Journal: Biological procedures online

Article Title: MBD3 promotes epithelial-mesenchymal transition in gastric cancer cells by upregulating ACTG1 via the PI3K/AKT pathway.

doi: 10.1186/s12575-023-00228-9

Figure Lengend Snippet: Fig. 2 MBD3 enhanced the migration ability of GC cells. A, B Transwell assays were used to detect the migration ability of MGC-803 and HGC-27 cells transfected with sh-EGFP and sh-MBD3. C, D Transwell assays were used to examine the migration ability of SGC-7901 and MGC-803 cells transfected with vector and Flag-MBD3. E Wound healing assay showed the migration ability of MGC-803 and HGC-27 cells transfected with sh-EGFP and sh-MBD3. F Histograms represent the analysis of the wound healing rate in Fig. 2E. G Wound healing assay showed the migration ability of SGC-7901 and MGC-803 cells transfected with vector and Flag-MBD3. H Histograms represent the analysis of the wound healing rate in Fig. 2H. (*P < 0.05)

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech, Cat No. 14258– 1-AP) antibody at a 1:800 dilution at 4 °C overnight.

Techniques: Migration, Transfection, Plasmid Preparation, Wound Healing Assay

Journal: Biological procedures online

Article Title: MBD3 promotes epithelial-mesenchymal transition in gastric cancer cells by upregulating ACTG1 via the PI3K/AKT pathway.

doi: 10.1186/s12575-023-00228-9

Figure Lengend Snippet: Fig. 3 MBD3 enhanced the invasion ability of GC cells. A, B Transwell invasion assays showed the invasion ability of MGC-803 and HGC-27 cells transfected with sh-EGFP and sh-MBD3. C, D MMP2 and MMP9 expression were tested by western blot and qRT-PCR in MGC-803 and HGC-27 cells transfected with sh-EGFP and sh-MBD3. E, F Transwell invasion assays were used to examine the invasion ability of SGC-7901 and MGC-803 cells transfected with vector and Flag-MBD3. G, H MMP2 and MMP9 expression were tested by western blot and qRT-PCR in SGC-7901 and MGC-803 cells transfected with vector and Flag-MBD3. (*P < 0.05)

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech, Cat No. 14258– 1-AP) antibody at a 1:800 dilution at 4 °C overnight.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Plasmid Preparation

Journal: Biological procedures online

Article Title: MBD3 promotes epithelial-mesenchymal transition in gastric cancer cells by upregulating ACTG1 via the PI3K/AKT pathway.

doi: 10.1186/s12575-023-00228-9

Figure Lengend Snippet: Fig. 4 MBD3 promoted GC cell proliferation. A, B MBD3 downregulation inhibited the proliferation of MGC-803 and HGC-27 cells. C, D MBD3 upregulation promoted the proliferation of SGC-7901 and MGC-803 cells. All experiments were performed using at least three biological replicates. E, F MBD3 downregulation weakened colony formation in MGC-803 and HGC-27 cells. G, H MBD3 upregulation enhanced colony formation in SGC-7901 and MGC-803 cells. I, J MGC-803 cells with MBD3 upregulation were injected (2 × 10.6 cells/site) subcutaneously into a mouse, and the tumor volume was measured weekly (n = 5 mice). (*P < 0.05)

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech, Cat No. 14258– 1-AP) antibody at a 1:800 dilution at 4 °C overnight.

Techniques: Injection

Journal: Biological procedures online

Article Title: MBD3 promotes epithelial-mesenchymal transition in gastric cancer cells by upregulating ACTG1 via the PI3K/AKT pathway.

doi: 10.1186/s12575-023-00228-9

Figure Lengend Snippet: Fig. 5 MBD3 promoted EMT in GC cells via activation of the PI3K/AKT pathway. A The expression of EMT proteins was examined in sh-MBD3-MGC-803 and HGC-27 cells by western blot. B The expression of EMT proteins was examined in Flag-MBD3-SGC-7901 and MGC-803 cells by western blot. C Western blotting was used to determine PI3K/AKT pathway protein expression in MGC-803 and HGC-27 cells after transfection with sh-EGFP or sh-MBD3. D Western blotting was used to test PI3K/AKT pathway protein expression in SGC-7901 and MGC-803 cells after transfection with vector or Flag-MBD3

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech, Cat No. 14258– 1-AP) antibody at a 1:800 dilution at 4 °C overnight.

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: Biological procedures online

Article Title: MBD3 promotes epithelial-mesenchymal transition in gastric cancer cells by upregulating ACTG1 via the PI3K/AKT pathway.

doi: 10.1186/s12575-023-00228-9

Figure Lengend Snippet: Fig. 7 MBD3 promoted GC cell migration and growth by upregulating ACTG1 expression. A, B The migration ability of MGC-803 and HGC-27 cells transfected with plasmids was tested by transwell assays. C, D Transwell assays were used to examine the migration ability of SGC-7901 and MGC-803 cells after transfection. E, F Colony formation was detected in MGC-803 and HGC-27 cells transfected with plasmids. G, H Colony formation was detected in SGC-7901 and MGC-803 cells after transfection. (*P < 0.05)

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech, Cat No. 14258– 1-AP) antibody at a 1:800 dilution at 4 °C overnight.

Techniques: Migration, Expressing, Transfection

Journal: Cell

Article Title: Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

doi: 10.1016/j.cell.2019.07.017

Figure Lengend Snippet: Figure 3. The LSALT Peptide Binds to DPEP1 (A) Shown are immunofluorescence (IF) images of LSALT (green) binding to DPEP1-COS1 cells. Expression of DPEP1 was validated using anti-DPEP1 (Sigma). A second in vivo isolated peptide was used as a control. n = 5 experiments. Scale bars, 50 mm. (B) DPEP1-COS1 cells were incubated with LSALT or ContP conjugated to Sulfo-SBED biotin, and transfer of biotin was activated by UV light. Cells were lysed, and proteins were recovered with NeutrAvidin-conjugated beads. The control was mock-transfected COS1 cells. Western blotting for DPEP1 confirmed transfer of biotin from LSALT to DPEP1. n = 3 experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human DPEP3 cDNA Origene GenBank: NM_022357 Cat# SC125567 Human DPEP1 E141D This paper NA Software and Algorithms cellSens Standard Olympus NA Imagescope v12.2.2.5015 Leica Biosystems RRID: SCR_014311 GraphPad Prism 8 Graphpad software RRID: SCR_002798 In Cell developer toolbox 1.9.1 GE healthcare RRID: SCR_015790 Softmax PRO Molecular Devices RRID: SCR_014240 LIF Leica Molecular Devices NA Volocity Perkin Elmer NA QuPath (0.1.2.) https://qupath.github.io/ NA R version 3.5.1 http://www.R-project.org/ RRID: SCR_001905

Techniques: Binding Assay, Expressing, In Vivo, Isolation, Control, Incubation, Transfection, Western Blot

Journal: Cell

Article Title: Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

doi: 10.1016/j.cell.2019.07.017

Figure Lengend Snippet: Figure 5. Reduced Neutrophil Recruitment to Pulmonary Vascular Compartments of DPEP1/ Mice (A) DPEP1/ mice were generated as described in the STAR Methods and Figure S3 using CRISPR/Cas9 technology. Lungs, liver, and kidneys were assessed for DPEP1 expression (brown) by IHC (left; scale bars, 20 mm) and enzymatic activity (right) from DPEP1+/+ mice (n = 4 lungs, 4 kidneys, 3 livers) and DPEP1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human DPEP3 cDNA Origene GenBank: NM_022357 Cat# SC125567 Human DPEP1 E141D This paper NA Software and Algorithms cellSens Standard Olympus NA Imagescope v12.2.2.5015 Leica Biosystems RRID: SCR_014311 GraphPad Prism 8 Graphpad software RRID: SCR_002798 In Cell developer toolbox 1.9.1 GE healthcare RRID: SCR_015790 Softmax PRO Molecular Devices RRID: SCR_014240 LIF Leica Molecular Devices NA Volocity Perkin Elmer NA QuPath (0.1.2.) https://qupath.github.io/ NA R version 3.5.1 http://www.R-project.org/ RRID: SCR_001905

Techniques: Generated, CRISPR, Expressing, Activity Assay

Journal: Cell

Article Title: Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

doi: 10.1016/j.cell.2019.07.017

Figure Lengend Snippet: Figure 6. Reduced Recruitment of Neutrophils to the Hepatic Vascular Beds in DPEP/ Mice (A) IHC analysis of livers from DPEP1/ or DPEP1+/+ mice 4 h after LPS were stained for neutrophils (Ly6G, brown). Representative liver images are shown. Scale bars, 20 mm. The graph shows quantitation of neutrophils/FOV. Each dot represents the average number of neutrophils from 3 non-sequential tissue sections (10 FOV/section) per mouse from experiments performed on 4 separate days. (B and C) Liver intravital images from DPEP1/ or DPEP1+/+ mice 4 h after LPS. Scale bars, (B) stitched image, 700 mm and (C) 50 mm. (D and E) Graphs showing the number of (D) adherent or (E) crawling neutrophils in the liver sinusoids. Each dot represents the average from 3 FOV/mouse from intravital imaging performed on 5 separate days. (F) Liver intravital images at 4 h from DPEP1/ injected without and with LSALT 5 min after LPS. Scale bars, 50 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human DPEP3 cDNA Origene GenBank: NM_022357 Cat# SC125567 Human DPEP1 E141D This paper NA Software and Algorithms cellSens Standard Olympus NA Imagescope v12.2.2.5015 Leica Biosystems RRID: SCR_014311 GraphPad Prism 8 Graphpad software RRID: SCR_002798 In Cell developer toolbox 1.9.1 GE healthcare RRID: SCR_015790 Softmax PRO Molecular Devices RRID: SCR_014240 LIF Leica Molecular Devices NA Volocity Perkin Elmer NA QuPath (0.1.2.) https://qupath.github.io/ NA R version 3.5.1 http://www.R-project.org/ RRID: SCR_001905

Techniques: Staining, Quantitation Assay, Imaging, Injection

Journal: Redox Biology

Article Title: Methylation reader MBD2-mediated GPX4 transcriptional repression drives ovarian granulosa cell ferroptosis in PCOS

doi: 10.1016/j.redox.2026.104034

Figure Lengend Snippet: GPX4 suppression is triggered by DNMT and MBD2-mediated Gpx4 promoter hypermethylation. (a) Schematic diagrams of the mouse Gpx4 promoter. The positions of the CpG island (gray area) and the methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) primers (boxes) are shown. (b) MSP analysis of the Gpx4 promoter in control (Ctrl) and DHEA-treated (DHEA) mice. (Left) Agarose gel images of MSP products from two randomly selected samples per group ( n = 6); (Right) Quantification of the methylated-to-unmethylated ratio, normalized to input. Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (c) BSP analysis of the Gpx4 promoter in Ctrl and DHEA mice. Quantification of the percentage of methylated CpG sites was on the right side. Data were presented as Box-and-whisker plots with data points. ∗ P < 0.05, Student's t -test. (d) Heatmap of normalized expression (FPKM) for 23 key transcription regulation-related genes from RNA-seq analysis of Ctrl and DHEA mice. The methylation-related methyltransferases DNMT1 and DNMT3a, significantly increase, and are highlighted in red. (e) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice stained for DNMT1, DNMT3a and MBD2 by immunohistochemistry (IHC) staining. Positively-stained granulosa cells were indicated by arrows. (f) Western blot analysis of DNMT1, DNMT3a, DNMT3b, MBD2, MeCP2, MBD4, MBD3 and MBD1 proteins in ovarian tissues. GAPDH served as a loading control. Blots are representative of two samples per group. Quantification was presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (g) MSP analysis of the Gpx4 promoter in ovarian tissues from four treatment groups: Ctrl, 5-Azacytidine (5-Aza, 1.5 mg/kg, the last 10 days), DHEA (60 mg/kg, 21 days), and 5-Aza/DHEA. Agarose gel showing one representative sample per group. Right panel: Quantification of the methylated-to-unmethylated ratio, normalized to input. Data are presented as mean ± SEM, n = 6. ∗ P < 0.05, one-way ANOVA. (h) BSP analysis of the Gpx4 promoter. CpG methylation patterns from three mice per group (M1-M3), with five clones sequenced per mouse. Each row is a clone; each circle is a CpG site (open: unmethylated; closed: methylated). One box encompasses the data from one mouse. Quantification of the percentage of methylated CpG sites was on the right side. Data were presented as Box-and-whisker plots with data points. ∗ P < 0.05, one-way ANOVA.

Article Snippet: The following primary antibodies were used: GPX4 (ab125066, Abcam, UK), 4HNE (A26085, ABclonal, China), α-SMA (BS70000, Bioworld), Col1α (BS1530, Bioworld), DNMT1 (MA5-16169, Invitrogen, USA), DNMT3a (49768S, CST, USA), DNMT3b (48488S, CST, USA), MBD2 (ab188474, Abcam, UK), MeCP2 (A5694, ABclonal, China), MBD1 (29998-1-AP, Proteintech, USA), MBD3 (A8905, ABclonal, China), MBD4 (11270-1-AP, Proteintech, USA), FSHR (AF04761, AiFang Biological, China), Cyp19a1 (AF0449, AiFang Biological, China), MAZ (21068-1-AP, Proteintech, USA), NCoR (20018-1-AP, Proteintech, USA), HDAC3 (A19537, ABclonal, China), and GAPDH (FD0063, FDbio).

Techniques: Methylation, Methylation Sequencing, Control, Agarose Gel Electrophoresis, Whisker Assay, Expressing, RNA Sequencing, Staining, Immunohistochemistry, Western Blot, CpG Methylation Assay, Clone Assay