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: α2,3-sialylation regulates the Hippo pathway through competing with α2,6-sialylation. A, the cell lysates from ST3GAL4-3xFLAG-overexpressing (ST3GAL4-OE) MDA-MB-231 cells were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, anti-FLAG, anti-ST6GAL1, and anti-GAPDH antibodies. B, the cell membrane fractions from Con and ST3GAL4-OE cells were blotted with ConA, RCA-I (recognizing terminal galactose), MAA, and SNA lectins. C, cell lysates from the MDA-MB-231 derivative cell lines as indicated (The ST6GAL1 KO + ST3GAL4 OE stable cell line was established by overexpressing ST3GAL4 in ST6GAL1 KO cells.) were immunoblotted with indicated antibodies as mentioned in ( A ). The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1) in ( A ) and ( C ) are shown as the mean ± SD ( n = 3 biological replicates, n.s. not statistically significant, p > 0.05, ∗∗∗, p < 0.001, ∗∗∗∗, p < 0.0001 are determined by two-tail unpaired t test and one-way ANOVA with Tukey's post hoc test, respectively). D, the cell membrane fractions from the cells mentioned in ( C ) were blotted with ConA, RCA-I, MAA, and SNA lectins. LATS, large tumor suppressor kinase; SNA, Sambucus nigra ; YAP, yes-associated protein; RCA-I, Ricinus communis agglutinin I; ConA, Concanavalin A; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; MAA, Maackia amurensis agglutinin; Con, control.

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, Stable Transfection, 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: 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: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of AMF30a on cell adhesion of corneal endothelial cells. ( A - C ) Cell adhesion assay was performed using crystal violet assay. Scale bar = 500 μm. ( D ) immunofluorescence staining of E-cadherin are shown. Scale bar = 100 μm. ( E and F ) F-actin was visualized using phalloidin staining. Scale bar = 100 μm. ( G and H ) pERK1/2 levels were evaluated using Western blot. ( I and J ) pYAP levels were evaluated using western blot. ( K and L ) Nuclear translocation of YAP was evaluated using immunofluorescence staining. Scale bar = 50 μm. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Article Snippet: Primary antibodies were as follows: rabbit anti-human CD166 antibody (ab109215, Abcam, 1∶500 dilution), rabbit anti-human ZO-1 antibody (sc-10804, Santa Cruz, 1∶500 dilution), mouse anti-human ERK1/2 antibody (sc-514032, Santa Cruz, 1∶500 dilution); mouse anti-human pERK1/2 antibody (sc-136521, Santa Cruz, 1∶500 dilution); mouse anti-human YAP antibody (sc-376830, 1∶500 dilution); rabbit anti-pYAP antibody (PA5-17481, Invitrogen, 1∶500 dilution); rabbit anti-human PAD2 antibody (12110-1-AP, Proteintech, 1∶500 dilution); mouse anti-ATF4 antibody (sc-390063, 1∶500 dilution); mouse anti-human ROCK1 antibody (sc-17794, 1∶500 dilution); mouse anti-ROCK2 antibody (sc-398519, 1∶500 dilution); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1∶5000 dilution).

Techniques: Cell Adhesion Assay, Crystal Violet Assay, Immunofluorescence, Staining, Western Blot, Translocation Assay

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of AMF30a on TGF-β-mediated ROCK/HIPPO signaling pathway of corneal endothelial cells. ( A and B ) pERK1/2 levels were evaluated using western blot. ( C and D ) pYAP levels were evaluated using western blot assays. ( E and F ) Nuclear translocation of YAP was evaluated using immunofluorescence staining. Scale bar = 100 μm. ( G - I ) ROCK1 and ROCK2 levels were evaluated by western blotting. (J and K) Nuclear translocation of NF-κB was evaluated using immunofluorescence staining. Scale bar = 100 μm. (L and M) ATF4 levels were evaluated by western blotting. * p < 0.05, and ** p < 0.01.

Article Snippet: Primary antibodies were as follows: rabbit anti-human CD166 antibody (ab109215, Abcam, 1∶500 dilution), rabbit anti-human ZO-1 antibody (sc-10804, Santa Cruz, 1∶500 dilution), mouse anti-human ERK1/2 antibody (sc-514032, Santa Cruz, 1∶500 dilution); mouse anti-human pERK1/2 antibody (sc-136521, Santa Cruz, 1∶500 dilution); mouse anti-human YAP antibody (sc-376830, 1∶500 dilution); rabbit anti-pYAP antibody (PA5-17481, Invitrogen, 1∶500 dilution); rabbit anti-human PAD2 antibody (12110-1-AP, Proteintech, 1∶500 dilution); mouse anti-ATF4 antibody (sc-390063, 1∶500 dilution); mouse anti-human ROCK1 antibody (sc-17794, 1∶500 dilution); mouse anti-ROCK2 antibody (sc-398519, 1∶500 dilution); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1∶5000 dilution).

Techniques: Western Blot, Translocation Assay, Immunofluorescence, Staining