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: Aging Cell

Article Title: Activated mTOR Signaling in the RPE Drives EMT , Autophagy, and Metabolic Disruption, Resulting in AMD ‐Like Pathology in Mice

doi: 10.1111/acel.70018

Figure Lengend Snippet: Generation and characterization of mLST8 KI mice. (a) Cartoon showing that in addition to mTOR, mLST8 is a major component of both mTOR complexes 1 and 2. (b) Schematic showing the strategy for generating mLST8 KI mice. Briefly, TGA stop codon in the mouse Best1 gene was replaced with the “T2A‐mouse mLST8 CDS (coding sequence)” cassette. The targeting vector was generated by PCR using BAC clone RP23‐340G9 from the C57BL/6 library as a template. The targeting vector had Neo cassette, which was flanked by SDA (self‐deletion anchor) sites. DTA (diptheria toxin A) was used for negative selection. C57BL/6 embryonic stem cells were used for gene targeting. (c) Western blot analysis indicating that RPE lysates shows overexpression of mLST8 relative to WT. Such changes were not seen in retina lysates from the same mice. Retina RPE lysate preparation was confirmed by evaluating the levels of GFAP and RPE65 in the respective lysates n = 3. ** p < 0.01. (d) Western blot from RPE lysates of mLST8 KI RPE cells further revealed an increased ratio of p‐S6K/S6K, p‐4EBP/4EBP, p‐ULK1/ULK1, and p‐Akt1/Akt1 in these cells, compared to controls (WT) n = 3. * p < 0.05. (e) Western blot showing that mLST8 overexpression (AAV2‐ mLST8 ) in Raptor or Rictor KO MEF cells activates mTORC2 target (p‐Akt1) and mTORC1 target (p‐P70S6K), respectively, compared to controls n = 3. **** p < 0.001, ns = not significant.

Article Snippet: The cells from all the experimental groups were lysed in the CHAPS lysis buffer, and Raptor and Rictor antibodies were used to immunoprecipitate mTOR bound complexes using protein Ig beads (Thermo Fisher, 88802). mTORC1 and mTORC2 target proteins S6K1 (Sino Biological, 10099‐H09B‐50) and Akt1 (EMD Milipore, 14–279) were given to the immune‐precipitated complexes and incubated for 15 min in 37°C.

Techniques: Sequencing, Plasmid Preparation, Generated, Selection, Western Blot, Over Expression

Journal: Aging Cell

Article Title: Activated mTOR Signaling in the RPE Drives EMT , Autophagy, and Metabolic Disruption, Resulting in AMD ‐Like Pathology in Mice

doi: 10.1111/acel.70018

Figure Lengend Snippet: βA3/A1‐crystallin overexpression in the RPE rescues autophagy and melanosome alterations and retinal structure/function in mLST8 KI mice. (a) Cartoon showing the strategy for subretinal injection of the AAV2‐m Cryba1 construct into one eye of 8‐month‐old mLST8 KI mice, with the contralateral eyes receiving PBS vehicle. Animals were euthanized 2 and 4 months after injection. (b, c) Western blot analysis and densitometry showing that Cryba1 overexpression in the RPE (confirmed by western blot in b) of mLST8 KI mice could rescue the abnormal levels of both mTORC1 (p‐S6K) and mTORC2 (p‐Akt1) targets (b), and the melanosome marker (PMEL; b), as well as rescued the levels of major regulators of autophagosome formation (Atg9b, Atg7; c) in these animals (b, c), relative to PBS injected eyes (b, c). n = 3. * p < 0.05, ** p < 0.01. AAV2‐m Cryba1 treatment (subretinal injection) to mLST8 KI mice for 4 months rescued retinal function as evident from increase in scotopic (d) a‐ and (e) b‐wave amplitudes after the treatment, compared to PBS‐injected contralateral eyes of the KI mouse. n = 4. **** p < 0.0001, * p < 0.05. (f) AAV2‐m Cryba1 treatment to mLST8 KI mice for 4 months also rescued early RPE changes (arrows) like the patchy appearance of the monolayer and decline in thickness (spider plot), compared to PBS–treated contralateral eyes of the mLST8 KI mouse. n = 4. Scale bar = 20 μm. * p < 0.05. (g) Cartoon depicting overexpression of mLST8 in RPE cells ( mLST8 KI mice) activated both mTORC1 and mTORC2, disrupting glucose metabolism, mitochondrial function, autophagy, and melanosome function, leading to debris accumulation, EMT activation, and age‐related retinal degeneration resembling AMD. Targeting mTOR with inhibitors or modulators rescued these changes, suggesting a potential therapeutic strategy for retinal diseases by modulating mTOR signaling. Created with BioRender.com .

Article Snippet: The cells from all the experimental groups were lysed in the CHAPS lysis buffer, and Raptor and Rictor antibodies were used to immunoprecipitate mTOR bound complexes using protein Ig beads (Thermo Fisher, 88802). mTORC1 and mTORC2 target proteins S6K1 (Sino Biological, 10099‐H09B‐50) and Akt1 (EMD Milipore, 14–279) were given to the immune‐precipitated complexes and incubated for 15 min in 37°C.

Techniques: Over Expression, Injection, Construct, Western Blot, Marker, Activation Assay

Journal: Molecular cell

Article Title: Multisite phosphorylation of S6K1 directs a kinase phospho-code that determines substrate selection

doi: 10.1016/j.molcel.2018.11.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Full-length human S6K1 cDNA in pCMV6-Entry , Origene Technologies , Cat# RC217324.

Techniques: Recombinant, Purification, Mutagenesis, Cell Culture, Staining, Protease Inhibitor, Western Blot, Silver Staining, Transfection, Affinity Chromatography, Plasmid Preparation, Software

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