Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a , IP 1 production in HEK293 cells transfected with either control siRNA or integrin β3 siRNA, along with the expressing of vector and G qi9 , or GABA B receptor and G qi9 , under either adhesion or suspension conditions. Data are present as mean ± s.e.m. from five biologically independent experiments and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, not significant (ns) > 0.05. b , Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β 3 antibody, under basal condition. Only GB1 in cell surface was labeled and visualized using an impermeable Snap fluorescent substrate. c-f, Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (HA-tagged GB1 and GB2) using anti-HA antibody, under conditions including suspension or adhesion (c) , without (control) or with shear stress (c) , Blebbistatin (50 μM, 30 min) treatment (d) , RGDS (10 μM, 12 h) treatment (e) , or CGP54626 (50 μM, 30 min) treatment (f) . Blots are representative from at least three biologically independent experiments ( c , suspension or adhesion, n = 3; control or with shear stress, n = 4; d , n = 3; e , n = 3; f , n = 4). The amount of integrin β 3 immunoprecipitated by IgG or HA antibody are present as mean ± s.e.m. in (e) and analyzed using unpaired t test (two-tailed) to determine significance. ** P < 0.01. g, Schematic representation of the BRET assay detecting direct interaction between GABA B receptor and integrin β 3 . Rluc was fused in C-terminal of GB1 or GB2 subunit (GB1 Rluc or GB2 Rluc ) as luminescence donor. Venus was fused in C-terminal of integrin β 3 or integrin α V subunit (integrin β 3Venus or integrin α Venus ) as fluorescence acceptor. h-i, Interaction of GABA B receptor and integrin β 3 interaction between GB1 and integrin β 3 or GB1 and integrin α V (h) , or GB2 and integrin β 3 (i) detected using BRET titration assay. Data were analyzed by nonlinear regression on a pooled data set from at least three biologically independent experiments each performed in triplicates, fitting with 1-site binding model in GraphPad Prism 8.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Suspension, Two Tailed Test, Immunoprecipitation, Construct, Labeling, Shear, Bioluminescence Resonance Energy Transfer, Fluorescence, Titration, Binding Assay

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a , Schematic representation of GABA B -ΔVFT truncation, in which GB1 subunit lacks of the VFT domain (GB1-ΔVFT), but retains the ability to be activated by positive allosteric modulator Rac BHFF (yellow square). Co-immunoprecipitation experiments were performed using anti-HA antibody targeting to the HA tag, which is fused in the N-terminal of GB1 or GB1-ΔVFT. b, Co-immunoprecipitation of GABA B receptor or GABA B -ΔVFT and integrin β 3 using anti-HA antibody. Blots are from one representative of five biologically independent experiments. c, IP 1 production in cells transfected with GABA B receptor, or GABA B -ΔVFT, along with G qi9 under suspension or adhesion conditions. Data are present as mean ± s.e.m. from four biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. * P < 0.05, not significant (ns) > 0.05. d, Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B -ΔVFT and G qi9 . After recording the basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which Rac BHFF was added for 200 seconds. Data are present as mean ± s.e.m. from 135 cells recorded.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Immunoprecipitation, Transfection, Suspension, Two Tailed Test, Expressing, Shear

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a , Immunofluorescent staining of GFAP (red), GFP (green) and DAPI (cyan) in astrocytes transfected with control siRNA or GABA B receptor siRNA along with GFP, treatment with or without shear stress (15 dyn/cm , 30 min). Images are representative from three biologically independent experiments. Scale bar: 10 μm. b-c, Analysis of the cell size and GFAP expression of astrocytes with the same treatment in (a) . Measurements are made on each cell by cell basis (ROI) from three biologically independent experiments (number of cells from left to right: n = 47, 37, 32, 39). Data are present as mean ± s.e.m and analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test to determine significance. *** P < 0.001, ** P < 0.01, ns: non-significant. d, RNA interference efficacy of the GB1 in astrocytes in (b-c) using qPCR detecting gbb1 expression. Data are present as mean ± s.e.m. from three biologically independent experiments and analyzed with unpaired t test (two-tailed) to determine significance. *** P < 0.001.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Staining, Transfection, Control, Shear, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a , Real-time recording of intracellular Ca 2+ release in astrocytes under shear stress. Cells were transfected with control siRNA and treated with shear stress, baclofen (100 μM) or ATP (100 μM) in the indicated time point. Data are representative from six biologically independent experiments. b, Percentage of astrocytes in response to shear stress or baclofen in 236 recorded cells. c, Real-time recording of intracellular Ca 2+ release in astrocytes under shear stress. Cells are transfected with siRNA knocking down GABA B receptor and administrated to shear stress, baclofen (100 μM) or ATP (100 μM) in the indicated time point. Data are representative from six biologically independent experiments. d, Percentage of astrocytes with the calcium response (sensitive) or without the calcium response (insensitive) to shear stress (control siRNA: 236 cells; GABA B siRNA: 314 cells). Data are analyzed with χ2 test to determine significance. **** P < 0.0001.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Shear, Transfection, Control

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: Antagonist CGP54626-bound GABA B receptor fails to interact with integrin β3 whereas integrin β3 interacts with GB1 VFT of GABA B receptor with constitutive activity. Mechanical force promotes GB1 VFT and integrin β3 interaction to stabilize the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact, further inducing an allosteric re-arrangement of the GABA B receptor 7TM towards an TM6-TM6 active conformation, culminating in the asymmetric activation of the receptor with G protein under GB2. Mechanical force acts as a positive allosteric modulator to boost up GABA-induced GABA B receptor activation through interaction between GB1 VFT and integrin β3.

Article Snippet: The integrin β 3 siRNA (#sc-63292) and control siRNA (#sc-37007) were purchased from Santa Cruz (Shanghai, China) and the GB1 siRNAs were synthesized from GenePharma (Suzhou, China) and the sequences are: GCGGUUUCCAACGUUCUUUTT (Forward), AAAGAACGUUGGAAACCGCTT (Reverse); GCUACAAGAAGAUCGGCUATT (Forward), UAGCCGAUCUUCUUGUAGCTT (Reverse); GGGAGAAGCCAGUUCCCAUTT (Forward), AUGGGAACUGGCUUCUCCCTT (Reverse).

Techniques: Activity Assay, Activation Assay

Journal: Journal of Functional Biomaterials

Article Title: LXW7 Peptide Modification of Acellular Liver Scaffolds Improves Endothelialization and Hemocompatibility in Bioengineered Liver

doi: 10.3390/jfb17030122

Figure Lengend Snippet: Thrombogenicity evaluation of endothelialized scaffolds ( A ) schematic of the experimental design of ex vivo blood perfusion. ( B ) Gross appearance of DLS, endothelialized, and LXW7 endothelialized scaffolds after blood perfusion, showing markedly reduced clot deposition in the LXW7 group (yellow circles highlight thrombi). Scale bar = 2 cm. ( C ) IF staining of platelet marker integrin αIIb (red) with DAPI (blue) showing reduced platelet adhesion in endothelialized and LXW7endothelialized scaffolds compared to DLS. Scale bar = 100 µm. ( D ) Quantification of fluorescence intensity confirming significantly decreased platelet adhesion in LXW7 endothelialized scaffolds ( n = 4 fields, * p < 0.05 vs. DLS). ( E ) Time-dependent changes in platelet count (%) in the blood perfusate during ex vivo blood perfusion ( n = 3 samples each time point, * p < 0.05 vs. DLS).

Article Snippet: For IF staining, sections were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 2% bovine serum albumin (Sigma-Aldrich) for 45 min. Tissue sections were incubated overnight with primary antibodies including anti-human CD31 (1:100, MA5-13188, Invitrogen), Albumin (1:200, PA5-89332, Invitrogen), anti-Integrin αIIb (1:100, sc-21783, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TGF-β1 (1:200, ab170874, Abcam).

Techniques: Ex Vivo, Staining, Marker, Fluorescence

Journal: Oncotarget

Article Title: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

doi: 10.18632/oncotarget.10757

Figure Lengend Snippet: ( A ) mRNA levels of ERα , TRβ1 , ITG αv and ITG β3 from MCF-7, SKOV-3 and OVCAR-3 were measured by quantitative real-time PCR and normalized with 18Sr RNA. Results were expressed as folds of increase. ( B ) SKOV-3 cells were treated in the presence or absence of indicated peptides with T 3 or T 4 for 3 days, cells in 96 wells were subjected to the MTT assay. ( C ) scramble or shRNA of integrin αv or β3 were transiently transfected to SKOV-3 cells and treated with thyroid hormone (T 4 ) for 30 min, and whole cell lysates were harvested for western blot analysis. Indicated antibodies were used. Compared to sc: p < 0.05 :* p < 0.01 :** p < 0.001 :*** ; Compared to sc treated with T 4 : p < 0.05: † p < 0.01: †† p < 0.001: †††

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology).

Techniques: Real-time Polymerase Chain Reaction, MTT Assay, shRNA, Transfection, Western Blot

Journal: Oncotarget

Article Title: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

doi: 10.18632/oncotarget.10757

Figure Lengend Snippet: SKOV- 3 cells were treated with ( A ) thyroid hormone, ( B ) estrogen, for different periods of time as indicated. ( C ) SKOV-3 cells treated with thyroid hormone or estrogen for 10 min in the absence or presence of ICI were fixed and stained with anti-integrin αv and phospho-ERα (S167) antibodies, and subsequently with fluorescent secondary antibodies. Nuclear punctate of phosphorylated ERα-induced by T 4 (shown in green) were increased with time and co-localized with integrin αvβ3 (shown in red) to yield a yellow color. Nuclei were stained with DAPI and showed as blue. The phosphorylation of ERα and nuclear translocation of integrin αv were inhibited by ICI. Quantitative fluorescence intensities are shown as average intensity per cell. p < 0.05: * p < 0.01: **

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology).

Techniques: Staining, Phospho-proteomics, Translocation Assay, Fluorescence

Journal: Oncotarget

Article Title: Crosstalk between integrin αvβ3 and ERα contributes to thyroid hormone-induced proliferation of ovarian cancer cells

doi: 10.18632/oncotarget.10757

Figure Lengend Snippet: SKOV-3 were pre-treated in the presence or absence of ICI for 30 min prior to another 30 min of T 4 treatment and harvested for ChIP. Total cell lysate was immunoprecipitated with anti-integrin αv and pulled-down DNA was measured with qPCR. Compared to control: p < 0.01: **; Compared to T 4 alone: p < 0.001: †††

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology).

Techniques: Immunoprecipitation, Control

Journal: Cell death & disease

Article Title: ID4-dependent secretion of VEGFA enhances the invasion capability of breast cancer cells and activates YAP/TAZ via integrin β3-VEGFR2 interaction.

doi: 10.1038/s41419-024-06491-2

Figure Lengend Snippet: Fig. 6 ID4/VEGFA axis stimulates nuclear YAP localization via integrin β3/VEGFR2 signaling in BC cells. A immunofluorescence analysis of YAP in M6 Control cells treated with vehicle or Cilengitide at 0.1 µM, with the relative mean fluorescence intensity quantification in nuclei and cytoplasm. B immunofluorescence analysis of M6 ID4-KO cells treated with vehicle, Cilengitide, and/or VEGFA, with the relative mean fluorescence intensity quantification in nuclei and cytoplasm. Scale bar: 50 µm. Data are presented as mean ± SD. **P < 0.01, ****P < 0.0001 calculated by Student’s t test (A) or One-way Anova (B) on n = 3 experiments.

Article Snippet: The following primary antibodies were used: ID4 (B5) (sc-365656), GAPDH (sc-32,233), VEGFR2 (sc-6251), p-Integrin β3 (sc-136458) (Santa Cruz Biotechnology, Dallas, TX, USA); p-FAK (Tyr397) (3283), FAK (3285), integrin β3 (13166), integrin αV (4711), YAP (14074), TAZ (83669), p-AKT (9271), AKT (9272), p-p38 (9211), p38 (9212), p-ERK 1/2 (9102), ERK 1/2 (9102) (Cell Signaling Technology, Danvers, MA, USA); VEGFA (ab46154) (Abcam, Cambridge, UK), alpha-Tubulin (Sigma, St. Louis, MO, USA).

Techniques: Control