Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 3 In vitro CRISPR/Cas9-mediated screen to identify E2F members important for HSPC expansion. A FACS analysis of the percentage of GFP+ (Cas9+) cells within the mCherry+ (sgRNA+) and mCherry- (sgRNA-) HSPCs on day two (top) and six (bottom) post transduction. B Heatmap of survival/proliferation scores in HSPCs treated with the indicated sgRNAs (n = 2, biological replicates). The color indicates decreased (orange) and increased (blue) survival/proliferation scores. C Western blot of TFDP1 and E2F4 three days post targeting with sgRNAs against Tfdp1 and E2f4. SgRosa26-1 was used as a negative control. Actin was used as a loading control. D Co-immunoprecipitation (Co-IP) using TFDP1 (top) and E2F4 as precipitating antibody (bottom) (n = 2, biological replicates).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: In Vitro, CRISPR, Transduction, Western Blot, Negative Control, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 5 TFDP1 and E2F4 regulate HSPC proliferation, but not apoptosis. A Scheme of assessment of HSPC proliferation and apoptosis. Cas9- HSPCs were labeled with Celltrace, cultured for one day and transduced with lentiviral particles expressing sgRNAs targeting Tfdp1, E2f4, or the Rosa26. B Representative FACS analysis of the percentages of Annexin V+DAPI- (early) and Annexin V+DAPI+ (late) apoptotic cells within mCherry- (sgRNA-, upper panel) or mCherry+ (sgRNA+, lower panel) HSPCs treated with the indicated sgRNAs. C Representative FACS analysis of active Caspase 3+ apoptotic HSPCs treated with the indicated sgRNAs three days post puromycin selection (top) and summary of the data (bottom) based on HSPCs from three mice (n = 3). D Representative FACS analysis of the proliferation rates of mCherry- (sgRNA-) and mCherry+ (sgRNA+) HSPCs infected with the indicated sgRNAs two and four days post cell-trace labeling. The number of cell divisions is indicated. E Percentage of cell division in mCherry+ (upper) and mCherry- (below) HSPC subpopulations treated with the indicated sgRNAs on day two and day four post cell-trace labeling (n = 3 independent experiments).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Labeling, Cell Culture, Transduction, Expressing, Selection, Infection

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 6 Transcriptional regulation of TFDP1 in HSPCs. A Experimental scheme of the RNA-seq experiment in Tfdp1-KO HSPCs. As a negative control, sgRosa26 was used. B Volcano plot depicting changes in gene expression in Tfdp1-KO HSPCs; y-axis represents the log10 transformation of the adjusted p value and x-axis the log2 transformation of the fold change. Red and blue dots represent up and downregulated genes, respectively. REACTOME pathway (C) and transcription factor enrichment analysis (D) of the downregulated genes in Tfdp1-KO HSPCs. The bubble plots depict the top 10 most significantly enriched gene sets. The bubble size corresponds to the number of genes and the color intensity reflects the adj. p value for each geneset. The total number of genes found within each dataset and the number of genes present in the downregulated genes are shown on the right.

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: RNA Sequencing, Negative Control, Gene Expression, Transformation Assay

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 7 Meta-analysis of the role of TFDP1 and E2F4 in gene activation in mouse HSPCs. A Venn diagrams depicting the overlap between the differentially expressed genes in Tfdp1-KO HSPCs (downregulated genes in blue and upregulated genes in red) and human TFDP1- (left; GSE80661; GSE105217; GSE127368), human E2F4- (middle; GSE31477; GSE170651), and mouse E2F4- (right; GSE48666) bound target genes. B Intersection between human TFDP1- and E2F4-bound genes downregulated in Tfdp1-KO HSPCs. C Density plots (upper panel) and heatmaps (lower panel) depicting the average tag densities around TSSs (−2/+2 kb) of up- and downregulated genes in Tfdp1 KO HSPCs. Data are derived from the ChIP-seq of RNA polymerase II S5P (RnapolII S5P; GSE34518), H3K4Me3 (GSE75426), and E2F4 (GSE48666) (together with a negative control) in mouse ES cells. Right panel: ATAC-seq signals (GSE100738) from mouse short-term (ST) HSCs in the same genomic regions. D Example of RnapolII S5P, H3K4Me3, E2F4 tracks in mouse ES cells and ATAC-seq in ST-HSCs at the mouse Cdk1 locus. E Example of E2F4 and TFDP1 ChIP-seq signals at the CDK1 locus in various human cell types. Mouse and human E2F4 and TFDP1 DNA binding sites derived from the Unibind database are shown and the core nucleotides involved in DNA binding are highlighted.

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Activation Assay, Derivative Assay, ChIP-sequencing, Negative Control, Binding Assay

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: The mRNA expression analysis of MFSD12 and Its association with Clinical Features. (A) Differential expression analysis of MFSD12 between pan-cancer tissues and adjacent normal tissues in TCGA and GETx database. (B) Differential expression analysis of MFSD12 between tumor tissues and normal tissues in LIHC based on TCGA and GETx database. (C) Association of MFSD12 expression with clinical parameters in LIHC. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. MFSD12, Major Facilitator Superfamily Domain-containing 12; LIHC, liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus, AFP, Alpha-fetoprotein. "ns" stands for "not significant".

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Expressing, Quantitative Proteomics, Gene Expression

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: The protein expression analysis of MFSD12. (A) Pan-cancer protein expression profile of MFSD12 and representative IHC staining of tissue microarrays in HPA database. (B) IHC analysis of MFSD12 in LIHC tumor tissues and paired adjacent non-tumor liver tissues. (C) Quantification of immunostains for MFSD12 by IOD analysis. * P < 0.05, ** P < 0.01. IHC, immunohistochemistry; HPA, Human Protein Atlas; LIHC, liver hepatocellular carcinoma; IOD, integrated optical density;.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: Prognostic significance of MFSD12 expression across cancers and validation in LIHC Cohorts. (A) A pan-cancer Cox regression analysis was performed to assess MFSD12 expression. (B) OS analysis of MFSD12 in TCGA-LIHC data. (C) PFS analysis of MFSD12 in TCGA-LIHC data. (D) DFS analysis of MFSD12 in TCGA-LIHC data. (E) The prognostic significance of MFSD12 expression in LIHC patients was evaluated through both univariate and multivariate analyses. (F–H) Independent validation using external GEO cohorts corroborated the prognostic significance of MFSD12 in LIHC. AUC, Area Under Curve; CI, Confidence Interval; DFS, Disease-Free Survival; GEO, Gene Expression Omnibus; HR, Hazard Ratio; LIHC, Liver Hepatocellular Carcinoma; OS, Overall Survival; PFS, Progression-Free Survival; RFS, Relapse-Free Survival; ROC, Receiver Operating Characteristic; TCGA, The Cancer Genome Atlas; TPM, Transcripts Per Million.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Expressing, Biomarker Discovery, Gene Expression

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: Genomic alteration landscape of MFSD12 in LIHC. (A) Mutation spectrum of MFSD12 in pan-cancer analysis. (B) CNV analysis of MFSD12 in LIHC. (C) Genomic Landscape of mutations in MFSD12 within LIHC. (D) Classification profile of MFSD12 genetic variants in LIHC. (E) Comparative mutation profiling in MFSD12 low- and high-expressing subpopulations. (F) Protein-protein interaction network analysis of MFSD12 in LIHC. (G) Gene Co- Expression Network Correlated with MFSD12 Expression Patterns in LIHC. * P < 0.05, ** P < 0.01. CC, Cholangiocarcinoma; CNV, Copy Number Variation; LIHC, Liver Hepatocellular Carcinoma; MFSD12, Major Facilitator Superfamily Domain Containing 12; SNV, Single Nucleotide Variant; TCGA, The Cancer Genome Atlas; WES, Whole Exome Sequencing.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Mutagenesis, Expressing, Variant Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: DNA methylation analysis of the MFSD12 genomic features in LIHC. (A) The chromosomal localization of MFSD12 within the human genome. (B) The genomic architecture of MFSD12 and its adjacent regions. (C) The dynamics of promoter methylation in LIHC and normal liver tissues. (D) MFSD12 methylation levels in tumor tissue samples compared to normal tissues. (E) Analysis of the correlation between MFSD12 expression and its methylation status. (F) The identification of tumor stage-specific methylation alterations. (G) The relationship between MFSD12 individual CpG site methylation values and CNV status (deep deletion, loss, neutral, gain, amplification). (H) The association of MFSD12 methylation with patient survival outcomes. *** P < 0.001. CpG, Cytosine-phosphate-Guanine dinucleotide; LIHC, Liver Hepatocellular Carcinoma; TCGA, The Cancer Genome Atlas; TNM, Tumor-Node-Metastasis staging system.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: DNA Methylation Assay, Methylation, Expressing, Amplification

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: MFSD12 functional enrichment analysis across immune-related pathways and biological processes in LIHC. (A) GO enrichment analysis of MFSD12-associated biological processes. (B) KEGG pathway enrichment of MFSD12. (C) The GSEA-GO enrichment profile of MFSD12 in the context of immune regulation, as indicated by the enrichment score. (D) The GSEA-KEGG enrichment profile of MFSD12 in the context of immune regulation, as indicated by the enrichment score. (E) Hallmark gene set enrichment of MFSD12 in LIHC. ES, Enrichment Score; GSEA, Gene Set Enrichment Analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MFSD12, Major Facilitator Superfamily Domain Containing 12.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: Integrative analysis of MFSD12 expression correlation with tumor microenvironment immunocytes in LIHC. (A) Correlation of MFSD12 with tumor microenvironment scores using algorithm of ESTIMATE database: association of MFSD12 with immune score, stromal score, and ESTIMATE score in LIHC. (B) Correlation of MFSD12 expression level with immune cell across 33 cancer types. (C) Comparison of immune cell proportions stratified by MFSD12 expression levels (Low vs. High) in TCGA_LIHC. (D) Relationship between MFSD12 expression and immune infiltration in LIHC, as analyzed by the ssGSEA algorithm. (E) Relationship between MFSD12 expression and immune infiltration in LIHC across a range of immune infiltration analysis tools and multiple genomic datasets. ** P < 0.01, *** P < 0.001. CIBERSORT, cell-type identification by estimating relative subsets of RNA Transcripts; Cor, Pearson correlation coefficient; ESTIMATE, estimation of stromal and immune cells in malignant tumor tissues using expression data; LIHC, Liver Hepatocellular Carcinoma; Pval, p-value; TCGA, The Cancer Genome Atlas; xCell, cell type enrichment analysis tool.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: Integrated analysis of the link between MFSD12 expression and immune-related genes. (A) The relationship between the expression levels of MFSD12 and Immune-related genes in pan-cancers; (B) The relationship between the MFSD12 expression levels and immune checkpoints in LUSC. (C) Relationship between MFSD12 expression and Immune-related genes in LIHC across a range of immune infiltration analysis tools and multiple genomic datasets. * P < 0.05, ** P < 0.01. LIHC, Liver Hepatocellular Carcinoma; Pearson, Pearson correlation coefficient; Cor, Correlation coefficient.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: Single-Cell analysis of MFSD12 in LIHC by scRNA-seq. (A) UMAP visualization of cell type distribution in LIHC. (B) UMAP expression profile of MFSD12 in LIHC. (C) Relative expression levels of MFSD12 across cell types. (D) UMAP visualization of cell distribution by location. (E) UMAP visualization of cell distribution by cancer subtype (Normal, HCC, CC). (F) Heatmap of G1/S and G2/M phase transition gene expression across cell types. (G) Cell number and proportion statistics for each cell type. (H) Expression Proportion of MFSD12 in Different Cell Types and Cancer Subtypes. UMAP, Uniform Manifold Approximation and Projection; MFSD12, Major Facilitator Superfamily Domain Containing 12; CD4T_conv, Conventional CD4+ T cells; CD8T_typical, Typical CD8+ T cells; CD8T_exhausted, Exhausted CD8+ T cells; T_prolif, Proliferating T cells; Treg, Regulatory T cells; NK_cell, Natural Killer cell; B_cell, B lymphocyte; Mono/Macro, Monocyte/Macrophage; HCC, Hepatocellular Carcinoma; CC, Cholangiocarcinoma; G1/S, G1/S phase transition genes; G2/M, G2/M phase transition genes.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Single-cell Analysis, Expressing, Sublimation, Gene Expression

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: A thorough analysis of the correlation between MFSD12 expression and drug response across various databases, as well as its association with survival outcomes. (A) Correlation between MFSD12 expression and drug resistance/sensitivity in the CTRP dataset. (B) Correlation between MFSD12 expression and drug resistance/sensitivity in the PRISM dataset. (C) Correlation between MFSD12 expression and drug resistance/sensitivity in the GDSC1 database. (D) Correlation between MFSD12 expression and drug resistance/sensitivity in the GDSC2 database. (E) Overall survival analysis of Hugo cohort 2016 (Anti-PD-1) and Nathanson cohort 2017 (Anti-CTLA-4). CTRIP, Cancer Therapeutics Response Portal; PRISM, Preclinical Repurposing of Medicines; GDSC1/GDSC2, Genomics of Drug Sensitivity in Cancer 1/2; Anti-PD-1, Anti-Programmed Cell Death Protein 1; Anti-CTLA-4, Anti-Cytotoxic T-Lymphocyte-Associated Protein 4; Log-rank, Log-rank test; Number at risk, Number of patients at risk at each time point.

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: MFSD12 promotes proliferation, metastasis and invasion of hepatocellular carcinoma cells and its potential correlation with HAVCR2/LGALS9 immune checkpoint axis

doi: 10.3389/fimmu.2025.1681887

Figure Lengend Snippet: The knockdown of MFSD12 inhibited the proliferation, migration, and invasion of LIHC cells, as well as the TIM-3/Galectin-9 signaling pathway. (A, B) RT-qPCR and Western blot validation of MFSD12 silencing efficiency using siRNAs (si-MFSD12–1 to −4) with GAPDH as loading control. (C) CCK-8 cell viability assay showing reduced HEP 3B2.1–7 cells proliferation after MFSD12 knockdown (si-MFSD12-3). (D) Transwell assay revealed a reduction in the migratory and invasive capabilities of HEP 3B2.1–7 cells following the knockdown of MFSD12. (E) Immunoblot analysis of EMT markers and TIM-3 axis components showing up-regulation of E-cadherin and down-regulation of Vimentin, MMP-2, MMP-9, HAVCR2 (TIM-3) and LGALS9 in si-MFSD12-treated cells. * P < 0.05, ** P < 0.01, *** P < 0.001. CTRL, control untreated; si-NC, negative control siRNA; si-MFSD12, MFSD12-targeting siRNA; E-cadherin, epithelial cadherin; MMP-2/9, matrix metalloproteinase-2/9; HAVCR2, hepatitis A virus cellular receptor 2 (TIM-3); LGALS9, lectin galactoside-binding soluble 9 (Galectin-9).

Article Snippet: The primary antibodies utilized in the Western blot analysis included: anti-rabbit MFSD12 (1:1000, Boster, China), anti-mouse GAPDH (1:30000, Proteintech, China), anti-rabbit E-cadherin (1:40000, Proteintech, China), anti-mouse Vimentin (1:40000, Proteintech, China), anti-rabbit MMP2 (1:1000, BIOSS, China), anti-rabbit MMP9 (1:1000, Affinity, China), anti-rabbit LGALS9 (1:1000, Abmat, China), and anti-rabbit HAVCR2 (1:1000, Boster, China).

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot, Biomarker Discovery, Control, CCK-8 Assay, Viability Assay, Transwell Assay, Negative Control, Virus, Binding Assay

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A Up-regulated and down-regulated differentially expression genes (DEGs) in five mRNA expression profiles. B The GO category for DEGs. BP biological process, CC cellular component, MF molecular function. The color represents the P value, and the size indicates the enrichment gene number of each pathway. C KEGG enrichment pathways of DEGs. EIP Environmental Information Processing, CP Cellular Processes, OS Organismal Systems, GIP Genetic Information Processing, HD Human Diseases, M Metabolism. D Protein–protein interaction network of DEGs. E Up-regulated gene expression of lipid metabolism and tumor progression. F Representative images of IHC staining for SOAT1 of normal liver and HCC tissues cited from The Human Protein Atlas. G SOAT1 expression level in normal tissues and HCC tissues based on the TCGA dataset. H , I Analysis of the SOAT1 expression levels in TCGA HCC samples based on the individual clinical stage ( H ) and pathological grade ( I ). J High SOAT1 expression is positively correlated with poor survival ( P = 0.0175). K Representative images of positive and negative SOAT1 expression in different HCC tissues detected by IHC. L Analysis of the expression levels of SOAT1 in HCC patient liver tissues based on ES grade and MVI grade.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Expressing, Gene Expression, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A SOAT1 expression level in different HCC cell lines cited from CCLE database. B Western blot analysis of SOAT1 expression in HepG2 and PLC/PRF/5 cell lines. C Western blot analysis of EMT related markers in SOAT1 overexpressed or knocked down cells. D Immunofluorescence assay of E-cadherin and Vimentin in cells treated with SOAT1 overexpression or shRNA vectors. E Cell phenotype changes under SOAT1 overexpressed or knocked down treatment. F , G Migration ( F ) and invasion ( G ) of HepG2 cells transfected with SOAT1 or PLC/PRF/5 cells transfected with shSOAT1. H Cell proliferation under SOAT1 overexpressed or knocked down treatment.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Expressing, Western Blot, Immunofluorescence, Over Expression, shRNA, Migration, Transfection

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A , B Oil red O ( A ) and BODIPY 493/503 ( B ) staining of lipid droplets in SOAT1 overexpressed HepG2 cells and SOAT1 knocked down PLC/PRF/5 cells. The relative Oil red O and intensity of BODIPY493/503 were analyzed. C SOAT1 increased accumulation of cholesterol esters. D Cellular cholesterol distribution by Filipin III staining. E Western blot analysis of SOAT1, SREBP2, LDLR, ITGAV, and ITGB4 expression levels under SOAT1 overexpression or knockdown.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Staining, Western Blot, Expressing, Over Expression, Knockdown

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A Prediction docking score between small-molecule compounds and SOAT1. B Predicted interaction of nootkatone with cavity residues of SOAT1. C Cell viability of HCC cells with nootkatone treatment for 48 h. D , E Micrographs of Oil red O ( D ) and BODIPY ( E ) staining of lipid droplets in PLC/PRF/5 induced with cholesterol (200 μg/mL) for 24 h and then treated with nootkatone (150 and 300 µM) for 48 h. F Nootkatone decreased the content of cholesterol esters in different groups. G , H Expression of SOAT1 in different groups.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Staining, Expressing

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A BODIPY493/503 staining of lipid droplets in Control, NK, SOAT1 and SOAT1 + NK groups. B Content of cholesterol esters in different groups. C Cholesterol distribution was determined by Filipin III staining. D , E Invasion ( D ) and migration ( E ) of PLC/PRF/5 cells with different treatments. F Immunofluorescence assay of E-cadherin and Vimentin of cells in different group. G Cell phenotype under nootkatone treatment with different concentration (150 and 300 µM). H Western blot analysis of SOAT1, SREBP2, LDLR, E-cadherin, Occludin, Vimentin, Twist1, N-cadherin, Snail1, Slug, and Fibronectin expression level in different groups.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Staining, Control, Migration, Immunofluorescence, Concentration Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A Representative images of subcutaneous tumor xenografts in Control, SOAT1, shSOAT1, NK and SOAT1 + NK groups. B Tumor volume in different groups. C WB analysis of SOAT1, SREBP2, LDLR, E-cadherin, Occludin, Vimentin, Twist1, N-cadherin, Snail1, Slug, and Fibronectin expression levels in tumor tissue of different groups. D Visible metastatic nodules on the surface of lungs in different groups.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Control, Expressing

Journal: Cell Death & Disease

Article Title: SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

doi: 10.1038/s41419-024-06711-9

Figure Lengend Snippet: A Schematic illustration of experimental procedure. B Representative macroscopic images of liver in Control, Model, NK-L, and NK-H groups. C AFP expression in serum and liver tissue of mice in different groups. D Body weight of mice in different groups. E , F Liver weight ( E ) and liver weight-to-body weight ratio ( F ). G Serum TC level in four groups. H Contents of hepatic free cholesterol and cholesterol esters in four different groups. I Serum ALT and AST level in different groups. J Morphological observations of the liver and liver tissue with Oil red O, H&E and Sirius red staining. The relative Oil red O and Sirius red were obtained through the Image J Pro software. K The protein expression level of SOAT1, SREBP2, LDLR, E-cadherin, Occludin, Vimentin, Twist1, N-cadherin, Snail1, Slug, and Fibronectin expression in liver tissue of mice in different groups.

Article Snippet: After being blocked with 3% H 2 O 2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400).

Techniques: Control, Expressing, Staining, Software