Journal: Bioactive materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis.

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Fig. 3. DDR1 mediates substrate stiffness-induced downregulation of DNMT1 expression. Western blot assay to determine the expressions of DNMT1, p-DDR1, DDR1 and the internal control GAPDH in vSMCs with the indicated treatments. Shown are representative blots from at least 3 independent experiments. Semi- quantification of indicated proteins were performed by using the ImageJ software based on the analysis of the gray band intensity. One dot represents a biolog ical repeat. (A) The protein level of DNMT1 in SMCs cultured for 24 h on collagen type I (Col) coated-gels with the indicated stiffness. (B) The protein level of DNMT1 in SMCs grown in dishes that have been pre-coated with Col at variable concentrations. (C) and (D) The expressions of DNMT1, p-DDR1, and DDR1 were determined in SMCs, which were either pretreated with DDR1-IN-1 or the control reagent (DMSO) (C) or transfected with siRNA targeting DDR1 (siDDR1) or scrambled siRNA (siCL) (D). (E) The expression of DNMT1 was determined in SMCs grown on fibronectin (Fn)- or Col-coated gels. (F) The expressions of DNMT1, p- DDR1, and DDR1 were determined in SMCs transfected with siDDR1 or siCL on Fn-coated gels. *P < 0.05 vs. the indicated group.

Article Snippet: For DNMT1 overexpression, cells were transfected with pcDNA3/Myc-DNMT1 that express full length of DNMT1, gifts from Arthur Riggs (Addgene plasmid # 36939) [27], or control plasmid pcDNA3, using lipofectamine 2000 transfection agent.

Techniques: Expressing, Western Blot, Control, Software, Cell Culture, Transfection

Journal: Bioactive materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis.

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Fig. 4. The ERK-p53 pathway is involved in the DDR1-mediated DNMT1 regulation by matrix stiffness. (A) Schematic diagram of the promoter regions of human DNMT1 gene. Two p53 binding sites (−868~-862 and + 47~+53) are indicated. (B) Representative Western blots (left panel) and their quantification (right panel) indicating the phosphorylated p53 at Ser15 (p-p53) and total p53 levels in SMCs grown on gels. (C) Representative Western blots indicating the DNMT1, p- p53 and p53 levels in SMCs grown on gels. The cells were pre-treated with PFT-α (a reagent inhibiting p53 activity) or DMSO. (D) Luciferase assay to determine p53 activity in SMCs grown on gels. The schematic illustration of reporter plasmid construction is shown in the left panel. Each dot means one independent experiment. (E) Western blots (left panels) and their quantification (right panels) indicating the p53 accumulation in the nucleus and cytoplasm of SMCs cultured on gels. (F) Chromatin immunoprecipitation to determine the binding of p53 to the promoter regions of DNMT1 in SMCs grown on gel substrates. Left panel: a DNMT1 promoter region. Right panel: a DNMT1 exon region. Each dot means one independent experiment. (G) Immunofluorescent staining of p53 in SMCs grown on gel substrates. The cells were pre-treated for 3 h with DDR1-IN-1 or DMSO. Quantification of the ratio of nuclear p53 signal versus the cytoplasmic p53 is shown in the lower panel. Each dot represents one cell. (H) and (I) Western blots and their quantification indicating the levels of the indicated proteins in SMCs grown on gels. The cells were pretreated with DDR1 inhibitor DDR1-IN-1, ERK inhibitor PD98059, or DMSO. For Western blotting assay, shown are representative blots from at least 3 independent experiments. Semi-quantification of indicated proteins were performed by using the ImageJ software. *P < 0.05 vs. the indicated group.

Article Snippet: For DNMT1 overexpression, cells were transfected with pcDNA3/Myc-DNMT1 that express full length of DNMT1, gifts from Arthur Riggs (Addgene plasmid # 36939) [27], or control plasmid pcDNA3, using lipofectamine 2000 transfection agent.

Techniques: Binding Assay, Western Blot, Activity Assay, Luciferase, Plasmid Preparation, Cell Culture, Chromatin Immunoprecipitation, Staining, Software

Journal: Bioactive materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis.

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Fig. 6. DDR1 inhibition alleviates the acute vascular injury-induced arterial inflammation and stiffening. (A) Schematic diagram of the acute vascular injury model in mouse. The mice were subjected to repetitive intraperitoneal injections with DDR–IN–1 or DMSO. (B) Representative immunofluorescent staining and the quantification of p-DDR1 in the abdominal aorta from the CaCl2-incubated or sham-operated mice. (C) Representative Western blots and the quantification of DNMT1 in arterial segments from the CaCl2-incubated or sham-operated mice. (D) Representative immunofluorescent staining and the quantification of MCP1 and SMMHC in mouse aortas with or without CaCl2-incubation. The mice were subjected to applications with DDR1-IN-1 or DMSO. (E) Representative immunofluo rescent staining and the quantification of CD68 in mouse aortas with or without CaCl2-incubation. The mice were subjected to applications with DDR1-IN-1 or DMSO. The border between the adventitia and the media is denoted by a dashed line. (F) The elastic moduli of the operated vessels in the indicated mice were measured by nanoindentation. Each dot represents one mouse. (G) Representative Alizarin red S staining and its quantification in the indicated aortas. Shown are representative images from at least 3 mice in each group. L: lumen. *P < 0.05 vs. the indicated group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For DNMT1 overexpression, cells were transfected with pcDNA3/Myc-DNMT1 that express full length of DNMT1, gifts from Arthur Riggs (Addgene plasmid # 36939) [27], or control plasmid pcDNA3, using lipofectamine 2000 transfection agent.

Techniques: Inhibition, Staining, Incubation, Western Blot

Journal: Bioactive materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis.

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Fig. 7. DDR1-DNMT1 signaling regulates the adenine diet-induced arterial inflammation and stiffening. (A) Schematic diagram of the adenine diet-induced arterial stiffening model in mouse. The mice were subjected to repetitive intraperitoneal injections with DDR–IN–1 and/or 5-Aza or DMSO. (B) to (D) Representative immunofluorescent staining and the quantification of p-DDR1 (B), DNMT1 (C), MCP1 and SMMHC (D) in the abdominal aorta from mice with the indicated treatments. (E) The elastic moduli of the aortas in the indicated mice were measured by nanoindentation. Each dot represents one mouse. (F) Pulse wave velocity (PWV) in the carotid arteries of mice with the indicated treatments. One dot represents a mouse. (G) Representative Alizarin red S staining and its quantification in aortas from the indicated treatments. Shown are representative images from at least 3 mice in each group. EL: elastic lamina, L: lumen. *P < 0.05 vs the indicated group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For DNMT1 overexpression, cells were transfected with pcDNA3/Myc-DNMT1 that express full length of DNMT1, gifts from Arthur Riggs (Addgene plasmid # 36939) [27], or control plasmid pcDNA3, using lipofectamine 2000 transfection agent.

Techniques: Staining

Journal: Science translational medicine

Article Title: Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer

doi: 10.1126/scitranslmed.adf6732

Figure Lengend Snippet: (A) DNMT1, DNMT3A, and DNMT3B mRNA expression in benign prostate (n = 29), PCA (n = 75), CRPC-Adeno (n = 73), and NEPC (n = 36). Two-tailed t test was used. FPKM, fragments per kilobase of exon per million mapped reads. (B) DNMT1, DNMT3A, and DNMT3B mRNA expression in NEPC. Two-tailed t test was used. (C) A survey of DNMT mRNA expression across six prostate cell lines, organoids, and PDX models. (D) Correlation of DNMT expression and NEPC score in the International SU2C/PCF Dream Team dataset (11). (E) Representative IHC images of PCA, CRPC-Adeno, and NEPC stained by hematoxylin and eosin (H&E) and with CHGA, AR, DNMT1, DNMT3A, and DNMT3B antibodies. Scale bars, 100 μm. *P < 0.05, **P < 0.01, ****P < 0.0001. TPM, transcripts per million.

Article Snippet: Cells were transfected with nontargeting control pool or SMARTpool siRNAs (Horizon) to knock down DNMT1 and B7-H3 using electroporation (4D-Nucleofector Core Unit, Lonza). pcDNA3 vectors were used to overexpress DNMT1 [pcDNA3/Myc-DNMT1 was a gift from A. Riggs (Addgene, plasmid #36939; http://n2t.net/addgene:36939 ; RRID:Addgene_36939)] and DNMT3A [pcDNA3/Myc-DNMT3A was a gift from A. Riggs (Addgene, plasmid #35521; http://n2t.net/addgene:35521 ; RRID: Addgene_35521)] by an electroporation method ( 60 , 61 ).

Techniques: Expressing, Two Tailed Test, Staining

Journal: Science translational medicine

Article Title: Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer

doi: 10.1126/scitranslmed.adf6732

Figure Lengend Snippet: (A) DNMT1/DNMT3A protein expression after DNMT1/3A knockout in WCM154 (NEPC) cells. GAPDH, glyceraldehyde phosphate dehydrogenase. (B) Cell viability analyses in WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. A two-way ANOVA test was used. (C) Subcutaneous injection of mice with 1 × 107 cells and tumor appearance ratio in WCM154 sgGFP, sgDNMT1, and sgDNMT3A. (D) Postinjection time and tumor volumes in WCM154 sgGFP, sgDNMT1, and sgDNMT3A. A two-way ANOVA test was used. (E) Representative IHC images of WCM154 sgGFP, sgDNMT1, and sgDNMT3A tumors stained by H&E. Scale bars, 100 μm. (F) Representative IHC images of WCM154 sgGFP, sgDNMT1, and sgDNMT3A tumors stained by DNMT1 and DNMT3A. Scale bars, 100 μm. (G) Orthotopic injection in murine prostate with 2 × 105 cells and tumor appearance and liver metastasis ratio in WCM154 sgGFP, sgDNMT1, and sgDNMT3A. (H) Representative images of murine prostate injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. (I) Weights of gGU system injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. A two-tailed t test was used. (J) Representative IHC images of murine prostate injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells stained by H&E. Scale bars, 100 μm. (K) Representative IHC images of murine prostate injected with WCM154 sgGFP and sgDNMT3A cells stained by DNMT3A. Scale bars, 100 μm. (L) Multiple liver metastatic lesions generated from murine prostate injected with WCM154 sgGFP cells (left). Representative IHC images of liver metastatic tumors stained by H&E, DNMT1, DNMT3A, CHGA, and SYP. Scale bars, 100 μm. **P < 0.01, ****P < 0.0001.

Article Snippet: Cells were transfected with nontargeting control pool or SMARTpool siRNAs (Horizon) to knock down DNMT1 and B7-H3 using electroporation (4D-Nucleofector Core Unit, Lonza). pcDNA3 vectors were used to overexpress DNMT1 [pcDNA3/Myc-DNMT1 was a gift from A. Riggs (Addgene, plasmid #36939; http://n2t.net/addgene:36939 ; RRID:Addgene_36939)] and DNMT3A [pcDNA3/Myc-DNMT3A was a gift from A. Riggs (Addgene, plasmid #35521; http://n2t.net/addgene:35521 ; RRID: Addgene_35521)] by an electroporation method ( 60 , 61 ).

Techniques: Expressing, Knock-Out, Injection, Staining, Two Tailed Test, Generated

Journal: Science translational medicine

Article Title: Targeting DNA methylation and B7-H3 in RB1-deficient and neuroendocrine prostate cancer

doi: 10.1126/scitranslmed.adf6732

Figure Lengend Snippet: (A) Heatmap of DNMT mRNA gene expression, NEPC markers, apoptosis marker, and cell proliferation markers in WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. (B) GO enrichment analysis in down-regulated genes after DNMT1 knockout. WT, wild type; MAPK, mitogen-activated protein kinase. (C) Gene set enrichment analysis in down-regulated genes after DNMT3A knockout. NES, normalized enrichment score. (D) WB analyses of NE markers in WCM154 sgGFP, sgDNMT1, and sgDNMT3A cells. (E) Representative IHC images of subcutaneous tumors injected with WCM154 sgGFP, sgDNMT1, and sgDNMT3A stained by H&E, CHGA, SYP, and INSM1. Scale bars, 200 μm. (F) WB analyses of NE markers after overexpression of DNMT1/3A in DNMT1/3A knockout cells. (G) Cell viability analyses in WCM154 si_Control and si_DNMT1. A two-tailed t test was used. (H and I) WB analyses of NE markers after DNMT1 silencing in WCM154 and WCM155 cells. ****P < 0.0001.

Article Snippet: Cells were transfected with nontargeting control pool or SMARTpool siRNAs (Horizon) to knock down DNMT1 and B7-H3 using electroporation (4D-Nucleofector Core Unit, Lonza). pcDNA3 vectors were used to overexpress DNMT1 [pcDNA3/Myc-DNMT1 was a gift from A. Riggs (Addgene, plasmid #36939; http://n2t.net/addgene:36939 ; RRID:Addgene_36939)] and DNMT3A [pcDNA3/Myc-DNMT3A was a gift from A. Riggs (Addgene, plasmid #35521; http://n2t.net/addgene:35521 ; RRID: Addgene_35521)] by an electroporation method ( 60 , 61 ).

Techniques: Expressing, Marker, Knock-Out, Injection, Staining, Over Expression, Two Tailed Test