Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: MUCL1 gene expression analysis in CRC. (A) MUCL1 expression in CRC (fold-change) compared with adjacent normal tissue based on microarray data. Data are presented as the mean ± SD. (n=13). (B) Boxplot comparing the expression of MUCL1 in a cohort of COAD (n=275) compared with normal colon tissue (n=349) from the TCGA and GTEx datasets. (C) Boxplot comparing the MUCL1 expression in a cohort of READ (n=92) compared with normal rectal tissue (n=318) from the TCGA and GTEx datasets. (D) Stage plot of MUCL1 expression in colon cancer stages in COAD. (E) Stage plot showing MUCL1 expression in rectal cancer stages in READ. * P<0.05 vs. normal. MUCL1, mucin-like 1; CRC, colorectal cancer; COAD, colon adenocarcinoma; READ, rectal adenocarcinoma; TCGA, The Cancer Genome Atlas; GTEx, Genotype Tissue Expression.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Gene Expression, Expressing, Microarray

Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: MUCL1 transcription expression in various clinicopathological parameters of COAD. (A) Primary tumors and cancer stages. (B) Histological subtype and nodal metastasis. (C) Sex and race based. (D) Weight and age group based and (E) Based on TP-53 mutation status. * P<0.05, ** P<0.01 vs. normal. MUCL1, mucin-like 1; COAD, colon adenocarcinoma; TCGA, The Cancer Genome Atlas.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis

Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: MUCL1 protein expression in human CRC cell lines. (A) Soluble protein of whole cell lysate from HT-29, SW480 and SW620 cells was immunoblotted against the indicated antibodies. (B) Total cell lysate from HT-29-Control siRNA and HT-29-MUCL1siRNA clones ML1 and 2 was immunoblotted against the indicated antibodies. (C) Total cell lysate from SW620 Control siRNA and SW620-MUCL1siRNA clones ML1 and 2 was immunoblotted against the indicated antibodies. (D) Total cell lysate from HT-29-Control siRNA and HT-29-MUCL1 siRNA clones ML1 and 2 was evaluated for the expression of Bcl2, BclxL and caspase-3. (E) Total cell lysate from SW620-Control siRNA and SW620-MUCL1siRNA clones ML1 and 2 was immunoblotted against the indicated antibodies (Bcl2, BclxL and caspase-3). Densitometric analysis was conducted as follows: Intensity of protein bands were semi-quantified and plotted as relative protein expression to control. The bar graphs are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01 and ***P<0.001 vs. control. MUCL1, mucin-like 1; CRC, colorectal cancer; si-, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Control, Clone Assay

Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: MUCL1 promotes cell proliferation. (A) HT-29-Control siRNA and HT-29-MUCL1 siRNA clones ML1 and 2 and (B) SW620-Control siRNA and SW620-MUCL1 siRNA clones ML1 and 2 cells were seeded (500/well) in 6-well plate and incubated at 37°C. After 10-12 days of incubation crystal violet staining was performed, colonies were quantified and images were captured by Bio-Rad gel-doc system. (C) HT-29-Control siRNA and HT-29-MUCL1 siRNA clones ML1 and 2 were seeded at 5,000 cells/well in 96-well plates and incubated at 37°C. Cell proliferation was determined by Cell Counting Kit-8 assay on 24, 48, 72 and 96 h. (D) SW620-Control siRNA and SW620-MUCL1siRNA clone ML1 and 2 cells were seeded at 5,000 cells/well in 96-well plate for incubation at 37°C. Cell proliferation was measured by CCK-8 assay on 24, 48, 72 and 96 h. The results are expressed as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. MUCL1, mucin-like 1; si-, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Control, Clone Assay, Incubation, Staining, Cell Counting, CCK-8 Assay

Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Targeting MUCL1 inhibits migration and invasion. Transwell migration and invasion assay was performed using 24-well plates. Migration and invasion activity were evaluated after 48 h by crystal violet staining. Quantification of migration was measured as relative migration and compared with control. (A) Migration ability of HT-29-Con siRNA and HT-29-MUCL1siRNA clones ML1 and 2. (B) Migration ability of SW620-Con siRNA and SW620-MUCL1siRNA clones ML1 and 2 plated in Transwell plate inserts. (C) Invasion ability of HT-29-Con siRNA and HT-29-MUCL1siRNA clones ML1 and 2. (D) Invasion ability of SW620-Con siRNA and SW620-MUCL1siRNA clones ML1 and 2 plated in Transwell plate inserts. Invasive activity was determined as relative invasion. All assays were carried out in triplicate. Scale bar, 100 µ m. The results were expressed as the mean ± SD of three independent experiments. *** P<0.001 vs. control. MUCL1, mucin-like 1; si-, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Migration, Invasion Assay, Activity Assay, Staining, Control, Clone Assay

Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: MUCL1 induces epithelial-mesenchymal transition by activating β-catenin. Total cell lysates from (A) HT-29-Control siRNA and HT-29-MUCL1 siRNA clones ML1 and 2 or (B) from SW620-Control siRNA and SW620-MUCL1 siRNA clones ML1 and 2 cells were immunoblotted against the indicated antibodies (E-cadherin and vimentin). Total cell lysates from (C) HT-29-Control siRNA and HT-29-MUCL1 siRNA clones ML1 and 2 or (D) from SW620-Control siRNA and SW620-MUCL1 siRNA clones ML1 and 2 cells were immunoblotted against the indicated antibodies (phosphor-β-catenin-Ser-552 and β-catenin). Nuclear and cytosolic extracts from (E) HT-29-Control siRNA and HT-29-MUCL1 siRNA clones ML1 and 2 or (F) from SW620-Control siRNA and SW620-MUCL1 siRNA clones ML1 and 2 were immunoblotted against the indicated antibodies (β-catenin, β-actin and Lamin B). Densitometric analysis was conducted as follows: Intensity of the protein bands was semi-quantified and plotted as relative protein expression to control. The bar graphs are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. MUCL1, mucin-like 1; si-, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Control, Clone Assay, Expressing

Journal: International Journal of Oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β-catenin and increases irinotecan sensitivity in colorectal cancer

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Targeting MUCL1 increases sensitivity towards IRI. (A) HT-29-Control siRNA and HT-29-MUCL1siRNA clones ML1 and 2 cells were treated with IRI (50 and 100 µ M) for 24 h. (B) Percentage of total cell death was analyzed by Annexin V/PI staining using flow cytometry. (C) SW620-Control siRNA and SW620-MUCL1siRNA clones ML1 and 2 cells were exposed to IRI (50 and 100 µ M) for 24 h. (D) Total cell death (%) was analyzed by Annexin V/PI staining using flow cytometry. Total cell death (%) shown is representative of three independent experiments (n=3). * P<0.05, ** P<0.01 and *** P<0.001 vs. control. HT-ML1/HT-ML2 vs. control (black); HT-ML1-IRI-50/HT-ML2-IRI-50 vs. HT-IRI-50 (blue). SW-ML1/SW-ML2 vs. control (black); SW-ML1-IRI-50/SW-ML2-IRI-50 vs. SW-IRI-50 (blue); SW-ML1-IRI-100/SW-ML2-IRI-100 vs. SW-IRI-100 (red). MUCL1, mucin-like 1; si-, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: MUCL1 (cat. no. NBP1-92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc-492; 1:1,000), BclxL (cat. no. sc-56021; 1:1,000), caspase-3 (cat. no. sc-56053; 1:1,000), E-cadherin (cat. no. sc-8426; 1:1,000), vimentin (cat. no. sc-6260; 1:1,000), Lamin B (cat. no. sc-374015; 1:1,000) and β-actin (cat. no. sc-47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Control, Clone Assay, Staining, Flow Cytometry

Journal: Cell

Article Title: A white-box machine learning approach for revealing antibiotic mechanisms of action

doi: 10.1016/j.cell.2019.04.016

Figure Lengend Snippet: (A) Overall experimental design for measuring metabolite effects on antibiotic lethality. Overnight cultures of E. coli MG1655 were inoculated into MOPS minimal medium, grown to early exponential phase, and back-diluted to OD600 = 0.1. Cells were dispensed into Biolog phenotype microarray plates (PMs) 1–4 (Bochner, 2009) with different concentrations of ampicillin (AMP), ciprofloxacin (CIP) or gentamicin (GENT) added. OD600 was measured after 4 hours of incubation at 37°C and 900 rpm shaking. Antibiotic IC50s were estimated for each antibiotic-metabolite combination.

Article Snippet: Cells were dispensed into Biolog phenotype microarray plates (PMs) 1–4 ( Bochner, 2009 ) with different concentrations of ampicillin (AMP), ciprofloxacin (CIP) or gentamicin (GENT) added.

Techniques: Microarray, Incubation

Journal: Cell

Article Title: A white-box machine learning approach for revealing antibiotic mechanisms of action

doi: 10.1016/j.cell.2019.04.016

Figure Lengend Snippet: Key Resources Table

Article Snippet: Cells were dispensed into Biolog phenotype microarray plates (PMs) 1–4 ( Bochner, 2009 ) with different concentrations of ampicillin (AMP), ciprofloxacin (CIP) or gentamicin (GENT) added.

Techniques: Virus, Recombinant, Microarray, Software, Combined Bisulfite Restriction Analysis Assay

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: Upregulation of CARM1 is correlated with breast cancer progression. (A) Heatmap showing the gene expression status of the PRMT family in different subtypes of breast cancer according to TCGA. (B) Analysis of GSE65194 for the expression of PRMT family in normal or TNBC tissues (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant; two-tailed unpaired t -test). (C) MDA-MB-231 cells were transfected with siControl or siRNAs targeting the PRMT family. Knockdown efficiencies were verified by RT-qPCR. (D) EdU cell proliferation assays of MDA-MB-231 cells transfected with siControl and siRNAs specifically targeting PRMT family members. Scale bar, 100 μm. (E) Analysis of GSE104549 for the expression of CARM1 in distinct subtypes of breast cancer (** P < 0.01; two-tailed unpaired t -test). (F) Detection of CARM1 in the breast cancer tissue cDNA array by RT-qPCR (*** P < 0.001; two-tailed unpaired t -test). (G) Immunohistochemical staining of CARM1 in normal breast and breast tissues carcinomas (histological grades I, II, and III). The images represent fields under microscopy from two different cases in each grade. Positively stained nuclei in the grouped samples were calculated as percentages (*** P < 0.001; two-tailed unpaired t -test). (C and D) Error bars represent the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Expressing, Two Tailed Test, Transfection, Knockdown, Quantitative RT-PCR, Immunohistochemical staining, Staining, Microscopy

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: CARM1 promotes proliferation, invasion, epithelial–mesenchymal transition (EMT), and stemness in TNBC. (A) Representative photos of colony formation assays were performed on MDA-MB-231 and Hs 578T cells transfected with shSCR, two different CARM1 shRNAs, or vector, or FLAG-CARM1. Scale bar, 100 μm. C1, CARM1. (B) Cell invasion assays were carried out using Matrigel transwell filters in MDA-MB-231 and and Hs 578T cells. The images represent one field under microscopy in each group. Scale bar, 100 μm. C1, CARM1. (C and D) Expression of the indicated epithelial or mesenchymal markers was measured by RT-qPCR (C) and Western blot (D). α-cate, α-Catenin; γ-cate, γ-Catenin; Vim, Vimentin; N-cad, N-cadherin. (E) Representative images of sphere numbers in mammosphere assays. Scale bar, 100 μm. C1, CARM1. (F) Expression of the indicated stemness markers was measured by western blot. (G) MDA-MB-231 cells stably transfected with shSCR or shCARM1 were implanted into the fourth mammary fat pad of NOD SCID mice with various dilutions ( n = 5). The frequency of CSCs was calculated using the online tool extreme limiting dilution analysis (ELDA). (H) The tumor growth curves of different numbers (1000, 500, and 100) of shSCR or shCARM1 cells were monitored during the specified time period. The tumor volume at the end point is shown on the right. (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test). (A–E) Error bars represent the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Transfection, Plasmid Preparation, Microscopy, Expressing, Quantitative RT-PCR, Western Blot, Stable Transfection, Two Tailed Test

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: Identification of genome-wide transcription targets for CARM1. (A) Genomic distribution of CARM1 sites. (B) KEGG pathway analysis of CARM1 ChIP-seq promoter genes arranged into functional groups. Data were analyzed using KOBAS website. (C) Binding profiles of CARM1 on representative target genes Cyclin D1 and HIF1A . (D and E) qChIP verification of the ChIP-seq results on the promoter of the indicated genes with antibodies against the indicated proteins in MDA-MB-231 cells. Data are presented as fold-change relative to that of β-Actin (negative control). (F) Heatmap representation of differentially expressed genes in control siRNA (siControl-1, siControl-2) and CARM1 knockdown (KD) (siCARM1-1, siCARM1-2) MDA-MB-231 cells. Two independent samples were used for the RNA-seq analysis. Data were analyzed using R package (v3.6.3). The rows show the Z -scores calculated for each group. (G) KEGG pathway analysis of the differentially expressed genes of CARM1 arranged into functional groups. (H) GSEA of RNA-seq data. NES: normalized enrichment score. (I) The promoter genes in ChIP-seq were overlapped with downregulated mRNAs in CARM1 mRNA-seq ( P < 0.001). The overlapped gene numbers are shown in the venn diagram. (J) RT-qPCR experiments were performed to verify the results of RNA-seq. (K) Verification of RNA-seq results by Western blot. (D, E, and J) Error bars represent the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Genome Wide, ChIP-sequencing, Functional Assay, Binding Assay, Negative Control, Control, Knockdown, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: CARM1 is physically associated and directly interacts with HIF1A. (A) Immunoaffinity purification and mass spectrometry analysis of CARM1-associated proteins in MDA-MB-231 cells. (B and C) Association of CARM1 and HIF1A in MDA-MB-231 and Hs 578T cells under normoxia (B) or hypoxia (cells treated with 1% O 2 for 24 h) (C) Whole cell lysates were prepared, and co-immunoprecipitation experiments were performed with antibodies against the indicated proteins. Immunocomplexes were then immunoblotted using antibodies against the indicated proteins. IgG served as the negative control. (D) Molecular interactions between CARM1 and HIF1A. GST pull-down assays using bacterially expressed GST-fused proteins and in vitro transcribed/translated proteins are shown. (E and F) Identification of essential domains required for the interaction with HIF1A of CARM1. GST pull-down experiments with bacterially expressed series of truncation vectors of CARM1 [EVH1, catalytic core and C-ter] to generate GST fusion proteins and in vitro transcribed/translated indicated proteins. (G and I) Identification of essential domains required for the interaction with CARM1 of HIF1A. GST pull-down experiments with bacterially expressed series of truncation vectors of HIF1A (bHLH, PAS, ODD, and TAD) to generate GST fusion proteins and in vitro transcribed/translated indicated proteins. (J) MDA-MB-231 cells were transfected with shSCR or two different shRNAs targeting CARM1 or vector or FLAG-CARM1. Efficiency was verified using RT-qPCR and Western blot. (K) HeLa cells stably infected with lentivirus were transfected with pGL4.42[luc2P/HRE/Hygro] plasmid and treated with 1% O 2 for 24 h. Then, luciferase assays were performed. (L) Schematic representation of the promoter region of CDK4, Cyclin D1, β-Catenin, HIF1A, MALAT1, and SIX1. (J and K) Error bars represent the mean ± SD of three independent experiments (** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Immunoaffinity Purification, Mass Spectrometry, Immunoprecipitation, Negative Control, In Vitro, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Stable Transfection, Infection, Luciferase, Two Tailed Test

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: HIF1A recruits CARM1 for transcriptional activation in TNBC. (A and B) qChIP experiments with antibodies against IgG, CARM1, and HIF1A in MDA-MB-231 cells under normoxia (A) or hypoxia (1% O 2 for 24 h) (B) with MALAT1 and VEGFA as positive controls. (C) MDA-MB-231 cells were transfected with shSCR and three different shRNAs targeting HIF1A. Knockdown efficiencies were verified by RT-qPCR and Western blot. The most efficient shHIF1A#1 was used for the subsequent experiments. (D) qChIP analysis of selected promoters in MDA-MB-231 cells stably transfected with control shRNA or shRNAs targeting CARM1 or HIF1A under hypoxia (1% O 2 for 24 h) using the indicated antibodies. Data are presented as the fold-change relative to β-Actin, the negative control. (E) ChIP-Re-ChIP experiments were performed in MDA-MB-231 cells under hypoxia. C1, CARM1; H1, HIF1A; R17, H3R17me2a. (F) MDA-MB-231 cells stably infected with control shRNA or shRNAs targeting CARM1 or HIF1A treated with 1% O 2 for 24 h, and the mRNA and protein levels of CDK4, Cyclin D1, β-Catenin, HIF1A, MALAT1, and SIX1 were measured and normalized to those of β-Actin. (A–D, and F) Error bars represent the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Activation Assay, Transfection, Knockdown, Quantitative RT-PCR, Western Blot, Stable Transfection, Control, shRNA, Negative Control, Infection, Two Tailed Test

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: CARM1 coordinates with HIF1A to promote the proliferation, invasion, and stemness in TNBC. (A) Western blot analysis of the indicated proteins in MDA-MB-231 cells transfected with different lentiviruses. (B) Representative photos of growth curve assays performed on MDA-MB-231 cells transfected with vector, wild type CARM1 (CARM1 WT), EVH1 domain deletion (CARM1 ∆EVH1), or single point mutation R168A (CARM1 R168A) viruses. (C) Representative photos of transwell assays performed on MDA-MB-231 cells transfected with different lentiviruses. Scale bar, 100 μm. (D) Representative photos of mammosphere formation assays performed on MDA-MB-231 cells transfected with different lentiviruses. Scale bar, 100 μm. (E) Western blot of protein expression using antibodies against the indicated proteins in MDA-MB-231 cells transfected with different lentiviruses. (F) Representative photos of growth curve assays performed on MDA-MB-231 cells transfected with shSCR, shCARM1 (shC1), and shCARM1 together with vector, WT res, ∆EVH1 res, or R168A res. (G) Representative photos of transwell assays performed on MDA-MB-231 cells transfected with different lentiviruses. Scale bar, 100 μm. (H) Representative photos of mammosphere formation assays performed on MDA-MB-231 cells transfected with different lentiviruses. Scale bar, 100 μm. (I) CARM1 and HIF1A collaboratively enhanced the proliferation and invasion of breast cancer cells. MDA-MB-231 cells were stably transfected with CARM1, HIF1A, shRNAs, and expression vectors as indicated for the transwell assays. (J) Representative photos of colony formation assays performed on MDA-MB-231 cells transfected with the indicated specific shRNA and/or expression constructs. (K) Western blot was used to determine the protein expression in these cells using antibodies against the indicated proteins. (B–D, F–J) Error bars represent the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Stable Transfection, shRNA, Construct, Two Tailed Test

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: CARM1 inhibitor ellagic acid suppresses proliferation and invasion in TNBC. (A) Efficiencies of shRNA targeting CARM1, TP-064 (12 h), or ellagic acid (24 h) in MDA-MB-231 cells were evaluated by Western blot. (B) Western blot of indicated proteins in MDA-MB-231 cells treated with shRNA targeting CARM1, TP-064 (1 μmol/L, 12 h), or ellagic acid (100 μmol/L, 24 h). (C) Transwell assays of MDA-MB-231 cells. Scale bar, 100 μm. (D) Colony formation assays of MDA-MB-231 cells. Scale bar, 100 μm. (E) Heatmaps showing DEGs (| FC | > 1.2, P < 0.001) in the control and ellagic acid treatment of MDA-MB-231 cells. Three independent samples were used in RNA-seq analysis. Data were analyzed using R package (version 3.6.3). Rows show the Z -scores calculated for each group. EA, ellagic acid. (F) The up-regulated genes in CARM1 RNA-seq (| FC | > 1.2, P < 0.001) were overlapped with those in RNA-seq cells treated with ellagic acid (| FC | > 1.2, P < 0.001). The down-regulated genes in CARM1 RNA-seq seq (| FC | > 1.2, P < 0.001) were overlapped with those in RNA-seq cells treated with ellagic acid (| FC | > 1.2, P < 0.001). The numbers of overlapped genes are shown in Venn diagram. (G) KEGG pathway analysis of the DEGs of the overlapped genes arranged into functional groups (| FC | > 1.2, P < 0.001). Data were analyzed using KOBAS website. (H) GSEA of the overlapped data ( P < 0.001). (I) Verification of RNA-seq results by RT-qPCR. (J) BALB/c nude mice bearing MDA-MB-231 cells were treated with the control or ellagic acid (50 mg/kg/day) by gavage. Representative mice with MDA-MB-231 xenografts (left) and tumors dissected from the control or ellagic acid-treated mice (right) are shown ( n = 5). (K) Tumor growth of xenograft nude mice treated with the control or ellagic acid were measured every three days (** P < 0.01; two-tailed unpaired t -test). (L) Tumor tissues were prepared for IHC detection with specific antibodies against CARM1, CDK4, Cyclin D1, HIF1A, and Ki67. (M) RT-qPCR (left) and Western blot analysis (right) were performed. (C, D, I, and M) Each bar represents the mean ± SD of triplicate experiments (* P < 0.05, ** P < 0.01; two-tailed unpaired t -test).

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: shRNA, Western Blot, Control, RNA Sequencing Assay, Functional Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Protein & Cell

Article Title: CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A

doi: 10.1093/procel/pwae010

Figure Lengend Snippet: CARM1 is upregulated in multiple cancers and is a potential cancer biomarker. (A) Representative images of immunostained paired samples of cerebrum, esophagus, liver, lymph, ovary, pancreas, prostate, and rectum cancer tissues vs. adjacent normal tissue samples. (B) Positively stained nuclei were calculated as percentages (* P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test). (C) CARM1 expression in multiple cancer microarray datasets available from Oncomine (*** P < 0.001; two-tailed unpaired t -test). (D) Detection of CDK4 , HIF1A , and MALAT1 in the breast cancer tissue cDNA array by RT-qPCR (Each bar represents the mean ± SD of triplicate experiments (*** P < 0.001; two-tailed unpaired t -test). (E) Correlation analysis of public TCGA datasets for the expression of CARM1, HIF1A, and CDK4 in BRCA and BLCA. (F) Proposed mechanism of the coordinated role of CARM1 and HIF1A in TNBC tumorigenesis.

Article Snippet: The following antibodies were used: anti-PRMT1 (2449S), anti-CARM1 (12495S), anti-PRMT6 (14641S), anti-KLF4 (12173S), anti-CDK4 (12790S), anti-Rpb1 NTD (14958S) (Cell Signaling Technology, Danvers, MA, USA); anti-FLAG (F1408), anti-Vimentin (V6630), and anti-β-Actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-PRMT2 (ab154154), anti-PRMT3 (ab191562), anti-PRMT5 (ab109451), anti-PRMT8 (ab168134), anti-OCT4 (ab19857), anti-SOX2 (ab97959), anti-MYC (ab32072), anti-HIF1A (ab2185, ab51608), anti-H3R17me2a (ab8284), anti-H3R26me2a (ab194679), anti-H3 (ab1791) (Abcam, Cambridge, UK); anti-CARM1 (NB200-342), anti-PRMT7 (NBP2-19939) (Novus Biologicals, Littleton, Colorado, USA); anti-α-Catenin (610193), anti-γ-Catenin (610253), anti-N-cadherin (610920), anti-β-Catenin (610153) (BD Biosciences, Franklin Lakes, NJ, USA); anti-CDK4 (66950-1-Ig),anti-SIX1 (10709-1-AP) (Proteintech, Rocky Hill, NJ, USA); and anti-Cyclin D1 (0407-27), anti-MDM2 (ER1902-14) (Huabio, Hangzhou, China).

Techniques: Biomarker Assay, Staining, Two Tailed Test, Expressing, Microarray, Quantitative RT-PCR

Journal: Cancers

Article Title: A Systemic and Integrated Analysis of p63-Driven Regulatory Networks in Mouse Oral Squamous Cell Carcinoma.

doi: 10.3390/cancers15020446

Figure Lengend Snippet: Figure 11. Immunohistochemical staining in HNSCC tumor microarray tissues. Staining of p63 (A), COTL1 (B), and K14 (C) across normal, malignant tumor stage II, and malignant tumor stage III tissues at 10× magnification.

Article Snippet: After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. RoseAnne Romano) [29], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Trisbuffered saline with 0.05% Tween-20.

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Frontiers in endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: FIGURE 1 | GCM1 is expressed in equine trophoblast and expression correlates with LHB. (A) mRNA expression of LHB in ChG and chorion tissues at days 27, 30, 31, and 34 of pregnancy. (B) mRNA expression of GCM1 in ChG and chorion tissues at days 27, 30, 31, and 34 of pregnancy. (C) Correlation of mRNA expression of LHB and GCM1 in individual ChG tissues. ***p < 0.001, ****p < 0.0001 relative to day 27 ChG, ###p < 0.001, ####p < 0.0001 relative to CH at same time point (linear mixed-effects modeling, SPSS). (D) Glial cells missing 1 (GCM1) protein expression in untransfected COS7 cells and COS7 cells transfected with 250 ng of equine pCMV-myc-GCM1 expression vector. B-Actin expression was used as a loading control. (E) GCM1 and B-Actin protein expression in primary equine day 34 conceptus tissues: chorionic girdle (ChG), chorion (CH), allantochorion (ALC), and yolk sac (YS).

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Expressing, Transfection, Plasmid Preparation, Control

Journal: Frontiers in endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: FIGURE 2 | Glial cells missing 1 (GCM1) transactivates the LHB promoter. (A) Western blotting of protein extracted from human choriocarcinoma BeWo cells (n = 3) using an antihuman GCM1 monoclonal antibody (c-terminal). β-actin was used as a loading control. (B) BeWo cells were transfected with specific lengths of the LHB promoter in a pGL3-basic luciferase reporter vector (n = 3). Activity of promoter constructs was expressed as fold-change firefly/renilla compared to pGL3-basic. **p < 0.01, ***p < 0.001 (one-way ANOVA). (C) pGL3-basic and pGL3-335 were co-transfected with ratios of pCMV-myc-Empty: pCMV-myc-GCM1 to assess ability of GCM1 to drive promoter activity in COS7 cells (n = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001 relative to pGL3-335 alone, ##p < 0.01 relative to pGL3-335 + 250 ng pCMV-myc-Empty, ^^^p < 0.001 relative to pGGl-335 + 150 ng pCMV-myc-GCM1 (two-way ANOVA). (D) Specific lengths of the LHB promoter were transfected with 150 ng of empty pCMV-myc-Empty vector, or with 150 ng of pCMV-myc-GCM1 (n = 4). *p < 0.05, **p < 0.0.01, ***p < 0.001, ****p < 0.0001 (two-way ANOVA).

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Western Blot, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct

Journal: Frontiers in endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: FIGURE 3 | Glial cells missing 1 (GCM1) binds to the proximal LHB promoter in primary equine chorionic girdle (ChG) trophoblast cells and mutation of GCM1 site 1 results in loss of promoter activity. (A) Chromatin ImmunoPrecipitation was carried out on formaldehyde fixed chromatin complexes from passage 1 day 34 primary ChG trophoblasts, using an antihuman GCM1 antibody for immunoprecipitation. Binding of GCM1 to predicted GCM1-binding sites was expressed as fold-change enrichment over binding to a control region within the coding sequence of the LHB gene (n = 2 conceptuses). (B,C) The two GCM1-binding sites in the pGL3-335 promoter construct were mutated. Red boxes depict GCM1 binding sites. Red box with a cross depicts a mutated site. Wild-type pGL3-335 and its two mutant constructs, pGL3-335-mut1 and pGL3-335-mut2, were co-transfected (B) into BeWo cells (n = 3) (C) into COS7 cells, with 150 ng of pCMV-myc-GCM1 or pCMV-myc- Empty as a control (n = 4). Promoter activity was assessed by luciferase assay. *p < 0.05, **p < 0.01, ****p < 0.0001 [(B) one-way ANOVA, (C) two-way ANOVA].

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Mutagenesis, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Control, Sequencing, Construct, Transfection, Luciferase

Journal: Frontiers in endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: FIGURE 4 | Glial cells missing 1 (GCM1) binding partners are differentially expression in vivo during differentiation of chorionic girdle (ChG) trophoblast cells. (A) mRNA expression of ETV1, ETV7, HOXA13, and PITX1 in ChG and chorion tissues between days 27 and 34 of pregnancy, as determined by microarray analysis (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 relative to day 27 ChG, #p < 0.05, ##p < 0.01, ###p < 0.001, relative to time matched chorion. (B) Schematic of the LHB promoter showing directly bound GCM1-binding site and region of potential binding by other transcription factors. TLSS is translational start site, TSS shown by black arrow is transcriptional start site. Numbers are relative to TLSS.

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Binding Assay, Expressing, In Vivo, Microarray