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: Nucleic Acids Research

Article Title: Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells

doi: 10.1093/nar/gkab1184

Figure Lengend Snippet: Loss of MED12 in BRCA-deficient cells promotes chemoresistance. (A, B) Clonogenic survival experiments showing that depletion of MED12, but not of MED13 or CDK8, increases the resistance of HeLa-BRCA2 KO cells to olaparib ( A ) and cisplatin ( B ). The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance calculated using two-way ANOVA (A) or t -test two-tailed, unequal variance (B). ( C ) Cellular viability assay showing that depletion of MED12 increases the resistance of DLD1-BRCA2 KO cells to cisplatin. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance calculated using two-way ANOVA. (D, E) Cellular viability assay showing that depletion of MED12, but not of MED13, increases the resistance of RPE1-BRCA1 KO cells to olaparib ( D ) and cisplatin ( E ). The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (two-way ANOVA). ( F ) Annexin V assays showing a reduction in cisplatin-induced apoptosis upon MED12 depletion in RPE-BRCA1 KO cells. The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (t-test two-tailed, unequal variance). ( G ) Representative clonogenic assay showing that MED12 depletion in PEO1 cells promotes olaparib resistance. (H, I) Analyses of BRCA2-mutant ovarian ( H ) and bladder ( I ) TCGA cancer datasets showing that high MED12 levels are associated with increased survival, while low MED12 levels are associated with reduced survival. Mantel-Cox log ranked t test was used for statistical analyses ( n = 31, P -value = 0.3234 for the ovarian dataset; n = 38, P -value = 0.4424 for the bladder dataset). The differences observed are not significant, likely because of the small number of BRCA2-mutant samples in the datasets.

Article Snippet: Antibodies used for Western blot were: RAD51 (Abcam ab133534), ATM (Cell Signaling Technology 2873S), MED12 (Santa Cruz Biotechnology sc-515695, and Cell Signaling Technology 4529S), phospho-SMAD2 Ser465/Ser467 (Cell Signaling Technology 18338T), TGFBR2 (Cell Signaling Technology 79424S), MED7 (Abcam ab187146), MED13 (Invitrogen PA5-35924), CDK8 (ProteinTech 220671AP), ZRANB3 (Invitrogen PA5-65143), GAPDH (Santa Cruz Biotechnology sc-47724), Vinculin (Santa Cruz Biotechnology sc-73614), BRCA1 (Santa Cruz Biotechnology sc-6954), BRCA2 (Calbiochem OP95), TONSL (Invitrogen MA5-27769), RAD52 (Santa Cruz Biotechnology sc-365341), phospho-ATM Ser1981 (Cell Signaling Technology 4526S), SMAD2 (Santa Cruz Biotechnology sc-393312).

Techniques: Two Tailed Test, Cell Viability Assay, Clonogenic Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells

doi: 10.1093/nar/gkab1184

Figure Lengend Snippet: Impact of MED12-knockout on olaparib sensitivity of BRCA2-knockout cells. ( A ) Western blots confirming the loss of BRCA2 and MED12 expression in four independent double-knockout clones. (B, C) Clonogenic survival ( B ) and cellular viability ( C ) experiments showing that all four HeLa-BRCA2 KO MED12 KO double knockout clones show olaparib resistance. The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (two-way ANOVA). ( D ) Western blots showing the complementation of HeLa-BRCA2 KO MED12 KO#3 clone by exogenous re-expression of MED12. ( E ) Clonogenic survival experiments showing that complementation of HeLa-BRCA2 KO MED12 KO double knockout cells by exogenous re-expression of MED12 partly restores olaparib sensitivity. The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (two-way ANOVA).

Article Snippet: Antibodies used for Western blot were: RAD51 (Abcam ab133534), ATM (Cell Signaling Technology 2873S), MED12 (Santa Cruz Biotechnology sc-515695, and Cell Signaling Technology 4529S), phospho-SMAD2 Ser465/Ser467 (Cell Signaling Technology 18338T), TGFBR2 (Cell Signaling Technology 79424S), MED7 (Abcam ab187146), MED13 (Invitrogen PA5-35924), CDK8 (ProteinTech 220671AP), ZRANB3 (Invitrogen PA5-65143), GAPDH (Santa Cruz Biotechnology sc-47724), Vinculin (Santa Cruz Biotechnology sc-73614), BRCA1 (Santa Cruz Biotechnology sc-6954), BRCA2 (Calbiochem OP95), TONSL (Invitrogen MA5-27769), RAD52 (Santa Cruz Biotechnology sc-365341), phospho-ATM Ser1981 (Cell Signaling Technology 4526S), SMAD2 (Santa Cruz Biotechnology sc-393312).

Techniques: Knock-Out, Western Blot, Expressing, Double Knockout, Clone Assay

Journal: Nucleic Acids Research

Article Title: Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells

doi: 10.1093/nar/gkab1184

Figure Lengend Snippet: MED12 depletion promotes chemoresistance through activation of the TGFβ pathway. ( A) Western blots showing that treatment of HeLa-BRCA2 KO cells with TGFβRi for 24h abolishes the SMAD2 phosphorylation induced by MED12 depletion. (B, C) Clonogenic survival experiments showing that treatment with 20 μM LY2109761 (TGFβRi) restores olaparib ( B ) and cisplatin ( C ) sensitivity in MED12-depleted HeLa-BRCA2 KO cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (two-way ANOVA). (D, E) Cellular viability experiments showing that treatment with 20 μM LY2109761 (TGFβRi) restores olaparib ( D ) and cisplatin ( E ) sensitivity in MED12-depleted HeLa-BRCA2 KO cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (2-way ANOVA).

Article Snippet: Antibodies used for Western blot were: RAD51 (Abcam ab133534), ATM (Cell Signaling Technology 2873S), MED12 (Santa Cruz Biotechnology sc-515695, and Cell Signaling Technology 4529S), phospho-SMAD2 Ser465/Ser467 (Cell Signaling Technology 18338T), TGFBR2 (Cell Signaling Technology 79424S), MED7 (Abcam ab187146), MED13 (Invitrogen PA5-35924), CDK8 (ProteinTech 220671AP), ZRANB3 (Invitrogen PA5-65143), GAPDH (Santa Cruz Biotechnology sc-47724), Vinculin (Santa Cruz Biotechnology sc-73614), BRCA1 (Santa Cruz Biotechnology sc-6954), BRCA2 (Calbiochem OP95), TONSL (Invitrogen MA5-27769), RAD52 (Santa Cruz Biotechnology sc-365341), phospho-ATM Ser1981 (Cell Signaling Technology 4526S), SMAD2 (Santa Cruz Biotechnology sc-393312).

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells

doi: 10.1093/nar/gkab1184

Figure Lengend Snippet: Loss of MED12 restores Homologous Recombination in BRCA-deficient cells. ( A , B ) DR-GFP assay showing that MED12 co-depletion with two different siRNAs restores HR efficiency in BRCA1-depleted and BRCA2-depleted cells. The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (t-test two-tailed, unequal variance). ( C ) DR-GFP assay showing that TGFβ pathway inhibition with LY2109761 (TGFβRi) suppresses the restoration of HR induced by MED12 depletion in BRCA2-knockdown cells. The average of three experiments is presented, with standard deviations shown as error bars. Asterisks indicate statistical significance (t-test two-tailed, unequal variance). ( D ) RAD51 immunofluorescence experiment showing that MED12 depletion partly restores RAD51 foci formation upon CPT treatment in HeLa-BRCA2 KO cells. At least 75 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance (t-test two-tailed, unequal variance). (E, F) Western blots showing that MED12 depletion in HeLa-BRCA2 KO cells does not affect the levels of RAD51 or ATM upon CPT ( E ) or HU ( F ) treatment.

Article Snippet: Antibodies used for Western blot were: RAD51 (Abcam ab133534), ATM (Cell Signaling Technology 2873S), MED12 (Santa Cruz Biotechnology sc-515695, and Cell Signaling Technology 4529S), phospho-SMAD2 Ser465/Ser467 (Cell Signaling Technology 18338T), TGFBR2 (Cell Signaling Technology 79424S), MED7 (Abcam ab187146), MED13 (Invitrogen PA5-35924), CDK8 (ProteinTech 220671AP), ZRANB3 (Invitrogen PA5-65143), GAPDH (Santa Cruz Biotechnology sc-47724), Vinculin (Santa Cruz Biotechnology sc-73614), BRCA1 (Santa Cruz Biotechnology sc-6954), BRCA2 (Calbiochem OP95), TONSL (Invitrogen MA5-27769), RAD52 (Santa Cruz Biotechnology sc-365341), phospho-ATM Ser1981 (Cell Signaling Technology 4526S), SMAD2 (Santa Cruz Biotechnology sc-393312).

Techniques: Homologous Recombination, Two Tailed Test, Inhibition, Knockdown, Immunofluorescence, Western Blot

Journal: Nucleic Acids Research

Article Title: Loss of MED12 activates the TGFβ pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells

doi: 10.1093/nar/gkab1184

Figure Lengend Snippet: MED12 depletion promotes TGFβ-dependent fork protection in BRCA-deficient cells. (A–C ) DNA fiber combing assays showing that MED12 knockdown, but not knockdown of MED7 or CDK8, suppresses HU-induced nascent strand degradation in HeLa-BRCA2 KO ( A ), DLD1-BRCA2 KO ( B ) and RPE1-BRCA1 KO ( C ) cells. Co-depletion of TGFBR2 restores fork degradation in MED12-depleted BRCA1/2-knockout cells, indicating that activation of the TGFβ pathway upon MED12 depletion is essential for fork stability. Knockdown of ZRANB3, which suppresses fork reversal, is shown as control. For all panels, the ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph and listed at the top. At least 100 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitney test). Schematic representations of the assay conditions are shown at the top. ( D ) BrdU alkaline comet assay showing that MED12 depletion reduces replication-associated ssDNA gaps accumulation upon HU treatment in HeLa-BRCA2 KO cells. At least 75 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann-Whitney test). Schematic representations of the assay conditions are shown at the top.

Article Snippet: Antibodies used for Western blot were: RAD51 (Abcam ab133534), ATM (Cell Signaling Technology 2873S), MED12 (Santa Cruz Biotechnology sc-515695, and Cell Signaling Technology 4529S), phospho-SMAD2 Ser465/Ser467 (Cell Signaling Technology 18338T), TGFBR2 (Cell Signaling Technology 79424S), MED7 (Abcam ab187146), MED13 (Invitrogen PA5-35924), CDK8 (ProteinTech 220671AP), ZRANB3 (Invitrogen PA5-65143), GAPDH (Santa Cruz Biotechnology sc-47724), Vinculin (Santa Cruz Biotechnology sc-73614), BRCA1 (Santa Cruz Biotechnology sc-6954), BRCA2 (Calbiochem OP95), TONSL (Invitrogen MA5-27769), RAD52 (Santa Cruz Biotechnology sc-365341), phospho-ATM Ser1981 (Cell Signaling Technology 4526S), SMAD2 (Santa Cruz Biotechnology sc-393312).

Techniques: Knockdown, Knock-Out, Activation Assay, Control, MANN-WHITNEY, Alkaline Single Cell Gel Electrophoresis

Journal: Oncotarget

Article Title: TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma

doi: 10.18632/oncotarget.24883

Figure Lengend Snippet: (A) TP-064 inhibits the dimethylation of PRMT4 substrates. HEK293 cells were treated with indicated concentrations of TP-064 for 3 days and dimethylation levels of BAF155 and MED12 in whole cell extracts were analyzed by western blotting. (B) Quantitation of data in (A). Graphs represent nonlinear curve fits of dimethyl-BAF155 and dimethyl-MED12 signal intensities normalized to total BAF155 or MED12, respectively. Data represent mean ± SEM of two independent experiments prepared in triplicate.

Article Snippet: Blots were blocked for 1 h in blocking buffer composed of 5% milk and 0.1% Tween 20 in phosphate-buffered saline (PBS) and then incubated overnight at 4°C in blocking buffer containing primary antibodies against MED12 (1:1000) (Abnova, Taipei, Taiwan; H00009968-A01), MED12 with asymmetrically dimethylated arginine (1:1000; gift from Dr. Mark Bedford and Cell Signaling Technology, Danvers, MA, USA), BAF155 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc32763), or R1064-dimethylated BAF155 (1:2000) (Millipore; ABE1339).

Techniques: Western Blot, Quantitation Assay