Med12/p300/Notch-IC–binding sites are labeled with red rectangles, including the Tcf7 enhancer (−31 kb) and T cell–specific Gata3 enhancer (+280 kb) regions. Data are representative of two independent experiments. SD, standard deviation.

Techniques: Binding Assay, ChIP-sequencing, Labeling, Standard Deviation

Effects of RUNX, CTCF, or Med12 deletion on DN2 cell generation before and after Notch stimulation in primary BM progenitors. (A) Experimental scheme. BM progenitors from Cas9;Bcl2 Tg mice were transduced with sgRNA and cultured without stromal cells for 1 day. Then, they were transferred onto OP9 stromal cells and cocultured for 4 days. CD45 + hNGFR + sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45 + hNGFR + sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45 + hNGFR + Lin − CD19 + cells (lower panel). (B and C) Percentage (B) and relative number (C) of CD25 + cells among CD45 + hNGFR + Lin − CD19 + sgRNA-transduced cells (from A) are shown with SD. (D) Alternative experimental scheme. sgRNA-transduced BM progenitors were cultured without stromal cells for 2 days. Then, they were transferred onto OP9-DLL1 stromal cells and cocultured for 4 days. CD45 + hNGFR + sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45 + hNGFR + sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45 + hNGFR + Lin − CD19 − cells (lower panel). (E and F) Percentage and relative number of CD25 + cells among hNGFR + CD45 + Lin − CD19 − sgRNA-transduced cells (from D) are shown with SD. (G) Working model of Notch-dependent functional conversion of RUNX TFs regulating the initiation of the T-lineage program. The RUNX/CTCF complex in the LP stage represses T-signature genes. Notch signaling induces the dissociation of RUNX from CTCF and facilitates the redirection of the RUNX/Mediator/p300 complex, thereby triggering T cell differentiation. Results shown in A and D are representative of three independent experiments, whereas data in B, C, E, and F represent the mean values of three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (B, C, E, and F). For Notch-: B, *adjusted P = 0.0224 for sgCbfb; **adjusted P < 0.0001 for sgCTCF. For Notch-: C, *adjusted P = 0.0208 for sgCbfb; **adjusted P = 0.0083 for sgCTCF. For Notch+; E, **adjusted P = 0.0014 for sgCbfb; *adjusted P = 0.0337 for sgMed12. For Notch+: F, **adjusted P = 0.0006 for sgCbfb; **adjusted P = 0.0029 for sgMed12. SD, standard deviation.

Journal: The Journal of Experimental Medicine

Article Title: Notch interaction with RUNX factors regulates initiation of the T-lineage program

doi: 10.1084/jem.20250911

Figure Lengend Snippet: Effects of RUNX, CTCF, or Med12 deletion on DN2 cell generation before and after Notch stimulation in primary BM progenitors. (A) Experimental scheme. BM progenitors from Cas9;Bcl2 Tg mice were transduced with sgRNA and cultured without stromal cells for 1 day. Then, they were transferred onto OP9 stromal cells and cocultured for 4 days. CD45 + hNGFR + sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45 + hNGFR + sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45 + hNGFR + Lin − CD19 + cells (lower panel). (B and C) Percentage (B) and relative number (C) of CD25 + cells among CD45 + hNGFR + Lin − CD19 + sgRNA-transduced cells (from A) are shown with SD. (D) Alternative experimental scheme. sgRNA-transduced BM progenitors were cultured without stromal cells for 2 days. Then, they were transferred onto OP9-DLL1 stromal cells and cocultured for 4 days. CD45 + hNGFR + sgRNA-transduced cells were gated and analyzed for Lin markers, CD19, CD44, and CD25 expression. Representative plots show Lin/CD19 profiles in CD45 + hNGFR + sgRNA-transduced cells (upper panel) and CD44/CD25 profiles in CD45 + hNGFR + Lin − CD19 − cells (lower panel). (E and F) Percentage and relative number of CD25 + cells among hNGFR + CD45 + Lin − CD19 − sgRNA-transduced cells (from D) are shown with SD. (G) Working model of Notch-dependent functional conversion of RUNX TFs regulating the initiation of the T-lineage program. The RUNX/CTCF complex in the LP stage represses T-signature genes. Notch signaling induces the dissociation of RUNX from CTCF and facilitates the redirection of the RUNX/Mediator/p300 complex, thereby triggering T cell differentiation. Results shown in A and D are representative of three independent experiments, whereas data in B, C, E, and F represent the mean values of three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (B, C, E, and F). For Notch-: B, *adjusted P = 0.0224 for sgCbfb; **adjusted P < 0.0001 for sgCTCF. For Notch-: C, *adjusted P = 0.0208 for sgCbfb; **adjusted P = 0.0083 for sgCTCF. For Notch+; E, **adjusted P = 0.0014 for sgCbfb; *adjusted P = 0.0337 for sgMed12. For Notch+: F, **adjusted P = 0.0006 for sgCbfb; **adjusted P = 0.0029 for sgMed12. SD, standard deviation.

Techniques: Transduction, Cell Culture, Expressing, Functional Assay, Cell Differentiation, Standard Deviation

(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.

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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

Techniques: Variant Assay, Gene Expression

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

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

Techniques: Variant Assay, Cell Differentiation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Techniques: Mutagenesis, Activation Assay

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