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: Translational Oncology

Article Title: MTHFD1L, A Folate Cycle Enzyme, Is Involved in Progression of Colorectal Cancer ☆ ☆☆

doi: 10.1016/j.tranon.2019.07.011

Figure Lengend Snippet: Elevated expression of MTHFD1L in CRC tissues. (A) Gene expression analysis of CRC datasets showed upregulated expression of MTHFD1L in CRC tissues . (B) Dataset from Skrzypczak et al., 2010 showing overexpression of MTHFD1L in CRCs (n = 36) as compared to normal colon tissues (n = 24). (C) TCGA data acquired from UALCAN with MTHFD1L expression in normal colon (n = 41) and CRCs tissues (n = 286). (D) qRT-PCR analysis showing mRNA levels of MTHFD1L in CRCs (n = 110) and matched adjacent non-cancerous tissue (n = 110). (E) Stage-wise expression of MTHFD1L transcripts from UALCAN in CRC tissues Stage I (n = 45), Stage II (n = 110), Stage (n = 80), and Stage IV (n = 39) and normal (n = 41). (F) MTHFD1L mRNA expression in paired samples from different stages of CRC [(Stage 1 (n = 18), Stage 2 (n = 30), Stage 3 (n = 42), and Stage 4 (n = 20)].

Article Snippet: Deparaffinization, rehydration, antigen retrieval, reduction of endogenous activity, and blocking steps were performed sequentially before probing with an antibody against MTHFD1L (Cat# 16113–1-AP, Proteintech, Irvine, CA).

Techniques: Expressing, Gene Expression, Over Expression, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: MTHFD1L, A Folate Cycle Enzyme, Is Involved in Progression of Colorectal Cancer ☆ ☆☆

doi: 10.1016/j.tranon.2019.07.011

Figure Lengend Snippet: MTHFD1L protein overexpression in CRC tissues. (A) Immunoblot analysis to showing MTHFD1L protein expression in different stages of CRCs by probing of lysates of paired CRCs and matched adjacent non-cancerous tissues with MTHFD1L antibody. β-Actin was used as a loading control. (B) Photographs of MTHFD1L immunostaining using MTHFD1L antibody in tissue containing CRC cells and normal cells.

Article Snippet: Deparaffinization, rehydration, antigen retrieval, reduction of endogenous activity, and blocking steps were performed sequentially before probing with an antibody against MTHFD1L (Cat# 16113–1-AP, Proteintech, Irvine, CA).

Techniques: Over Expression, Western Blot, Expressing, Control, Immunostaining

Journal: Translational Oncology

Article Title: MTHFD1L, A Folate Cycle Enzyme, Is Involved in Progression of Colorectal Cancer ☆ ☆☆

doi: 10.1016/j.tranon.2019.07.011

Figure Lengend Snippet: Silencing of MTHFD1L reduced malignant phenotypes of colon cancer. (A) Colon cancer cells, SW480 and HT29, were transiently transfected with MTHFD1L siRNA, and immunoblot analyses were performed to show knockdown of MTHFD1L. (B) Proliferation assay of cells transfected with MTHFD1L siRNA or control siRNA at 2, 4, and 6 days (∗ P < .001). (C) Representative images of colony formation after transfection of cells with control or MTHFD1L siRNA. (D) Cells transfected with MTHFD1L siRNA or control siRNA and plated in 8-μm pore invasion chambers with Matrigel. After 48 h, cells that invaded through the pores were fixed and stained. Photographs of invaded cells are shown. (E) Spheroid formation assay after transfection of MTHFD1L siRNA or control siRNA. (F) Wound healing assay after transfection with MTHFD1L siRNA or control siRNA.

Article Snippet: Deparaffinization, rehydration, antigen retrieval, reduction of endogenous activity, and blocking steps were performed sequentially before probing with an antibody against MTHFD1L (Cat# 16113–1-AP, Proteintech, Irvine, CA).

Techniques: Transfection, Western Blot, Knockdown, Proliferation Assay, Control, Staining, Tube Formation Assay, Wound Healing Assay

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 3. Identification of CAT1 as a specific surface antigen on CEC-derived EVs. A, The result of differential analysis based on paired t test between proteome from tumor region–derived EVs (tumor Te-EVs) and that from normal mucosa-derived EVs (normal Te-EVs) is displayed as the volcano plot. Significantly upregulated 487 proteins (adjusted P< 0.05 and fold-change >5.0) or downregulated 88 proteins (adjusted P< 0.05 and fold-change <0.2) were indicated in red or blue dots. B, Principal component analysis was performed for top 100 upregulated proteins in (A). C, The line chart shows LC/MS-based relative protein abundances of CAT1 on EVs in 17 paired samples. D, A representative image of transmission electron microscopy (TEM) for tumor Te-EVs. The black dot on the surface of the EV indicates the signal of the gold particle-labeled anti-CAT1 antibody; bar, 200 nm. The expression of CAT1 was positive in 24 out of 74 (32.4%) of tumor Te-EVs whereas none (0/66) of normal Te-EVs were CAT1 positive. E, Expression of CAT1 and CD9 in normal (N) and tumor (T) Te-EVs was confirmed by western blotting. F, The CD9-normalized protein abundances of CAT1 in (E) were displayed with the box plot. G, Representative images of IHC staining of CAT1 in colorectal cancer tissues. The expression levels of CAT1 were classified into 3 groups as indicated; bars, 50 mmol/L. H, The result of IHC staining of CAT1 for 75 samples was summarized with the violin plot. N, normal colon mucosa tissue; I–IV, tissues of pathological stage I–IV colorectal cancer.

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Transmission Assay, Electron Microscopy, Labeling, Expressing, Western Blot, Immunohistochemistry

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 4. The diagnostic potential of plasma EV-CAT1 for detection of colorectal cancer. A, The framework of anti-CAT1 and anti– CD81 EV-sandwich ELISA isshown. B, biotin; SA, streptavidin; HRP, horseradish peroxidase; TMB, 3,30,5,50-tetramethylben- zidine. B, The result of EV-CAT1 sandwich ELISA measuring 119 plasma samples is displayed as the box plot. N, normal donors; I–IV, plasma from pathological stage I–IV patients with colorectal cancer. The P values were calculated by Student t test. C, The diagnostic potential to distinguish patients with colorectal cancer (n ¼ 94) from normal donors (n ¼ 25) was assessed by ROC curve analysis. In addition to the single usage of EV-CAT1 or CEA, the combination bio- marker model, EV-CAT1 þ CEA, was also evaluated based on logistic regression. AUC, area under the curve.

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Diagnostic Assay, Clinical Proteomics, Sandwich ELISA, Marker

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 5. CAT1-dependent promotion of cell growth and tubule formation of HUBEC via EVs. A, Representative images of IHC staining for CAT1 (A, C, E, G, I, K) or CD31 (B, D, F, H, J, L) are shown. CD31 (PECAM1) was stained as a marker for vascular endothelial cells. Serial sections were used for the comparison between the IHC staining of CAT1 and CD31. B, The rate of moderate to strong-CAT1 expression in vascular endothelial cells of or near colorectal cancer tissueswas significantly higher than that of adjacent normal tissues. C, The high expression of CAT1 in HCT116 cells transfected with pCAGGS-CAT1-FLAG (CAT1-overexpressing HCT116) compared with those transfected with pCAGGS-FLAG (Control HCT116) was observed. D, The expression level of CAT1 or CD9 was examined for EVs purified from CAT1-overexpressed HCT116 cells (CAT1þþ-EVs) or mock-transfected cells (mock-EV). E, A representative image of TEM for mock-EV or CAT1þþ-EV is shown. The black dot on the surface of the EV indicates the signal of the gold particle-labeled anti-CAT1 antibody; bar, 50 nm. F, Fluorescent microscopic images of HUVEC 4 hours after treatment with PBS or fluorescence-labeled CAT1þþ-EV; bar, 5 mm. G, Fluorescent microscopic images of HUVEC 72 hours after treatment with PBS or CAT1þþ-EV. The expression of CAT1 was detected by the Alexa 488-labeled secondary antibody; bar,5 mmol/L. H and I, The expression of CAT1 (H) or the growth activity (I) was measured in HUVEC 72 hours after treatment with PBS, mock-EV, or CAT1þþ-EV. Each error bars are presented as the mean SE (n ¼ 3). J, Representative images of tube formation assay. 72 hours after treatment with PBS, mock-EV, or CAT1þþ-EV, HUVEC was cultured in Matrigel plates for 24 hours. K, The relative total tube length in (J) was measured by ImageJ. Each error bar is presented as the mean SE (n ¼ 3).

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Immunohistochemistry, Staining, Marker, Comparison, Expressing, Transfection, Control, Labeling, Activity Assay, Tube Formation Assay, Cell Culture

Journal: Molecular Cancer Research

Article Title: Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

doi: 10.1158/1541-7786.mcr-20-0827

Figure Lengend Snippet: Figure 6. CAT1-dependent modulation of the NO metabolic pathway in vascular endothelial cells. A, Expression level of CAT1 was measured in mock-transfected HUVEC (HUVEC-mock) or CAT1-overexpressed HUVEC (HUVEC-CAT1). B, The results of quantitative metabolomic analysis for the arginine/NO/cGMP pathway were illustrated. Absolute quantification of the metaboliteswas performed for HUVEC-mock or HUVEC-CAT1 after stimulation with arginine for 15 minutes. Each error bar is presented as the mean SE (n ¼ 4). C, Cyclic GMP concentrations of HUVEC treated with CAT1þþ-EVs or mock-EVs were compared. Each error bars are presented as the mean SE (n ¼ 3). D, An illustration of CAT1 transfer from colorectal cancer cells to vascular endothelial cells via EVs in tumor microenvironment. Putative signaling pathway in a vascular endothelial cell from the arginine/NO/cGMP pathway to its downstream is shown. Arg, arginine; eNOS, endothelial nitric oxide synthase; NADPH, nicotinamide adenine dinucleotide phosphate; NADP, nicotinamide adenine dinucleotide phosphate; NHA, Nw-hydroxy-L-arginine; NO, nitric oxide; Cit, citrulline; sGC, soluble guanylatecyclase; GTP, guanosine triphosphate; cGMP, cyclic guanosine monophosphate; PRAK, p38-regulated/activated kinase; FAK, Focal adhesion kinase.

Article Snippet: The deparaffinization, rehydration, and IHC were automatically carried out on the Leica Bond III Automated IHC and ISH system (Leica Microsystems Ltd.) with polyclonal anti-CAT1 antibody (Proteintech) and anti-CD31 antibody.

Techniques: Expressing, Transfection

Journal: Frontiers in Immunology

Article Title: Integrated bioinformatic analysis of mitochondrial metabolism-related genes in acute myeloid leukemia

doi: 10.3389/fimmu.2023.1120670

Figure Lengend Snippet: Cox regression to prognosis MMRGs associated with OS in TCGA-LAML.

Article Snippet: Tissue sections were deparaffinized and rehydrated, and then stained with H&E, or incubated with antibodies, specifically ECHS1 (Cat No. 11305-1-AP; proteintech, China), and NUDFS2 (Cat No. R27071; Zenbioscience, China).

Techniques:

Journal: Frontiers in Immunology

Article Title: Integrated bioinformatic analysis of mitochondrial metabolism-related genes in acute myeloid leukemia

doi: 10.3389/fimmu.2023.1120670

Figure Lengend Snippet: Validation of the expression levels of key MMRGs by IHC. A-B. H&E and ECHS1 (A) and NDUFS2 (B) IHC staining of BM samples from nonneoplastic patients and AML patients. Original magnification, 400X. (C-D) . Statistics of ECHS1 (C) and NDUFS2 (D) mean IOD from IHC images of nonneoplastic patients (n = 10) and AML patients (n = 10). ****P-value< 0.0001.

Article Snippet: Tissue sections were deparaffinized and rehydrated, and then stained with H&E, or incubated with antibodies, specifically ECHS1 (Cat No. 11305-1-AP; proteintech, China), and NUDFS2 (Cat No. R27071; Zenbioscience, China).

Techniques: Biomarker Discovery, Expressing, Immunohistochemistry

Journal: Genes & Diseases

Article Title: Lactate transporter MCT4 regulates the hub genes for lipid metabolism and inflammation to attenuate intracellular lipid accumulation in non-alcoholic fatty liver disease

doi: 10.1016/j.gendis.2025.101554

Figure Lengend Snippet: Correlation analysis between the expression of MCT4 in hepatocytes and NAFLD. (A, B) The scatter plots for Mct4 TPM of Normal ( n = 31) and NAFLD ( n = 112) in the GSE162694 dataset (A), and of NAFL ( n = 51) and NASH ( n = 47) in the GSE167523 dataset (B). Unpaired, two-sided Mann–Whitney U test P -values are depicted in the plots, and the significant P -value cutoff was set at 0.05. The plots show the medians (black line), standard deviation, and P -values. (C) The scatter plots for Mct4 TPM in GSE174478 ( n = 94) in which patients were stratified by NAFLD activity score (NAS, left) or fibrosis score (F, right). P -values were obtained via a nonparametric two-stage Benjamini, Krieger, & Yekutieli false discovery rate (FDR) procedure. The mean expression (black line), standard deviation, and P -values are shown. (D) The immunohistochemical staining of protein levels of MCT4 in the livers of C57BL/6J mice fed with chow diet or high-fat diet (HFD). Representative positive stains are indicated with black arrows (100 × and 400 × ). (E) The immunohistochemical staining of MCT4 expression in hepatocellular carcinoma patients with NAFLD (HCC group) and adjacent non-tumor tissues without NAFLD (Para-Ca group). Representative positive stains are indicated with black arrows (100 × and 400 × ). Each assay condition was done in triplicate, and representative images were shown. (F) The expression of Mct4 in oleic acid (OA)-induced lipid accumulation in hepatocytes. Subconfluent iHPx cells were stimulated with 0.05 mM OA (methanol as a vehicle control), total RNA was isolated at 24 h and subjected to touchdown-quantitative PCR analysis of Mct4 expression. ∗∗ P < 0.01, OA versus Methanol. MCT4, monocarboxylate transporter 4; NAFLD, non-alcoholic fatty liver disease; TPM, transcript per million; NAFL, non-alcoholic fatty liver; NASH, non-alcoholic steatohepatitis.

Article Snippet: The sections were deparaffinized and subjected to hematoxylin & eosin staining (Solarbio, Cat# G1120) and immunohistochemical staining as described., For immunohistochemical staining, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked, and incubated overnight with primary antibodies against ARG2 (1:50–1:500 dilution; Santa; Cat# sc-393496) and MCT4 (1:1000–1:4000 dilution; Proteintech; Cat# 22787-1-AP), followed by staining with biotin-labeled goat anti-rabbit IgG or anti-mouse IgG/streptavidin-HRP kit (SP Kit, PV-9000, ZSGB-Bio, China).

Techniques: Expressing, MANN-WHITNEY, Standard Deviation, Activity Assay, Immunohistochemical staining, Staining, Control, Isolation, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Lactate transporter MCT4 regulates the hub genes for lipid metabolism and inflammation to attenuate intracellular lipid accumulation in non-alcoholic fatty liver disease

doi: 10.1016/j.gendis.2025.101554

Figure Lengend Snippet: Silencing MCT4 induces lipid accumulation while exogenous MCT4 leads to decreased lipid accumulation in hepatocytes. Subconfluent iHPx were infected with AdR-siMCT4 or Ad-RFP and stimulated with 0.05 mM oleic acid (OA) for the indicated time points. (A) The oil red O staining (a) and bodipy 493/503 staining (b) were done on day 5. Representative lipid droplets were indicated with arrows (200 × ). (B) Touchdown-quantitative PCR analysis of the expression of the genes involved in lipolysis and lipogenesis in hepatocytes at 48 h. Expression of each target gene was calculated as a relative expression to Gapdh . ∗ P < 0.05; ∗∗ P < 0.01, OA + AdR-siMCT4 versus OA + Ad-RFP. (C) The cellular triglyceride (TG), free fatty acid (FFA), glucose (Glu), lactate (LA), and pyruvate (PA) levels were measured at day 5. ∗∗ P < 0.01, OA + AdR-siMCT4 versus OA + Ad-RFP. Alternatively, subconfluent iHPx were infected with Ad-MCT4 or Ad-GFP and stimulated with 0.05 mM OA for the indicated time points. (D) The oil red O staining was done on day 7. Representative lipid droplets were indicated with arrows (200 × ). (E) Touchdown-quantitative PCR analysis of the expression of the genes involved in lipolysis and lipogenesis, and transcriptional regulation in hepatocytes at 48 h. Expression of each target gene was calculated as a relative expression to Gapdh . ∗∗ P < 0.01, OA + Ad-MCT4 versus OA + Ad-GFP. (F) The cellular TG, FFA, Glu, LA, and PA levels were measured on day 5. ∗∗ P < 0.01, ∗ P < 0.05, OA + Ad-MCT4 versus OA + Ad-GFP. Each assay condition was done in triplicate, and representative images were shown. MCT4, monocarboxylate transporter 4.

Article Snippet: The sections were deparaffinized and subjected to hematoxylin & eosin staining (Solarbio, Cat# G1120) and immunohistochemical staining as described., For immunohistochemical staining, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked, and incubated overnight with primary antibodies against ARG2 (1:50–1:500 dilution; Santa; Cat# sc-393496) and MCT4 (1:1000–1:4000 dilution; Proteintech; Cat# 22787-1-AP), followed by staining with biotin-labeled goat anti-rabbit IgG or anti-mouse IgG/streptavidin-HRP kit (SP Kit, PV-9000, ZSGB-Bio, China).

Techniques: Infection, Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Genes & Diseases

Article Title: Lactate transporter MCT4 regulates the hub genes for lipid metabolism and inflammation to attenuate intracellular lipid accumulation in non-alcoholic fatty liver disease

doi: 10.1016/j.gendis.2025.101554

Figure Lengend Snippet: MCT4 inhibits the progress of NAFLD in vivo . A NAFLD model was built as described. Ad-MCT4 or Ad-GFP was intrahepatically injected once every 5 days. The mice were sacrificed at week 8. (A) The dynamic weight changes of mice. ∗∗ P < 0.01, ∗ P < 0.05, HFD + Ad-MCT4 versus HFD + Ad-GFP. (B) The retrieved liver tissues were subjected to oil red O staining. Representative positive stains are indicated with black arrows (400 × ). (C) Total RNA was isolated from the liver tissues of the mice and touchdown-quantitative PCR analysis of the expression of the genes involved in lipolysis and lipogenesis, and transcriptional regulation. The expression of each target gene was calculated as a relative expression to Gapdh . ∗∗ P < 0.01, ∗ P < 0.05, HFD + Ad-MCT4 versus HFD + Ad-GFP. (D) The cellular triglyceride (TG), free fatty acid (FFA), glucose (Glu), lactate (LA), and pyruvate (PA) levels were measured. ∗∗ P < 0.01, ∗ P < 0.05, HFD + Ad-MCT4 versus HFD + Ad-GFP. Each assay condition was done in triplicate, and representative images were shown. MCT4, monocarboxylate transporter 4; NAFLD, non-alcoholic fatty liver disease; HFD, high-fat diet.

Article Snippet: The sections were deparaffinized and subjected to hematoxylin & eosin staining (Solarbio, Cat# G1120) and immunohistochemical staining as described., For immunohistochemical staining, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked, and incubated overnight with primary antibodies against ARG2 (1:50–1:500 dilution; Santa; Cat# sc-393496) and MCT4 (1:1000–1:4000 dilution; Proteintech; Cat# 22787-1-AP), followed by staining with biotin-labeled goat anti-rabbit IgG or anti-mouse IgG/streptavidin-HRP kit (SP Kit, PV-9000, ZSGB-Bio, China).

Techniques: In Vivo, Injection, Staining, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: Genes & Diseases

Article Title: Lactate transporter MCT4 regulates the hub genes for lipid metabolism and inflammation to attenuate intracellular lipid accumulation in non-alcoholic fatty liver disease

doi: 10.1016/j.gendis.2025.101554

Figure Lengend Snippet: MCT4 regulates hub genes interactions to influence hepatic lipid metabolism. Subconfluent iHPx cells were infected with Ad-MCT4, AdR-siMCT4, or Ad-RFP, and stimulated with 0.05 mM oleic acid for 48 h. Total RNA was collected for RNA-sequencing analysis. (A) Venn Diagram of the differentially expressed genes (DEGs). (B) Gene set enrichment analysis (GSEA) of all expressed genes (MCT4/siMCT4). (C) Clustering analysis of FPKM values of 556 DEGs. (D) Venn diagram of partial DEGs verified by touchdown-quantitative PCR. (E) The network landscape generated by Cytoscape indicates a potential interaction relationship (colors indicate the importance of genes). (F) Scatter plot for Arg2 TPM of Normal ( n = 31) and NAFLD ( n = 112) in GSE162694 dataset (a). Unpaired, two-sided Mann–Whitney U test P -value are depicted in the plot, and the plot shows the medians (black line), standard deviation, and P -value. Correlation analysis of Arg2 level with Mct4 in human NAFL liver samples ( GSE167523 , n = 51) (b). r and P -value were obtained via two-tailed nonparametric Spearman's test, and the plot shows the linear regression line (black line) and r and P -value. (G) Immunohistochemical staining of ARG2 expression in liver samples in E. Representative positive stains are indicated with black arrows (100 × and 400 × ). (H) Subconfluent iHPx cells were infected with Ad-MCT4 or Ad-GFP, and stimulated with oleic acid, and total cell lysate was subjected to western blotting analysis to assess MCT4 and ARG2 expression at 72 h. Each assay condition was done in triplicate, and representative images were shown. (I) Immunohistochemical staining of MCT4 and ARG2 expression in liver tissues retrieved in . Representative positive stains are indicated with red arrows (400 × ). MCT4, monocarboxylate transporter 4; NAFLD, non-alcoholic fatty liver disease; NAFL, non-alcoholic fatty liver; FPKM, fragments per kilo base per million mapped reads; TPM, transcript per million; ARG2, arginase 2.

Article Snippet: The sections were deparaffinized and subjected to hematoxylin & eosin staining (Solarbio, Cat# G1120) and immunohistochemical staining as described., For immunohistochemical staining, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked, and incubated overnight with primary antibodies against ARG2 (1:50–1:500 dilution; Santa; Cat# sc-393496) and MCT4 (1:1000–1:4000 dilution; Proteintech; Cat# 22787-1-AP), followed by staining with biotin-labeled goat anti-rabbit IgG or anti-mouse IgG/streptavidin-HRP kit (SP Kit, PV-9000, ZSGB-Bio, China).

Techniques: Infection, RNA Sequencing, Real-time Polymerase Chain Reaction, Generated, MANN-WHITNEY, Standard Deviation, Two Tailed Test, Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Genes & Diseases

Article Title: Lactate transporter MCT4 regulates the hub genes for lipid metabolism and inflammation to attenuate intracellular lipid accumulation in non-alcoholic fatty liver disease

doi: 10.1016/j.gendis.2025.101554

Figure Lengend Snippet: MCT4 regulates hub genes interactions to influence hepatic lipid metabolism. Subconfluent iHPx cells were infected with Ad-MCT4, AdR-siMCT4, or Ad-RFP, and stimulated with 0.05 mM oleic acid for 48 h. Total RNA was collected for RNA-sequencing analysis. (A) Venn Diagram of the differentially expressed genes (DEGs). (B) Gene set enrichment analysis (GSEA) of all expressed genes (MCT4/siMCT4). (C) Clustering analysis of FPKM values of 556 DEGs. (D) Venn diagram of partial DEGs verified by touchdown-quantitative PCR. (E) The network landscape generated by Cytoscape indicates a potential interaction relationship (colors indicate the importance of genes). (F) Scatter plot for Arg2 TPM of Normal ( n = 31) and NAFLD ( n = 112) in GSE162694 dataset (a). Unpaired, two-sided Mann–Whitney U test P -value are depicted in the plot, and the plot shows the medians (black line), standard deviation, and P -value. Correlation analysis of Arg2 level with Mct4 in human NAFL liver samples ( GSE167523 , n = 51) (b). r and P -value were obtained via two-tailed nonparametric Spearman's test, and the plot shows the linear regression line (black line) and r and P -value. (G) Immunohistochemical staining of ARG2 expression in liver samples in E. Representative positive stains are indicated with black arrows (100 × and 400 × ). (H) Subconfluent iHPx cells were infected with Ad-MCT4 or Ad-GFP, and stimulated with oleic acid, and total cell lysate was subjected to western blotting analysis to assess MCT4 and ARG2 expression at 72 h. Each assay condition was done in triplicate, and representative images were shown. (I) Immunohistochemical staining of MCT4 and ARG2 expression in liver tissues retrieved in . Representative positive stains are indicated with red arrows (400 × ). MCT4, monocarboxylate transporter 4; NAFLD, non-alcoholic fatty liver disease; NAFL, non-alcoholic fatty liver; FPKM, fragments per kilo base per million mapped reads; TPM, transcript per million; ARG2, arginase 2.

Article Snippet: The sections were deparaffinized and subjected to hematoxylin & eosin staining (Solarbio, Cat# G1120) and immunohistochemical staining as described., For immunohistochemical staining, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked, and incubated overnight with primary antibodies against ARG2 (1:50–1:500 dilution; Santa; Cat# sc-393496) and MCT4 (1:1000–1:4000 dilution; Proteintech; Cat# 22787-1-AP), followed by staining with biotin-labeled goat anti-rabbit IgG or anti-mouse IgG/streptavidin-HRP kit (SP Kit, PV-9000, ZSGB-Bio, China).

Techniques: Infection, RNA Sequencing, Real-time Polymerase Chain Reaction, Generated, MANN-WHITNEY, Standard Deviation, Two Tailed Test, Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Frontiers in Microbiology

Article Title: NF-κB/TWIST1 Mediates Migration and Phagocytosis of Macrophages in the Mice Model of Implant-Associated Staphylococcus aureus Osteomyelitis

doi: 10.3389/fmicb.2020.01301

Figure Lengend Snippet: S. aureus infection activates the expression of TWIST1 in macrophages. (A,B) Western blotting analysis (A) and quantification (B) of TWIST1 expression in the bone marrow from S. aureus infected bone and control bone. n = 6/group. (C) Representative images of immunohistochemistry staining for TWIST1 in bone from S. aureus infected mice and control mice. Scale bars form left to right represents 100 μm and 20 μm, respectively, and stars represent the implant track. (D) Quantitative analysis of the number of TWIST1 + cells per tissue area around implant ( N . TWIST1 + /mm 2 ). n = 6/group. (E) Representative images of double-immunofluorescence staining for TWIST1 and F4/80 in bone from S. aureus infected mice and control mice. Scale bar represents 100 μm. Stars locate the implant track white arrows point to positive stained cells. (F) Quantitative analysis of the number of F4/80 + TWIST1 + cells per tissue area around implant (N. F4/80 + TWIST1 + /mm 2 ). n = 4/group. * p < 0.05, ** p < 0.01, and Mann–Whitney U -test.

Article Snippet: After being deparaffinized and rehydrated, sections were incubated with primary antibodies to TWIST1 (Cat. 25465-1-AP, Proteintech, Wuhan, China), and F4/80 (Cat.71299, Cell Signal Technology, MA, United States) overnight at 4°C.

Techniques: Infection, Expressing, Western Blot, Control, Immunohistochemistry, Staining, Double Immunofluorescence Staining, MANN-WHITNEY

Journal: Frontiers in Microbiology

Article Title: NF-κB/TWIST1 Mediates Migration and Phagocytosis of Macrophages in the Mice Model of Implant-Associated Staphylococcus aureus Osteomyelitis

doi: 10.3389/fmicb.2020.01301

Figure Lengend Snippet: S. aureus infection activates the expression of TWIST1 in Raw 264.7 cells. (A) qPCR analysis of the mRNA expression of TWIAT1, NANOG, and ERBB2 in Raw 264.7 cells in response to S. aureus infection with MOI [the ratio of the S. aureus colonies number (CFU) to the Raw 264.7 cells number] at 0.01, 0.1, 1.0, and 10. ANOVA followed by Dunnett’s test, n = 3/group. (B) Immunoblot and (C) quantitative analysis of TWIST1 in Raw 264.7 cells after infection. Two-tailed Student’s t test, n = 3/group. (D) qPCR analysis of the effect of siRNA fragments on the mRNA expression of TWIST1 in Raw 264.7 cells. ANOVA followed by Dunnett’s test, n = 3/group. (E) Immunoblot and (F) quantitative analysis of the effect of siRNA fragments on the protein expression of TWIST1 in Raw 264.7 cells. ANOVA followed by Dunnett’s test, n = 3/group. (G) qPCR analysis of the effect of knocking down TWIST1 on the mRNA expression of marker genes for macrophage polarization in Raw 264.7 cells after S. aureus infection. ANOVA followed by Dunnett’s test, n = 3/group. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: After being deparaffinized and rehydrated, sections were incubated with primary antibodies to TWIST1 (Cat. 25465-1-AP, Proteintech, Wuhan, China), and F4/80 (Cat.71299, Cell Signal Technology, MA, United States) overnight at 4°C.

Techniques: Infection, Expressing, Western Blot, Two Tailed Test, Marker

Journal: Frontiers in Microbiology

Article Title: NF-κB/TWIST1 Mediates Migration and Phagocytosis of Macrophages in the Mice Model of Implant-Associated Staphylococcus aureus Osteomyelitis

doi: 10.3389/fmicb.2020.01301

Figure Lengend Snippet: TWIST1 mediates the migration and phagocytosis of macrophage in response to S. aureus infection. Wound-healing assay was used to evaluate the effect of S. aureus on macrophage migration. After 1 × 10 6 cells were seeded in 6-well plate and cultured for 12 h, they were infected with S. aureus at 0.01 MOI. (A) Representative images for wound-healing assay and (B) quantitative analysis of gap closure ratio of Raw 264.7 cells at 0, 24, 48, and 72 h after S. aureus infection. Scale bar represents 100 μm. (C) Transwell-based migration assays and (D) quantitative analysis of the migrated cells of Raw 264.7 cells at 24 h after S. aureus infection at 0.01 MOI. Scale bar represents 100 μm. (E) Wound-healing assay and (F) quantitative analysis of gap closure ratio of Raw 264.7 cells at 0, 24, 48, and 72 h after S. aureus infection. Scale bar represents 100 μm. NC represents negative control. Cells were transfected with negative random fragments. (G) Transwell-based migration assays and (H) quantitative analysis of the migrated cells of Raw 264.7 cells at 24 h after S. aureus infection. Scale bar represents 100 μm. Quantitative analysis of the effect of knocking down TWIST1 on Phagocytosis rate (PR; I ) and killing rate (KR; J ) of Raw 264.7 cells in response to S. aureus infection. * p < 0.05, ** p < 0.01, *** p < 0.001, and Two-tailed Student’s t test, n = 3/group.

Article Snippet: After being deparaffinized and rehydrated, sections were incubated with primary antibodies to TWIST1 (Cat. 25465-1-AP, Proteintech, Wuhan, China), and F4/80 (Cat.71299, Cell Signal Technology, MA, United States) overnight at 4°C.

Techniques: Migration, Infection, Wound Healing Assay, Cell Culture, Negative Control, Transfection, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: NF-κB/TWIST1 Mediates Migration and Phagocytosis of Macrophages in the Mice Model of Implant-Associated Staphylococcus aureus Osteomyelitis

doi: 10.3389/fmicb.2020.01301

Figure Lengend Snippet: NF-κB/TWIST1 mediates the effect of S. aureus on macrophages. (A) Immunoblot and (B) quantitative analysis of MMP9 and MMP13 expression in Raw 264.7 cells in response to S. aureus infection. Two-tailed Student’s t test, n = 3/group. (C) Immunoblot and (D) quantitative analysis of the effect of knocking down TWIST1 on the expression of MMP9 and MMP13 in Raw 264.7 cells after S. aureus infection. Two-tailed Student’s t test, n = 3/group. (E) Immunoblot and quantitative analysis (F) of NF-κB in Raw 264.7 cells at 0, 1, 3, 6, 12, and 24 h after S. aureus infection. ANOVA followed by Dunnett’s test, n = 3/group. (G) Immunoblot and quantitative analysis (H) of the effect of NF-κB inhibitor (Bay11-7082) on the expression of MMP9 and MMP13 in Raw 264.7 cells after S. aureus infection. Two-tailed Student’s t test, n = 3/group. * p < 0.05, and ** p < 0.01.

Article Snippet: After being deparaffinized and rehydrated, sections were incubated with primary antibodies to TWIST1 (Cat. 25465-1-AP, Proteintech, Wuhan, China), and F4/80 (Cat.71299, Cell Signal Technology, MA, United States) overnight at 4°C.

Techniques: Western Blot, Expressing, Infection, Two Tailed Test