Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Activation Assay, Membrane, Binding Assay, Incubation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 catalyzes the α2,6-sialylation of LPAR4, EGFR, integrin α5, and integrin β1 in MDA-MB-231 cells. A, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 cells were immunoprecipitated by SSA-agaroses and then blotted with antibodies against LPAR4, EGFR, integrin β1, and integrin α5. The whole-cell lysates were also subjected to WB with indicated antibodies. B, the cell lysates from Con- and ST3GAL4-OE- MDA-MB-231 cells were immunoprecipitated by SSA- and MAM-agaroses and then blotted with antibodies against integrin β1, EGFR, and integrin α5 separately. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the α2,6-sialylated LPAR4, EGFR, integrin α5, or integrin β1 versus total LPAR4, EGFR, integrin α5, or integrin β1, respectively) in ( A ) and (the α2,3-sialylated or α2,6-sialylated integrin β1, EGFR, or integrin α5 versus total integrin β1, EGFR, or integrin α5, respectively) in ( B ) are shown as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test and two-tail unpaired t test, respectively). WB, Western blot; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; SSA, Sambucus sieboldiana agglutinin; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; Maackia amurensis.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Immunoprecipitation, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 mediates integrin β1–LPAR4/EGFR complex formation. A and B, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 ( A ) and BT549 ( B ) cells were immunoprecipitated by anti-integrin β1 antibody and then blotted with antibodies against LPAR4, EGFR, and integrin β1. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the association of EGFR or LPAR4 with integrin β1, respectively) in ( A ) and ( B ) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). ST6GAL1, β-galactoside α2,6-sialyltransferase 1; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; WB, Western blot.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Immunoprecipitation, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Schematic diagram of the proposed molecular mechanism for negative regulation of Hippo signaling via ST6GAL1. Various upstream cell membrane receptors of the Hippo pathway have been identified, including the RTKs ( e.g. , EGFR), GPCRs ( e.g. , LPAR4), and integrins ( e.g. , integrin α5β1). The RTK, GPCR, and integrin signals transduced by growth factors (GFs, e.g. , EGF), extracellular factors ( e.g. , LPA), and the extracellular matrix (ECM, e.g. , FN) can facilitate Hippo pathway effectors ( e.g. , PI3K and FAK) association, which promote LATS1/2-mediated regulation of YAP. In the cells with ST6GAL1 expression ( left ), the cell membrane receptors, such as EGFR, LPAR4, and integrin α5β1, are modified by α2,6-sialylation, which mediate the integrin β1–EGFR/LPAR4 complex formation and in turn facilitate their responses to EGF, LPA, and FN, respectively. These signalings inactivate LATS1/2 kinases or induce the dephosphorylation of YAP, finally leading to hypophosphorylated YAP (p-YAP S127). Hypophosphorylated YAP accumulates in the nucleus, where it can bind to various transcription factors (TFs, e.g. , TEAD family) to enhance the expression of target genes ( e.g. , ANKRD1 , CTGF , and CYR61 ) expression that promote cell adhesion, spreading, proliferation, migration, and metastasis. The Hippo signaling can be inhibited by the verteporfin (VP) inhibitor, which targets YAP-TEAD activity. In the ST6GAL1 deficiency cells ( right ), the N -glycans on cell membrane receptors are without α2,6-sialylation, which exhibit weak integrin β1–EGFR/LPAR4 complex formation and delayed responses to EGF, LPA, and FN stimulation and activate the LATS1/2 kinases and phosphorylate YAP on S127. The phosphorylated YAP (p-YAP S127) is retained in the cytoplasm, inhibiting YAP/TEAD-dependent transcription. The p of the red background represents the activation of related proteins, while gray background represents the inactivation. LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FN, fibronectin; GPCR, G protein–coupled receptor; GT, glycosyltransferase; LPA, lysophosphatidic acid; FAK, focal adhesion kinase.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Membrane, Expressing, Modification, De-Phosphorylation Assay, Migration, Activity Assay, Activation Assay

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

Journal: Cell Death Discovery

Article Title: Macrophages mediate psoriasis via Mincle-dependent mechanism in mice

doi: 10.1038/s41420-023-01444-8

Figure Lengend Snippet: Specific primers for real-time PCR.

Article Snippet: 50 μg of protein was subjected to SDS-PAGE electrophoresis, transferred through PVDF membrane, and then locked with 5% skimmed milk at room temperature for 1 h. Mincle (sc-390806, Santa cruz), p-Sky (2717 S, Cell Signaling Technology), Sky (13198 S, Cell Signaling Technology), p-P65 (3033 S, Cell Signaling Technology), P65 (8242 S, Cell Signaling Technology), iNOS (13120 S, Cell Signaling Technology), F4/80 (sc-52664, Santa cruz), IL-1β (sc-52012, Santa cruz), IL-6 (sc-32296, Santa cruz), TNFα (sc-52746, Santa cruz), PU.1 (2258 S, Cell Signaling Technology), keratin-17 (17516-1-AP, Proteintech), Integrin β1 (bs-0486R, Bioss), CXCL9 (bs-2551R, Bioss) primary antibody (dilution ratio 1:1,000) were added and incubated overnight at 4 °C.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Effect of Mixed Transplantation of Autologous and Allogeneic Microskin Grafts on Wound Healing in a Rat Model of Acute Skin Defect

doi: 10.1371/journal.pone.0085672

Figure Lengend Snippet: Aa, Ab, and Ac show integrin β1 expression at 2 post-graft weeks (PGWs) in groups II,III, and IV, respectively, in Experiment A. Ad shows integrin β1 expression in the hair follicle in group IV in Experiment A. Ae and Af show integrin β1 expression at 2 PGWs in groups II and III, respectively, in Experiment B. Ag and Ah show expression of integrin β1 in the hair follicle and no expression of integrin β1 in the epidermis, respectively, of normal Sprague-Dawley (SD) rat skin. Ai, Aj, and Ak show negative control staining of 2 PGW sections from groups II, III, and IV, respectively, in Experiment A. Scale bar, 50 µm in Aa–Ah and 100 µm in Ai–Ak. In Experiment A, the mean integrated optical density (IOD) of integrin β1 expression at 2 PGWs was higher in groups III and IV than in group II (* p<0.05), and, more interestingly, was highest in group III among these 3 groups (* p<0.05, # p<0.05). At 3 and 4 PGWs, the integrin β1 expression remained higher in group III than in groups II and IV (* p<0.05, # p<0.05). In Experiment B, the integrin β1 expression at 2 PGWs was stronger in group III than in group I (* p<0.05), and no intergroup difference was found at 4 PGWs.

Article Snippet: The sections were then sequentially incubated with normal goat serum for 40 minutes to block nonspecific binding, with the primary rabbit anti-integrin β1 polyclonal antibody (1∶100, Wuhan Boster Biological Technology LTD, Hubei, China) for 15 hours at 4°C, and with the SABC kit (Wuhan Boster Biological Technology, LTD, Hubei, China) at 37°C to bind the primary antibody.

Techniques: Expressing, Negative Control, Staining