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 proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes, followed by being blocked and incubated overnight with the primary antibodies against β-ACTIN (1:5000–1:50,000 dilution; Proteintech; Cat# 66009-1-Ig), ARG2 (1:100–1:1000 dilution; Santa; Cat# sc-393496), and MCT4 (1:2000–1:20,000 dilution; Proteintech; Cat# 22787-1-AP).

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

Journal: Journal of Pharmaceutical Analysis

Article Title: A novel exploration of COL11A1 's role in regulating myeloid-derived suppressor cell activation within the colon cancer microenvironment

doi: 10.1016/j.jpha.2024.101181

Figure Lengend Snippet: Analysis of cell interactions in colon cancer (CC) tissues. (A) It shows the number and strength of cell-to-cell interactions in normal tissues. (B) It shows the number and strength of cell-to-cell interactions in CC tissues. (C) Shell plots illustrating interactions between fibroblasts and other cells in normal tissues. (D) Shell plots illustrating interactions between fibroblasts and other cells in CC tissues. (E) Proportions of different cell types in a single sample are computed based on the Seurat object. (F) Expression of known fibroblast-specific markers in each cluster. (G) Expression of known myeloid-derived suppressor cell (MDSC)-specific markers in each cluster. (H) Expression of characteristic markers of granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs) in clusters. Darker red indicates higher expression levels and larger circles represent more cells expressing the gene. (I) Immunohistochemical analysis of the expression and distribution of collagen type XI alpha 1 (COL11A1) and arginase 2 (ARG2) in colon tissues under normal and CC conditions ( n = 10 in each group). ∗∗ P < 0.01 (J) Immunofluorescence staining analysis of the co-localization of COL11A1 and ARG2 in human CC tissues ( n = 10 in each group).

Article Snippet: The following antibody markers were employed for the co-cultivation of BMCs and fibroblasts over 72 h: the primary antibody Rabbit Anti-Arginase 2 (ARG2) (55003, Cell Signaling Technology, Danvers, MA, USA), PE-CD11b (ab8878, Abcam, Cambridge, UK), and the secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150077, Abcam) were used to label activated MDSCs.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Immunofluorescence, Staining

Journal: Journal of Pharmaceutical Analysis

Article Title: A novel exploration of COL11A1 's role in regulating myeloid-derived suppressor cell activation within the colon cancer microenvironment

doi: 10.1016/j.jpha.2024.101181

Figure Lengend Snippet: Effects of cancer-associated fibroblasts (CAFs) in colon cancer (CC) tissues on myeloid-derived suppressor cell (MDSC) differentiation and activation. (A) Flow cytometry analysis of arginase 2 ( ARG2 ) and CD11b expression frequency in bone marrow cells (BMCs) co-cultured with normal fibroblasts (NFs) and CAFs. (B) ARG2 and CD11b messenger RNA (mRNA) expression levels in BMCs co-cultured with NFs and CAFs. (C) ARG2 and CD11b protein expression levels in BMCs co-cultured with NFs and CAFs. (D) Flow cytometry analysis of interferon gamma (IFN-γ) secretion in CD8 + T cells. (E) Flow cytometry analysis of granzyme B (GZMB) secretion in CD8 + T cells. (F) Flow cytometry analysis of tumor necrosis factor alpha (TNF-α) secretion in CD8 + T cells. (G) Flow cytometry analysis of apoptosis in CD8 + T cells. (H) Flow cytometry analysis of proliferation in CD8 + T cells. ns P > 0.05, ∗ P < 0.05, ∗∗ P < 0.01; all cell experiments were repeated three times. N-BMCs: non-fibroblast (NF) BMCs; C-BMCs: cancer-associated fibroblast (CAF) BMCs; FITC: fluorescein isothiocyanate; 7-AAD: 7-aminoactinomycin D.

Article Snippet: The following antibody markers were employed for the co-cultivation of BMCs and fibroblasts over 72 h: the primary antibody Rabbit Anti-Arginase 2 (ARG2) (55003, Cell Signaling Technology, Danvers, MA, USA), PE-CD11b (ab8878, Abcam, Cambridge, UK), and the secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150077, Abcam) were used to label activated MDSCs.

Techniques: Derivative Assay, Activation Assay, Flow Cytometry, Expressing, Cell Culture

Journal: Journal of Pharmaceutical Analysis

Article Title: A novel exploration of COL11A1 's role in regulating myeloid-derived suppressor cell activation within the colon cancer microenvironment

doi: 10.1016/j.jpha.2024.101181

Figure Lengend Snippet: Effects of collagen type XI alpha 1 ( COL11A1 ) in cancer-associated fibroblasts (CAFs) on myeloid-derived suppressor cells (MDSCs) differentiation and activation. (A) Enzyme-linked immunosorbent assay (ELISA) detection of matrix metalloproteinase (MMP)3 and MMP13 content in supernatant of bone marrow cells (BMCs) co-cultured with COL11A1 knockout ( COL11A1 KO ) CAFs. (B) Flow cytometry analysis of arginase 2 ( ARG2 ) and CD11b expression frequency in BMCs co-cultured with COL11A1 KO CAFs. (C) ARG2 and CD11b messenger RNA (mRNA) expression levels in BMCs co-cultured with COL11A1 KO CAFs. (D) ARG2 and CD11b protein expression levels in BMCs co-cultured with COL11A1 KO CAFs. (E) Flow cytometry analysis of interferon gamma (IFN-γ) secretion in CD8 + T cells. (F) Flow cytometry analysis of granzyme B (GZMB) secretion in CD8 + T cells. (G) Flow cytometry analysis of tumor necrosis factor alpha (TNF-α) secretion in CD8 + T cells. (H) Flow cytometry analysis of apoptosis in CD8 + T cells. (I) Flow cytometry analysis of proliferation in CD8 + T cells. ∗∗ P < 0.01; all cell experiments were repeated three times. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; FITC: fluorescein isothiocyanate; CFSE: carboxyfluorescein succinimidyl ester.

Article Snippet: The following antibody markers were employed for the co-cultivation of BMCs and fibroblasts over 72 h: the primary antibody Rabbit Anti-Arginase 2 (ARG2) (55003, Cell Signaling Technology, Danvers, MA, USA), PE-CD11b (ab8878, Abcam, Cambridge, UK), and the secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150077, Abcam) were used to label activated MDSCs.

Techniques: Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Knock-Out, Flow Cytometry, Expressing

Journal: Journal of Pharmaceutical Analysis

Article Title: A novel exploration of COL11A1 's role in regulating myeloid-derived suppressor cell activation within the colon cancer microenvironment

doi: 10.1016/j.jpha.2024.101181

Figure Lengend Snippet: Impact of collagen type XI alpha 1 ( COL11A1 ) activation through myeloid-derived suppressor cells (MDSCs) on colon cancer (CC) tumor growth and proliferation. (A) Growth of CC orthotopic xenografts in mice after COL11A1 knockout. (B) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to evaluate cell apoptosis in CC orthotopic xenografts in mice after COL11A1 knockout. (C) Ki67 staining to assess cell proliferation in CC orthotopic xenografts in mice after COL11A1 knockout. (D) Immunohistochemical detection of COL11A1 and arginase 2 ( ARG2 ) expression in CC orthotopic xenografts in mice after COL11A1 knockout. (E) Immunohistochemical detection of matrix metalloproteinase ( MMP )3 and MMP13 expression in CC orthotopic xenografts in mice after COL11A1 knockout. (F) Flow cytometry analysis of MDSC differentiation and activation in CC orthotopic xenografts in mice after COL11A1 knockout ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: The following antibody markers were employed for the co-cultivation of BMCs and fibroblasts over 72 h: the primary antibody Rabbit Anti-Arginase 2 (ARG2) (55003, Cell Signaling Technology, Danvers, MA, USA), PE-CD11b (ab8878, Abcam, Cambridge, UK), and the secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150077, Abcam) were used to label activated MDSCs.

Techniques: Activation Assay, Derivative Assay, Knock-Out, TUNEL Assay, Staining, Immunohistochemical staining, Expressing, Flow Cytometry

Journal: iScience

Article Title: APE1 promotes lung adenocarcinoma through G4-mediated transcriptional reprogramming of urea cycle metabolism

doi: 10.1016/j.isci.2025.112275

Figure Lengend Snippet: APE1 is required to sustain KRAS G12D -induced LUAD development in mice (A) Hematoxylin and eosin (H&E)-staining of tumor-bearing lungs from indicated genetically engineered mouse models (GEMMs). Lung tissues were collected at 17 weeks after Ad-CMV-Cre intranasal inhalation. Right panel showed the quantification of tumor burdens. For each mouse lung sample, the largest cross-section of the lung was selected as a representative for tumor quantification. n = 7 tissue sections from seven mice. Scale bars indicate 2 mm. Statistical significance was assessed using two-tailed unpaired Student’s t test. (B) Scatterplot showing the DEGs in Ape1 knockout (left) or Ape1 N211A mutant (right) early-stage cancer cells versus Ape1 +/− ones. Mice were intranasally administered with Ad-CMV-Cre. After 12 weeks, lungs were collected to sort tdTomato + /EPCAM + /DAPI − /CD45 − /CD31 − epithelial cells for RNA-seq analysis. n = 4 mice for Ape1 +/− , Kras G12D genotype; n = 3 mice for Ape1 −/− , Kras G12D or Ape1 N211A/- , Kras G12D genotype. nCount represents the mean of counts normalized by DESeq2. The dashed lines indicate the 2-fold change threshold for defining DEGs. Red and blue dots depict significantly changed genes (log2(fold change) ≥ 1 or ≤ −1 and p value ≤0.05) and gray dots depict genes without significant changes. The number of DEGs is indicated. (C and D) KEGG pathways analysis of DEGs in Ape1-deficient (C) or Ape1 N211A mutant (D) early-stage cancer cells. Pathways involved in amino acid biosynthesis and pyrimidine metabolism are indicated in red. (E and F) Gene set enrichment analysis (GSEA) plots showing pathway involved in biosynthesis of amino acids in Ape1-deficient (E) or Ape1 N211A mutant (F) early-stage cancer cells. NES, normalized enrichment score. (G) Representative immunohistochemistry (IHC) images for Arg2 in lung tumor tissues from indicated GEMMs. Lung tissues were collected at 17 weeks after Ad-CMVR-Cre infection. Scale bars indicate 200 μm (top) or 20 μm (bottom). All the data are presented as mean ± SEM.

Article Snippet: Primary antibody against ARG2 (Cell Signaling, 55003, 1:2000) was used in this study.

Techniques: Staining, Two Tailed Test, Knock-Out, Mutagenesis, RNA Sequencing, Immunohistochemistry, Infection

Journal: iScience

Article Title: APE1 promotes lung adenocarcinoma through G4-mediated transcriptional reprogramming of urea cycle metabolism

doi: 10.1016/j.isci.2025.112275

Figure Lengend Snippet: APE1 knockout hinders the presence of G4 structures in the CPS1 and ARG2 promoters (A) Promoter sequences of CPS1 (top) and ARG2 (bottom) identified by G4Hunter using a window of 20 nucleotides and a threshold of 1.25 with their corresponding score on both forward and reverse strands. The shaded boxes indicate the G4-forming sequences used in Figure 5 . (B) Genome browser representation of G4 structures and Observed-G4s (OGs) at the promoters of CPS1 (top) and ARG2 (bottom) in human cells. Data of G4P ChIP-Seq and G4-seq are from GSE133379 and GSE63874, respectively. (C) Reduced G4 signals in A549 cells upon APE1 deletion. Plot profile (top) and heatmaps (bottom) showing enrichment of G4 (RPGC, reads per genome coverage) in APE1 +/+ and APE1 −/− A549 cells. The signal is plotted in a 6 kb window flanking the transcriptional start sites (TSS). (D) Genome browser representation of G4 structures, H3K4me3 and chromatin accessibility at the promoters of CPS1 (top) and ARG2 (bottom). The shaded boxes highlight G4 structures co-occurring in the promoter regions enriched in H3K4me3.

Article Snippet: Primary antibody against ARG2 (Cell Signaling, 55003, 1:2000) was used in this study.

Techniques: Knock-Out, ChIP-sequencing

Journal: iScience

Article Title: APE1 promotes lung adenocarcinoma through G4-mediated transcriptional reprogramming of urea cycle metabolism

doi: 10.1016/j.isci.2025.112275

Figure Lengend Snippet: APE1 regulates the transcription of CPS1 and ARG2 by facilitating G4 structure formation (A) BG4 CUT&Tag sequencing profiles for the G4 edited sites at the promoters of CPS1 (upper) or ARG2 (bottom) and the STAT3 control site. (B) RT-qPCR analysis of CPS1 in CPS1 MUT (top) and ARG2 in ARG2 MUT (bottom) A549 cells. “MUT” denotes that the G4 sequence in the promoter of the respective genes was mutated by gene editing. (C) RT-qPCR analysis of CPS1 in CPS1 MYC G4 (top) and ARG2 in ARG2 MYC G4 (bottom) A549 cells. “MYC G4” denotes that the native G4 sequence in the promoter of the respective genes was replaced by MYC G4 sequence. (D) RT-qPCR analysis of CPS1 and ARG2 in APE1 +/+ and APE1 −/− A549 cells treated with G4-stabilizing ligand PDS (5 mM) for 2 h. (E) RT-qPCR analysis of CPS1 and ARG2 in APE1 −/− A549 cells with restoration of either APE1 WT or N212A mutant. (F) RT-qPCR analysis of CPS1 and ARG2 in APE1 +/+ and APE1 −/− A549 cells treated with OGG1 inhibitor TH5487 (5 μM) for 2 h. (G) The proliferation of APE1 +/+ and APE1 −/− A549 cells treated with OGG1 inhibitor TH5487 for 24 h. Cells were counted using the CCK8 assay. The expression levels were normalized to GAPDH , and then compared to the untreated APE1 +/+ control, which was set to 1.0. Data in (B–G) are presented as mean ± SEM from three (G) or four (B–F) biological replicates. The data presented in (B–D) and (F and G) are derived from four independent CRISPR clones. Statistical significance was assessed using two-tailed unpaired Student’s t test. ns, not significant.

Article Snippet: Primary antibody against ARG2 (Cell Signaling, 55003, 1:2000) was used in this study.

Techniques: Sequencing, Control, Quantitative RT-PCR, Mutagenesis, CCK-8 Assay, Expressing, Derivative Assay, CRISPR, Clone Assay, Two Tailed Test

Journal: iScience

Article Title: APE1 promotes lung adenocarcinoma through G4-mediated transcriptional reprogramming of urea cycle metabolism

doi: 10.1016/j.isci.2025.112275

Figure Lengend Snippet: APE1 shows strong binding affinity to G4 structures in CPS1 and ARG2 promoters (A and B) Schematic diagram of G4, non-G4 (control) regions and G4-mutated sequences at the CPS1 (A) and ARG2 (B) promoters. The underlined sequences denote G4-forming oligo containing the G4 motif or non-G4-forming oligo in which G residues are substituted with A (Mut, in blue). The location and length of each DNA fragment amplified in the real-time ChIP-PCR experiments ( Figure 5 E) are indicated. Arrows represent the positions of the primers. Red letters indicate the positions critical for G4 formation. TSS, transcription start site. (C) Binding curves illustrating the association and dissociation of recombinant APE1 protein to the immobilized, biotinylated ssDNA oligonucleotide containing the CPS1 (left) and ARG2 (right) G4 by biolayer interferometry (BLI) analysis. Apparent KD for the most abundant population is indicated. (D) BLI assay for non-G4-forming (MUT) oligonucleotides of CPS1 (left) and ARG2 (right). (E) Enrichment of APE1 on the CPS1 (left) and ARG2 (right) promoters in knock-in A549 cells with HA-tagged APE1 (HA-APE1 WT ) and in APE1 −/− expressing transgenic FLAG-tagged APE1 N212A (FLAG-APE1 N212A ). ChIP-qPCR was performed, with control regions located within the gene body lacking G4-forming ability ( Figure 5 A). Data are presented as mean ± SEM from four biological replicates. Statistical significance was assessed using two-tailed unpaired Student’s t test (E). ns, not significant.

Article Snippet: Primary antibody against ARG2 (Cell Signaling, 55003, 1:2000) was used in this study.

Techniques: Binding Assay, Control, Amplification, Recombinant, Knock-In, Expressing, Transgenic Assay, ChIP-qPCR, Two Tailed Test

Journal: iScience

Article Title: APE1 promotes lung adenocarcinoma through G4-mediated transcriptional reprogramming of urea cycle metabolism

doi: 10.1016/j.isci.2025.112275

Figure Lengend Snippet:

Article Snippet: Primary antibody against ARG2 (Cell Signaling, 55003, 1:2000) was used in this study.

Techniques: Virus, Recombinant, Purification, Cell Counting, Protein Extraction, Genome Wide, Binding Assay, Next-Generation Sequencing, CRISPR, Software