Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: SPP1 promotes Th17 cell differentiation via interactions with ITGB1 and CD44. (A) Flow cytometry revealed a dose-dependent facilitation of Th17 differentiation by SPP1, with a concentration of 0.2 μg/mL emerging as optimal for ensuing experimental endeavors. Th17 cells were identified as CD3+CD4+IL-17A+ cells, and these results were represented as the percentage of Th17 cells among CD3+ T cells. (B) Flow cytometry indicated that ursolic acid dose-dependently inhibited SPP1-induced Th17 cell differentiation, with a concentration of 5 μM emerging as optimal for subsequent investigative pursuits. (C) Western blot analyses were conducted to detect the phosphorylation level of ERK protein. (D) SPP1 KD decreased the ERK phosphorylation level and substantially curtailed Th17 cell differentiation. (E) Co-immunoprecipitation analyses suggested strong interactions between SPP1 and both ITGB1 and CD44. (F) Both ITGB inhibitors and CD44 antagonists suppressed the SPP1-driven Th17 cell differentiation, with an even more pronounced inhibitory effect upon their combined application. SPP1, secreted phosphoprotein 1; ITGB1, integrin β1; CD44A, CD44 antagonists; ERK, extracellular signal-regulated kinase; SD, standard deviation. Data are represented as mean±SD. n=3. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: To assess the influence of ITGB1 and CD44 modulation on Th17 differentiation, cells were additionally co-incubated with ITGB1 inhibitor GLPG0187 (1 nM, HY-100506; MedChemExpress, Monmouth, NJ, USA) or CD44 antagonist (1 μg/ mL, 553131; BD Biosciences, San Jose, CA, USA) at specific concentrations recommended by preliminary dose-response studies.

Techniques: Cell Differentiation, Flow Cytometry, Concentration Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Standard Deviation

Journal: Clinical and Molecular Hepatology

Article Title: Ursolic acid targets secreted phosphoprotein 1 to regulate Th17 cells against metabolic dysfunction-associated steatotic liver disease

doi: 10.3350/cmh.2024.0047

Figure Lengend Snippet: Ursolic acid modulates SPP1-mediated Th17 cell differentiation to ameliorate MASLD. (A–C) Mice were derived from the experiments of SPP1 KD, wherein (A) presented that the protein levels of ITGB1 and CD44 expressed in the pull-down products were conspicuously surged by HFD administration, whereas a precipitous decline in their expression was observed in the liver tissue lysates procured from the SPP1 KD mice, while (B) demonstrated that such alterations were concomitant with trends in the phosphorylation level of ERK protein; and concurrently, (C) displayed a similar trend in hepatic Th17 cell populations. (D, E) Mice were derived from the first part of experiments, those were fed with HFD and different concentrations of ursolic acid, wherein (D) suggested that ursolic acid dose-dependently ameliorated the escalated Th17 cell populations induced by the high-fat dietary regimen, and (E) revealed consistent protein levels of SPP1, ITGB1, CD44, as well as the phosphorylation level of ERK. Data are represented as mean±SD. n=3-6. * P <0.05, ** P <0.01, *** P <0.001. SPP1, secreted phosphoprotein 1; MASLD, metabolic dysfunction-associated steatotic liver disease; KD, knockdown; ITGB1, integrin β1; CD44A, CD44 antagonists; HFD, highfat diets; ERK, extracellular signal-regulated kinase; SD, standard deviation.

Article Snippet: To assess the influence of ITGB1 and CD44 modulation on Th17 differentiation, cells were additionally co-incubated with ITGB1 inhibitor GLPG0187 (1 nM, HY-100506; MedChemExpress, Monmouth, NJ, USA) or CD44 antagonist (1 μg/ mL, 553131; BD Biosciences, San Jose, CA, USA) at specific concentrations recommended by preliminary dose-response studies.

Techniques: Cell Differentiation, Derivative Assay, Expressing, Phospho-proteomics, Knockdown, Standard Deviation

Journal: Redox Biology

Article Title: TSPAN15 sustains ITGB1 stability to block gemcitabine-induced ferroptosis in pancreatic ductal adenocarcinoma through the FAK/AKT/Mtor-gpx4 cascade

doi: 10.1016/j.redox.2025.103721

Figure Lengend Snippet: TSPAN15 directly sustains the protein stability of ITGB1 and activates the p -FAK/p-AKT/p-mTOR-GPX4 signaling axis to promote gemcitabine-induced ferroptosis resistance in pancreatic cancer cells. (A) Gene set enrichment analysis (GSEA) demonstrating that PI3K-AKT pathway related genes were significantly enriched following TSPAN15 knockdown in PANC-1 cells (p = 0.029). (B) The protein levels of ITGB1, p -FAK, p -AKT, p -mTOR, GPX4, NANOG and CD44 in PANC-1-shNC/shTSPAN15 cells were assessed by Western blotting (left). The relative expression of proteins was quantified via normalization to that of β-actin (right). (C) The relative mRNA expression levels of ITGB1 in PANC-1-shNC and PANC-1-shTSPAN15 cells were detected via RT-PCR. (D) The physical interaction of endogenous TSPAN15 with ITGB1 was detected by co-IP with anti-TSPAN15 and anti-ITGB1 antibodies in PANC-1 cells. (E) The physical interaction of exogenous TSPAN15 with ITGB1 was monitored by co-IP with anti-Ha antibody or anti-Flag antibody in HEK293T cells transfected with TSPAN15-Ha- and/or ITGB1-Flag-expressing plasmids. (F) Immunofluorescence assay showing the colocalization (yellow) of TSPAN15 (green) and ITGB1 (red) in PANC-1-shNC and PANC-1-shTSPAN15 cells (scale bar: 10 μm). (G) The turnover of ITGB1 proteins in PANC-1-shNC/shTSPAN15 cells was measured by CHX (150 μg/ml) treatment and then detected by Western blot analysis. (H) Representative Western blot showing TSPAN15 and ITGB1 expression levels in PANC-1-shNC/shTSPAN15 cells treated with MG132 (20 μM) for 6 h (top). The relative expression of proteins was quantified by normalization to that of β-actin (bottom). (I) Co-IP with an anti-ITGB1 antibody and representative a Western blot for ubiquitin and ITGB1 in PANC-1-shNC/shTSPAN15 cells. (J) The protein levels of p -FAK, p -AKT, p -mTOR, GPX4, NANOG and CD44 in MIAPaCa-2-vector and MIAPaCa-2-TSPAN15 cells treated with or without 100 ng/ml ITGB1 neutralizing antibody OS2966 for 24 h were assessed by Western blotting (left). The relative expression of proteins was quantified by normalization to that of β-actin (right). All error bars represent the means ± SDs; ns: not significant; ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: Next, we employed TargetMol to conduct a virtual screening of 2808 compounds based on the predicted interaction sites of TSPAN15 and ITGB1 to identify potential inhibitors of TSPAN15 ( ).

Techniques: Knockdown, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Ubiquitin Proteomics, Plasmid Preparation

Journal: Redox Biology

Article Title: TSPAN15 sustains ITGB1 stability to block gemcitabine-induced ferroptosis in pancreatic ductal adenocarcinoma through the FAK/AKT/Mtor-gpx4 cascade

doi: 10.1016/j.redox.2025.103721

Figure Lengend Snippet: Venetoclax is a TSPAN15 inhibitor that promotes gemcitabine-induced ferroptosis by inhibiting the TSPAN15-dependent p -FAK/p-AKT/p-mTOR-GPX4 signaling axis in pancreatic cancer cells. (A) Predicted binding complex models of TSPAN15 and ITGB1 via the AlphaFold protein docking tool ( https://alphafold.ebi.ac.uk/ ). (B) Relative cell viability of PANC-1 cells was detected by CCK-8 assay following treatment with gemcitabine (Gem, 5 μM) alone or in combination with the top 10 compounds for 48 h (BC-48: demecarium bromide, AG1341: nelfinavir, RDX: tenapanor, TD: revefenacin, Vene: venetoclax, Dabig: dabigatran etexilate mesylate, Dacla: daclatasvir, Pante: pantethine, Saqui: saquinavir). (C) Venetoclax binds to the active pocket of TSPAN15 through the formation of three hydrogen bonds with residues C177, K201 and Y200. (D) The relative viability of PANC-1 and MIAPaCa-2 cells treated with 5 μM gemcitabine or 10 μM venetoclax alone or a combination of both for 48 h was detected via a CCK-8 assay. (E) Representative images (left) and quantification (right) of the results of the clonogenic assay of PANC-1 and MIAPaCa-2 cells treated with 5 μM gemcitabine or 10 μM venetoclax alone or a combination of both for 7 days. (F) Heatmaps depicting the combination drug responses of gemcitabine and venetoclax in PANC-1 cells. Relative cells viability was determined via a CCK-8 assay after treatment with gemcitabine and venetoclax for 48 h, and SynergyFinder ( https://synergyfinder.fimm.fi ) was subsequently used to estimate drug interactions via ZIP synergy scores. ZIP synergy scores < −10 indicated antagonism, scores from −10 to 10 suggested addition, and scores >10 indicated synergism. (G) Western blot assays were conducted to assess the expression of ITGB1, p -FAK, p -AKT, p -mTOR and GPX4 in MIAPaCa-2-vector cells and MIAPaCa-2-TSPAN15 cells with or without venetoclax treatment (25 μM, 48 h) (left). The relative expression of proteins was quantified by normalization to that of β-actin (right). (H) Western blot assays were conducted to assess the expression of ITGB1, p -FAK, p -AKT, p -mTOR and GPX4 in PANC-1 cells treated with 5 μM or 10 μM venetoclax for 48 h (left). The relative expression of proteins was quantified by normalization to that of β-actin (right). (I) Co-IP with an ITGB1 antibody and a representative Western blot for ubiquitin and ITGB1 in PANC-1 cells after treatment with or without 10 μM venetoclax for 24 h. (J) Cellular thermal shift assays (CETSAs) were performed to confirm the binding specificity of venetoclax with TSPAN15 in the cell lysates of PANC-1 cells. Plots represent the signal intensity of TSPAN15 normalized to the signal intensity at 46 °C. All error bars represent the means ± SDs; ns: not significant; ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: Next, we employed TargetMol to conduct a virtual screening of 2808 compounds based on the predicted interaction sites of TSPAN15 and ITGB1 to identify potential inhibitors of TSPAN15 ( ).

Techniques: Binding Assay, CCK-8 Assay, Clonogenic Assay, Western Blot, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Ubiquitin Proteomics

Journal: Redox Biology

Article Title: TSPAN15 sustains ITGB1 stability to block gemcitabine-induced ferroptosis in pancreatic ductal adenocarcinoma through the FAK/AKT/Mtor-gpx4 cascade

doi: 10.1016/j.redox.2025.103721

Figure Lengend Snippet: Venetoclax treatment enhances the sensitivity of PDAC xenograft tumors to gemcitabine in vivo. (A–C) Representative images (A) and statistical analysis of xenograft tumor volumes (B) and weights (C) in nude mice after implantation of 1 × 10 6 PANC-1 cells, followed by intraperitoneal injection of DMSO or gemcitabine (10 mg/kg), oral administration of venetoclax (100 mg/kg), or a combination of both treatments every 3 days (n = 10 mice per group). (D) Representative images of immunohistochemical staining of serial xenograft tumor samples stained for GPX4, ITGB1, CD44 and Ki67 are shown (scale bars, 50 μm). (E) The body weights of the mice were measured every three days. All error bars represent the means ± SDs; ns: not significant; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001.

Article Snippet: Next, we employed TargetMol to conduct a virtual screening of 2808 compounds based on the predicted interaction sites of TSPAN15 and ITGB1 to identify potential inhibitors of TSPAN15 ( ).

Techniques: In Vivo, Injection, Immunohistochemical staining, Staining

Journal: Neural Regeneration Research

Article Title: Pre-clinical study of human umbilical cord mesenchymal stem cell transplantation for the treatment of traumatic brain injury: safety evaluation from immunogenic and oncogenic perspectives

doi: 10.4103/1673-5374.317985

Figure Lengend Snippet: Identification, immunogenicity and immunomodulation of huMSCs . (A) The huMSCs cultured in our experiment. The huMSCs were spindle-shaped and grew in a whorl pattern. Original magnification 100×, scale bars: 200 μm. (B) Flow cytometry detection of CD29, CD44, CD73 and CD105. The rate of positive expression for all factors was greater than 95%. (C) Detection of huMSC HLAII expression in PBMCs and huMSCs by western blot. No HLAII expression band was detected in the huMSCs. (D) Relative expression of HLAII in PBMCs and huMSCs. The bar graph shows no HLAII expression in huMSCs. (E) PCR amplification curves of HLA-DPA1 , HLA-DQA1 and HLA-DRA1 genes in PBMCs and huMSCs. (F) Relative expression of HLA-DPA1 , HLA-DQA1 and HLA-DRA1 genes in PBMCs and huMSCs. PCR results showed no expression of HLA-DPA1 , HLA-DQA1 or HLA-DRA1 genes in huMSCs. (G) Expression curves of serum IL-6, IL-10, IL-12, TGF-β, and TNF-α detected by ELISA method ( n = 5 rats per group). Compared with the TBI group, the serum pro-inflammatory cytokines IL-6, IL-12 and TNF-α in the Tail Vein and In Situ group were lower (### P < 0.001), whereas the inflammation inhibiting factors IL-10 and TGF-β were much higher (### P < 0.001), indicating that huMSCs exert good immunoregulatory effects. Data are expressed as the mean ± SD. *** P < 0.001, vs . PBMCs (one-way analysis of variance followed by least significant difference test). ELISA: Enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HLAII: human leukocyte antigen II; huMSCs: human umbilical cord mesenchymal stem cells; IL: interleukin; PBMCs: peripheral blood mononuclear cells; PCR: polymerase chain reaction; TBI: traumatic brain injury; TGF-β: transforming growth factor beta; TNF-α: tumor necrosis factor alpha.

Article Snippet: The sections were thereafter incubated overnight at 4°C with primary antibodies, including rabbit anti-human CD29 polyclonal antibody (Cat# PB9086, 1:100, Boster), proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (Cat# 2586S, 1:4000, CST, Danvers, MA, USA), Caspase 3 (CASP3) rabbit polyclonal antibody (Cat# 19677-1-AP, 1:200, Proteintech) and F4/80 rabbit polyclonal antibody (Cat# 28463-1-AP, 1:200, Proteintech), diluted in 1% bovine serum albumin containing PBS.

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Expressing, Western Blot, Amplification, Enzyme-linked Immunosorbent Assay, In Situ, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Pre-clinical study of human umbilical cord mesenchymal stem cell transplantation for the treatment of traumatic brain injury: safety evaluation from immunogenic and oncogenic perspectives

doi: 10.4103/1673-5374.317985

Figure Lengend Snippet: Sites of accumulation of huMSCs in vivo in TBI rats . (A) Live imaging of the primary organs of experimental rats. (B, C) The average fluorescence intensity in the brain, liver and lung in the In Situ (B) and Tail Vein (C) groups. Data are expressed as the mean ± SD ( n = 5 rats at each time point). # P < 0.05, ## P < 0.01, vs . TBI group (one-way analysis of variance followed by least significant difference test). (D, E) CD29 (red, stained by CoraLite594) immunoreactivity in CFSE-labeled huMSCs in brain tissue (immunofluorescence staining, original magnification 200×, scale bars: 100 μm). CD29 and CFSE co-labeled huMSCs were identified in the In Situ group on days 1, 3 and 7 (D) in the brain lesions. A few CFSE-labeled huMSCs in the Tail Vein group were also observed in the brain lesions on days 1 and 3 (E). CFSE: Carboxyfluorescein succinimidyl ester; CON: TBI group; DAPI: 4′,6-diamidine-2′-phenylindole dihydrochloride; huMSCs: human umbilical cord mesenchymal stem cells; TBI: traumatic brain injury.

Article Snippet: The sections were thereafter incubated overnight at 4°C with primary antibodies, including rabbit anti-human CD29 polyclonal antibody (Cat# PB9086, 1:100, Boster), proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (Cat# 2586S, 1:4000, CST, Danvers, MA, USA), Caspase 3 (CASP3) rabbit polyclonal antibody (Cat# 19677-1-AP, 1:200, Proteintech) and F4/80 rabbit polyclonal antibody (Cat# 28463-1-AP, 1:200, Proteintech), diluted in 1% bovine serum albumin containing PBS.

Techniques: In Vivo, Imaging, Fluorescence, In Situ, Staining, Labeling, Immunofluorescence