Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Role of Long Chain Acyl-CoA Synthetases in MASH-driven Hepatocellular Carcinoma and Ferroptosis

doi: 10.1152/ajpgi.00096.2025

Figure Lengend Snippet: ( A ) UMAP plot of liver/tumor cell clusters identified by 10X Genomics Visium analysis. ( B ) Volcano plot of differentially expressed genes in MASH-HCC vs. MASH tissues identified by spatial transcriptomics. A log2 fold-change of 0.5 and p-value of 0.05 were used to identify statistically significant genes. ( C ) Gene sets enriched in the indicated experimental groups. ( D ) 10X Genomics spatial distribution of HCC marker GPC3, as well as ACSL4, ACSL5, and GPX4 in human MASH-HCC tissue, paired adjacent non-cancerous MASH tissue, and normal liver tissue. ( E ) Heatmap of gene expression levels in normal, MASH, and MASH-HCC tissues. ( F ) Violin plots of lipid metabolism genes identified as upregulated in MASH-HCC compared to MASH tissues.

Article Snippet: Animal-Free Blocker and Diluent (Vector Laboratories, cat. n. SP-5035–100) followed by incubation overnight at 4C with primary antibodies, including anti-glypican-3 (GPC3) (1:200, Novus Biologicals, cat. n. NBP2–44486), anti-ACSL4 (1:200, Invitrogen, cat. n. PA5–27137), anti-ACSL5 (1:100, Invitrogen, cat. n. MA5–24595) and anti-HepPar-1 (1:100, Invitrogen, cat. n. MA5–12417) diluted in blocking solution.

Techniques: Gene Expression, Marker

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Role of Long Chain Acyl-CoA Synthetases in MASH-driven Hepatocellular Carcinoma and Ferroptosis

doi: 10.1152/ajpgi.00096.2025

Figure Lengend Snippet: ( A ) Histopathological and immunohistochemistry staining of three human MASH-HCC subjects, including H&E staining (top), GPC3, ACSL4 and ACSL5. “T” = tumor; “NT” = non-tumor. Scale bars represent 100μm. ( B ) Heatmap of ACSL4 and ACSL5 gene expression across MASH-HCC cell lines obtained from the DepMap portal (DepMap Public 25Q2 release). ( C-D ) CellTiter-Glo cell viability assay in Huh7 and SNU475 HCC cell exposed to ferroptosis inducer RSL3 (5μM) for 24 hours and rescued by lipid peroxidation inhibitor ferrostatin-1 (1μM). One-way ANOVA was used for statistical analysis. ( E ) Western blot of GPX4 and FSP1 expression in Huh7 and SNU475 cells treated with RSL3 (0.1μM) for 6hr and rescued by ferrostatin-1 (1μM). β-actin was used as a loading control. Vehicle = 0.1% DMSO. All samples were run at the same time on the same membrane. Densitometry is indicated directly on the blot as fold change compared to vehicle-treated Huh7 cells. ( F ) Dose-response curves of SNU475 cells treated with ferroptosis-inducers RSL3 and iFSP1 individually or in combination.

Article Snippet: Animal-Free Blocker and Diluent (Vector Laboratories, cat. n. SP-5035–100) followed by incubation overnight at 4C with primary antibodies, including anti-glypican-3 (GPC3) (1:200, Novus Biologicals, cat. n. NBP2–44486), anti-ACSL4 (1:200, Invitrogen, cat. n. PA5–27137), anti-ACSL5 (1:100, Invitrogen, cat. n. MA5–24595) and anti-HepPar-1 (1:100, Invitrogen, cat. n. MA5–12417) diluted in blocking solution.

Techniques: Biomarker Discovery, Immunohistochemistry, Staining, Gene Expression, Viability Assay, Western Blot, Expressing, Control, Membrane

Journal: Nature Communications

Article Title: Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

doi: 10.1038/s41467-024-55140-x

Figure Lengend Snippet: A – D Immunofluorescence images of representative CTCs enriched from leukopak samples from patients with metastatic prostate cancer (GU-1 and GU-2, n = 2), triple-negative breast cancer (TNBC-1, n = 1), hepatocellular carcinoma (HCC-1 and HCC-2, n = 2), and uveal melanoma (UM-1 and UM-2, n = 2). Bulk cell populations are stained with DAPI nuclear marker (blue), the relevant tumor markers grouped within a single color (green) (see individual epitopes below), and with antibodies against the WBC markers CD45, CD16, CD66b (red). Tumor markers: A GU-1 and GU-2 (EpCAM, pan CK, CK19), B TNBC-1 (EpCAM, pan CK, CK19), C HCC-1 and HCC-2 (EpCAM, pan CK, CK19, ASGR1, GPC3), D UM-1 and UM-2 (Sox10, Melan-A, NG2). E Representative contaminating WBCs. F Table listing blood volumes processed and yield of CTCs obtained from patient-derived leukopaks (median 2799 CTCs per leukopak). G Droplet digital RNA-PCR (ddPCR) analysis of 0.5 to 1% of the bulk CTC products from all seven cases, quantifying expression of previously curated RNA signatures that denote either tissue lineage or cancer-specific transcripts within the background of normal blood cells. Of note, GU-1 CTCs express multiple neuroendocrine genes (CHGA, SYP, and DLL3), consistent with immunohistochemistry staining for synaptophysin and chromogranin A in a resected adrenal metastasis from this patient. Bar graphs showing expression of wild-type androgen receptor (AR-wt) and AR variant 7 (AR-V7) for GU-1 and GU-2 are shown in accompanying Supplementary Fig. . Healthy donor blood is shown as negative control, with positive controls drawn from either cultured prostate cancer cell lines (LNCaP, 22Rv1, and PC3), cultured breast CTCs (BRx-142 ), cultured liver cancer cells (HepG2), or cultured melanoma CTCs (Mel-167 ), respectively. (H) Measured whole cell and nucleus diameters of individual CTCs ( n = 5543), compared with WBCs ( n = 223). Substantial overlap in size is evident between CTC and WBC populations. I Variation across individual CTCs from cases GU-1, GU-2, TNBC-1, HCC-1, HCC-2, and UM-1 in their intensity of staining for the combined lineage markers (see above). Source data are provided as a Source Data file.

Article Snippet: The enriched leukopak by LP CTC-iChip were Fc blocked (Jackson ImmunoResearch Laboratories, Cat# 009-000-008) and then immunostained with AF488-conjugated EpCAM (Cell signaling, Cat# 5198) / PSMA (Invitrogen, Cat# MA5-18161) / GPC3 (Novus Biologicals, Cat# NBP2-47763AF488) / FITC-conjugated ASGPR1 (Novus Biologicals, Cat# NBP1-51109) antibodies and PE-Cy7-conjugated CD45 (Biolegend, Cat# 982310) / CD16 (Biolegend, Cat# 980110) / CD66b (Biolegend, Cat# 396910) antibodies, and LIVE/DEAD Red (Invitrogen, Cat# L34971).

Techniques: Immunofluorescence, Staining, Marker, Derivative Assay, Expressing, Immunohistochemistry, Variant Assay, Negative Control, Cell Culture

Journal: Scientific reports

Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody.

doi: 10.1038/s41598-017-14793-z

Figure Lengend Snippet: Figure 3. SDC2 and GPC3, representative HSPGs, act as endocytic receptors for 3D8 scFv on HeLa and HEK293 cells. (a) Schematic diagrams of SD2 and GPC3, which are expressed on the cell surface, and their recombinants, which are expressed intracellularly (controls). The parts of the core protein are shown without attached of GAGs. SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain; GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. (b–e) Confocal microscopy. HeLa cells were transfected with plasmids encoding SDC2-GFP (b) and HA-GPC3 (d) which are expressed on the cell surface, or with plasmids encoding SDC2ΔTM-GFP (c) and GPC3-nfGPI-GFP (nf indicates non-functional) (e), which are expressed intracellularly. At 18 h post-transfection, cells were incubated with 10 μM 3D8 scFv for 1 h at 4 °C or 6 h at 37 °C. Cells transfected with plasmids containing the GFP gene were incubated with a rabbit anti-3D8 scFv antibody, followed by a TRITC-conjugated goat anti-rabbit IgG antibody. Cells transfected with the plasmid encoding the HA-GPC3 gene were incubated with a rabbit anti-3D8 scFv antibody and a mouse anti-HA antibody, followed by TRITC-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG, respectively. Nuclei were stained with Hoechst 33342 (blue). Bar, 10 μm. (f,g) Cell surface expression of endogenous SDC2 and GPC3 was examined by flow cytometry using core protein-specific antibodies. (h,i) Co-immunoprecipitation. HeLa (h) and HEK293 cells (i) were transfected with plasmids encoding SDC2-GFP and HA-GPC3 and treated for 6 h at 37 °C with 5 μM 3D8 scFv 24 h later. Cells were collected and lysed for co-immunoprecipitation assays. Co-immunoprecipitation was performed using an anti-GFP antibody. Samples were analyzed by immunoblotting with antibodies specific for SDC2 and the His tag (left panels of h, i). Samples from the co-immunoprecipitation performed using the anti-GPC3 antibody were analyzed by immunoblotting with antibodies specific for GPC3 and the His tag (right panels of h, i). Cells not treated with the 3D8 scFv protein were used as a negative control. Proteins in the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting. Asterisks denote endogenous SDC2.

Article Snippet: To express GPC3 in the cytoplasm as an N-terminal GPC3-nfGPI-GFP fusion protein, pCMV6-AC-GPC3-turboGFP, in which the GPI anchor is non-functional, was purchased from Origene (cat# RG205911).

Techniques: Confocal Microscopy, Transfection, Functional Assay, Incubation, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Immunoprecipitation, Western Blot, Negative Control

Journal: Scientific reports

Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody.

doi: 10.1038/s41598-017-14793-z

Figure Lengend Snippet: Figure 4. CD44 and BCAN, representative CSPGs, act as endocytic receptors for 3D8 scFv on HeLa and HEK293 cells. (a) Schematic diagrams of CD44 and BCAN, which are expressed on the cell surface, and their recombinants, which are expressed intracellularly (controls). SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain; GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. (b–e) Confocal microscopy. HeLa cells were transfected with plasmids encoding CD44-GFP (b) and HA-BCAN (d), which are expressed on the cell surface, or with plasmids encoding CD44ΔTM-GFP (c) and BCAN-ΔGPI-GFP (e), which are expressed intracellularly. At 18 h post-transfection, cells were incubated with 10 μM 3D8 scFv under the specified conditions. After fixation and permeabilization, cells transfected with plasmids containing the GFP gene were incubated with a rabbit anti-3D8 scFv antibody, followed by a TRITC-conjugated goat anti-rabbit IgG antibody. Cells transfected with the plasmid encoding the HA-BCAN gene were incubated with a rabbit anti-3D8 scFv antibody and a mouse anti-HA antibody, which were detected by TRITC-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG, respectively. Nuclei were stained with Hoechst 33342 (blue). Bar, 10 μm. (f,g) Cell surface expression of endogenous CD44 and BCAN was examined by flow cytometry. (h,i) Co-immunoprecipitation. HeLa (h) and HEK293 cells (i) were transfected with plasmids encoding CD44-GFP and HA-BCAN and 24 h later treated with 5 μM 3D8 scFv for 6 h at 37 °C. Cells were collected and lysed for co-immunoprecipitation assays. Co-immunoprecipitation was performed using an anti-GFP antibody, Samples were analyzed by immunoblotting with antibodies specific for CD44 and the His tag (left panels of h, i). Samples from the co-immunoprecipitation performed with the anti-HA antibody, were analyzed by immunoblotting with antibodies specific for the HA tag and the His tag (right panels of h, i). Cells not treated with 3D8 scFv protein were used as a negative control. Proteins from the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting. Asterisks denote endogenous CD44.

Article Snippet: To express GPC3 in the cytoplasm as an N-terminal GPC3-nfGPI-GFP fusion protein, pCMV6-AC-GPC3-turboGFP, in which the GPI anchor is non-functional, was purchased from Origene (cat# RG205911).

Techniques: Confocal Microscopy, Transfection, Incubation, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Immunoprecipitation, Western Blot, Negative Control