Journal: Communications Biology

Article Title: HOXA2 exerts anti-renal fibrosis effects through reducing endoplasmic reticulum stress via the upregulation of SIRT1

doi: 10.1038/s42003-025-09453-2

Figure Lengend Snippet: A qRT-PCR shows that the mRNAs of HOXA2 are downregulated in TGF-β1-induced HK-2 cells. B Western blot analysis shows that the proteins of HOXA2 are downregulated in TGF-β1-induced HK-2 cells. C BSP results of the HOXA2 promoter methylation in HK-2 cells treated with or without TGF-β1. D DNMT1 activity detection. E The enrichment of DNMT1 on the HOXA2 promoter region in HK-2 cells treated with or without TGF-β1 by ChIP assays. F The mRNA expression levels of DNMT1 were assessed using qRT-PCR after forty-eight hours treatment with DNMT1 siRNA in TGF-β1-induced HK-2 cells. G The protein expression levels of HOXA2 were assessed using Western blot analysis after forty-eight hours treatment with DNMT1 siRNA in TGF-β1-induced HK-2 cells. H qRT-PCR shows that the HOXA2 mRNA is recovered after treatment with demethylation drug 5-Aza for 48 h in TGF-β1-induced HK-2 cells. I Western blot analysis shows that the HOXA2 protein is recovered after treatment with demethylation drug 5-Aza for 48 h in TGF-β1-induced HK-2 cells. In all panels, the data are representative of three independent experiments ( n = 3). Data are presented as the mean ± SD. ** P < 0.01, NS not significant.

Article Snippet: HK-2 cells were transfected with DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology, USA) or control siRNA (sc-37007; Santa Cruz Biotechnology, USA) in the TGF-β1 group and named the TGF-β1+siDNMT1 or TGF-β1+siNC group, respectively.

Techniques: Quantitative RT-PCR, Western Blot, Methylation, Activity Assay, Expressing

Journal: Communications Biology

Article Title: HOXA2 exerts anti-renal fibrosis effects through reducing endoplasmic reticulum stress via the upregulation of SIRT1

doi: 10.1038/s42003-025-09453-2

Figure Lengend Snippet: A JASPAR tool predicted HOXA2 binding sites on the promoter of SIRT1. B The mRNA expression levels of HOXA2 were assessed using qRT-PCR after forty-eight hours treatment with HOXA2 overexpression plasmids in TGF-β1-induced HK-2 cells. C The mRNA expression levels of SIRT1 were assessed using qRT-PCR after forty-eight hours treatment with HOXA2 overexpression plasmids in TGF-β1-induced HK-2 cells. D The proteins expression levels of HOXA2 and SIRT1 were assessed using western blot analysis after forty-eight hours treatment with HOXA2 overexpression plasmids in TGF-β1-induced HK-2 cells. E Luciferase reporter analysis elucidated the influence of HOXA2 overexpression on the luciferase activity of the SIRT1 promoter reporter in HK-2 cells treated with or without TGF-β1. F ChIP analysis of HOXA2-FLAG tagged fusion vector levels at the SIRT1 promoter in HK-2 cells. ChIP results were analyzed via qRT–PCR using SIRT1 promoter-specifc primers and are expressed as the percentage of the input. In all panels, the data are representative of three independent experiments ( n = 3). Data are presented as the mean ± SD. ** P < 0.01, NS not significant.

Article Snippet: HK-2 cells were transfected with DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology, USA) or control siRNA (sc-37007; Santa Cruz Biotechnology, USA) in the TGF-β1 group and named the TGF-β1+siDNMT1 or TGF-β1+siNC group, respectively.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Over Expression, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

Journal: Communications Biology

Article Title: HOXA2 exerts anti-renal fibrosis effects through reducing endoplasmic reticulum stress via the upregulation of SIRT1

doi: 10.1038/s42003-025-09453-2

Figure Lengend Snippet: A The proteins expression levels of CHOP, GRP78, E-cadherin, α-SMA, FN and COL I were assessed using Western blot analysis after forty-eight hours treatment with HOXA2 overexpression plasmids in TGF-β1-induced HK-2 cells. B Cell proliferation was detected with a CCK-8 assay after transfection with the HOXA2 overexpression plasmidsat 0, 24, 48 and 72 h. C Representative images of HOXA2 overexpression improved ER and mitochondrial injury in TGF-β1-induced HK-2 cells were observed by TEM (×20.0k). Bar = 500 nm. D H2-DCFDA staining showed that HOXA2 overexpression plasmids alleviated TGF-β1-induced ROS generation in HK-2 cells (×200). Bar = 100 μm. E The proteins expression levels of CHOP, GRP78, E-cadherin, α-SMA, FN and COL I were assessed using Western blot analysis after forty-eight hours treatment with HOXA2 overexpression plasmids in thapsigargin-induced HK-2 cells. In all panels, the data are representative of three independent experiments ( n = 3). Data are presented as the mean ± SD. ** P < 0.01, NS not significant.

Article Snippet: HK-2 cells were transfected with DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology, USA) or control siRNA (sc-37007; Santa Cruz Biotechnology, USA) in the TGF-β1 group and named the TGF-β1+siDNMT1 or TGF-β1+siNC group, respectively.

Techniques: Expressing, Western Blot, Over Expression, CCK-8 Assay, Transfection, Staining

Journal: Communications Biology

Article Title: HOXA2 exerts anti-renal fibrosis effects through reducing endoplasmic reticulum stress via the upregulation of SIRT1

doi: 10.1038/s42003-025-09453-2

Figure Lengend Snippet: A The proteins expression levels of total ATF6, nuclear ATF6, and cytoplasmic ATF6 were assessed using Western blot analysis after forty-eight hours treatment with HOXA2 overexpression plasmids in TGF-β1-induced HK-2 cells. B The proteins expression levels of total ATF6, nuclear ATF6, and cytoplasmic ATF6 were assessed using Western blot analysis after forty-eight hours treatment with HOXA2 overexpression plasmids in thapsigargin-induced HK-2 cells. C Co-IP assay confimed an interaction between SIRT1 and active form ATF6 in HK-2 cells nucleus. D The mRNA expression levels of SIRT1 were assessed using qRT-PCR after forty-eight hours treatment with SIRT1 siRNA in control HK-2 cells. E The protein expression levels of SIRT1 were assessed using Western blot analysis after forty-eight hours treatment with SIRT1 siRNA in control HK-2 cells. F SIRT1 deacetylase activity in each HK-2 cells group. G Acetylation of active form ATF6 in nucleus of each HK-2 cells group was determined by IP with an anti-ATF6 antibody, followed by the immunoblotting analysis with an acetylated-lysine and ATF6 antibody. In all panels, the data are representative of three independent experiments ( n = 3). Data are presented as the mean ± SD. ** P < 0.01, NS not significant.

Article Snippet: HK-2 cells were transfected with DNMT1 siRNA (sc-35204; Santa Cruz Biotechnology, USA) or control siRNA (sc-37007; Santa Cruz Biotechnology, USA) in the TGF-β1 group and named the TGF-β1+siDNMT1 or TGF-β1+siNC group, respectively.

Techniques: Expressing, Western Blot, Over Expression, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Control, Histone Deacetylase Assay, Activity Assay

Journal: NPJ Precision Oncology

Article Title: Loss of MicroRNA-29b promotes DNMT3b-mediated STING downregulation to attenuate radiotherapy-induced antitumor immunity in KRAS-mutated colorectal cancer

doi: 10.1038/s41698-026-01290-8

Figure Lengend Snippet: A WiDr cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. WiDr-Vec. and WiDr-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. B CoLo320 cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. CoLo320-Vec. and CoLo320-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. C HCT116 cells were infected with lentivirus carrying pLKO-scramble shRNA (shNC) or pLKO-shKRAS (shKRAS) and selected for three days. HCT116 shNC and HCT116 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. D SW620 shNC and SW620 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. E WiDr and Colo320DM (endogenous wild-type KRAS) cells were individually infected with vector (Vec.) and KRAS G12D . Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. F CT26, HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shNC and shKRAS. Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. G KRAS G12D -tranduced WiDr (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. H . KRAS G12D -tranduced CoLo320 (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. I HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shDNMT1, shDNMT3a and shDNMT3b. Cells were selected by puromycin for three days. The mRNA level of STING was evaluated by qRT-PCR ( n = 3). One-way ANOVA t test. * p < 0.05. J The protein level of STING was evaluated by immunoblotting ( n = 3).

Article Snippet: The following antibodies were used: phospho-STING-S365 (#19781, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), cleaved caspase-3 (#9661, Cell Signaling Technology and IR96–401, iReal Biotech.), calreticulin (#12238, Cell Signaling Technology), beta-actin (Ab8227, Abcam), DNMT1 (#5032, Cell Signaling Technology), DNMT3a (#49768, Cell Signaling Technology), DNMT3b (#57868, Cell Signaling Technology), KRAS (A1190, ABclonal) and GAPDH (IR3-8, iReal Biotech.)

Techniques: Infection, Plasmid Preparation, Irradiation, Quantitative RT-PCR, shRNA, Western Blot, Mutagenesis

Journal: Cell Genomics

Article Title: Genome-wide classification of epigenetic activity reveals regions of enriched heritability in immune-related traits

doi: 10.1016/j.xgen.2023.100469

Figure Lengend Snippet:

Article Snippet: Following blotting onto nitrocellulose membrane, anti-actin (clone C4, Millipore) and anti-DNMT1 (clone JF09-89, Novus Biologicals) were used for protein detection, with staining with IRDye 680LT- and 800CW-conjugated secondary antibodies and visualization using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Techniques: Recombinant, Sequencing, Positive Control, Software

Journal: Cancer research

Article Title: GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

doi: 10.1158/0008-5472.CAN-16-2289

Figure Lengend Snippet: Altered Tumor Cell GAD1 Promoter Methylation and DNMT1 Expression Induced by Brain Microenvironment. A, UCSC Genome Browser plot of ENCODE data track of CpG Islands located around the GAD1 promoter. B, Promoter methylation-specific PCR (MSP) assay detecting methylation status in human GAD1 promoter region in vitro. C, (left) Bisulfite sequencing of CpG island 122 located in human GAD1 promoter region, (right) Percentage of Methylated CpG sites in sequenced region. D, MSP assay detecting methylation status in the human GAD1 promoter region in vivo. E, Bioinformatics analysis of GSE19184 showing normalized DNMT1 probe intensity in primary tumors or brain metastases arising from indicated cell lines. F, qRT-PCR of DNMT1 mRNA levels in primary tumors or brain metastases arising from MDA-MB231.

Article Snippet: Lentiviral-based expression vectors pcDNA3/Myc-DNMT1 (36939), pcDNA3.1-Peredox-mCherry (32383) and packaging vectors were purchased from Addgene.

Techniques: Methylation, Expressing, MSP Assay, In Vitro, Methylation Sequencing, In Vivo, Quantitative RT-PCR

Journal: Cancer research

Article Title: GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

doi: 10.1158/0008-5472.CAN-16-2289

Figure Lengend Snippet: Brain Microenvironment-Induced Down-Regulation of DNMT1 Reactivates GAD1 Expression. A, qRT-PCR of DNMT1 mRNA expression after 48 hours co-culture with either CAF or glia cells. (left) MDA-MB-231; (right) A375SM. B, qRT-PCR of DNMT1 mRNA expression of MDA-MB-231 cultured either with 100% conditioned media from either CAF or glia cells or 50% mix of conditioned media and fresh media. C, Cytokine screen of glia and CAF conditioned media. (left) MA plot of Log [mean expression of Glia/CAF] of 73 cytokines analyzed. *: differentially expressed cytokines (adjusted p < 0.1) (right) Heatmap of differentially expressed cytokines. D, Network analysis of differentially expressed cytokines. E, Impact of extracellular clusterin on DNMT1 and GAD1 expression. (left) Cytokine expression profile of clusterin in conditioned media from CAFs or glia cells. (right) qPCR of GAD1 and DNMT1 mRNA expression in MDA-MB-231 cells treated with control or 200 ng of clusterin. F, qRT-PCR of GAD1 and DNMT1 mRNA expression in tumor cells genetic knockdown of glia derived-clusterin. (left) qRT-PCR of clusterin mRNA expression in glia cells. (right) qRT-PCR of GAD1 and DNMT1 mRNA expression in MDA-MB-231 cells co-cultured with control glia or siClusterin glia cells. G, qRT-PCR of mRNA levels in tumor cells after 48 hours co-culture with primary glia cells. Prior to co-culture, tumor cells were transfected with either vector control or DNMT1 over expression plasmid for 24 hours. (left) DNMT1 mRNA expression; (right) GAD1 mRNA expression under glia co-culture. H, Proliferation of MDA-MB-231 cells after DNMT1 overexpression and co-culture with glia cells for 48 hours.

Article Snippet: Lentiviral-based expression vectors pcDNA3/Myc-DNMT1 (36939), pcDNA3.1-Peredox-mCherry (32383) and packaging vectors were purchased from Addgene.

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Cell Culture, Derivative Assay, Transfection, Plasmid Preparation, Over Expression