Journal: Nature

Article Title: Extracellular fluid viscosity enhances cell migration and cancer dissemination

doi: 10.1038/s41586-022-05394-6

Figure Lengend Snippet: a , ( Left ) Representative western blot of SC and shTRPV4 cells using two shRNA sequences. Image is representative of 2 independent biological replicas. ( Right ) Relative TRPV4 mRNA levels of SC and shTRPV4 cells. Data are normalized to the levels of SC cells, and represent the mean ± range from 2 experiments. b , Representative whole-cell TRPV4 current traces in SC ( left ) and shTRPV4 ( right ) cells exposed to 0.77 cP (pink) or 8 cP in the absence (blue) or presence (grey) of the TRPV4 inhibitor HC-067047 (HC). c , GCaMP6s activity in SC, shTRPV4 or shNHE1 cells at 0.77 or 8 cP. d , e , Confined migration speeds of SC and shTRPV4 (sequence 1) cells ( d ), or wild-type cells under GSK 2193874 (GSK2) or vehicle control treatment ( e ) at prescribed extracellular viscosities. Data are mean ± s.d. for n ≥140 cells from 3 experiments. f , Effect of TRPV4 inhibition via GSK 2193874 (GSK2) in SUM159, HOS and U87 cells or TRPV4 knockdown (sequence 1) in brain metastatic MDA-MB-231 cells (BrM2) on confined migration speeds at the prescribed viscosities. Data are mean ± s.d. for n ≥83 cells from ≥2 experiments. g , Representative whole-cell TRPV4 current traces in SC ( left ) and shNHE1 ( right ) cells exposed to 0.77 cP (pink) or 8 cP in the absence (blue) or presence (grey) of the TRPV4 inhibitor HC-067047 (HC). h , GCaMP6s activity in SC or shRNA β1-integrin (shITGB1) cells at the prescribed viscosities. Data are mean ± s.d. for n ≥32 cells from 2 experiments. Tests performed: one-way ANOVA followed by Tukey’s multiple comparisons ( f (only HOS)) and after log transformation of data ( d , e ), Kruskal-Wallis followed by Dunn’s multiple comparison ( f (except HOS), h ). For gel source data, see Supplementary Fig. . Cell model: MDA-MB-231 unless otherwise indicated.

Article Snippet: The following primary antibodies were used: anti-TRPV4 antibody (raised in mouse; 1B2.6; Millipore Sigma; MABS466; 1:1,000), anti-ARP3 (mouse; FMS338; Abcam; ab49671; 1:5,000), anti-ARPC4 (rabbit; Abcam; ab217065; 1:2,000), anti-integrin β1 antibody (rabbit; Cell Signaling; 4706S; 1:1,000) and anti-NHE1 (raised in mouse; 54; Santa Cruz Biotechnology; sc-136239; 1:200).

Techniques: Western Blot, shRNA, Activity Assay, Migration, Sequencing, Control, Inhibition, Knockdown, Transformation Assay, Comparison

Journal: Nature

Article Title: Extracellular fluid viscosity enhances cell migration and cancer dissemination

doi: 10.1038/s41586-022-05394-6

Figure Lengend Snippet: a , Representative western blot image of SC and shITGB1 (β1-integrin) cells ( left ) and their quantification ( right ) from 3 independent biological replicas. For gel source data, see Supplementary Fig. . b , Percentage of SC and shITGB1 cells migrating with blebbing versus protrusive phenotypes at 0.77 cP and 8 cP. Data are mean ± s.e.m. for n ≥20 cells per experiment from 3 independent experiments. c , Confined migration speeds of SC and shITGB1 cells at prescribed extracellular viscosities. Data are mean ± s.d. for n ≥89 cells from 3 experiments. d , e , Front to rear NHE1 ( d ) or ezrin ( e ) intensity ratio in confined cells migrating at 8 cP in the presence of vehicle control or LatA (2 µM). Data are mean ± s.d. for n ≥25 cells from 2 experiments. f , Rate of pH recovery after intracellular acidosis due to NH 4 Cl pulse treatment of cells expressing pHRed. Data are mean ± s.d. from n ≥19 cells in each condition pooled from 3 experiments. g , Confined migration velocity of wild-type or NHE1-GFP-overexpressing (NHE1+) MDA-MB-231 cells ( n ≥35) at 8 cP in the presence of vehicle control or Lat A from ≥3 experiments. The y axis is discontinued from 17–25 µm/h to highlight differences in velocity. Data are mean ± s.d. Tests performed: two-way ANOVA followed by Tukey’s multiple comparisons ( b ), Kruskal-Wallis followed by Dunn’s ( c , g ), unpaired t-test on log transformed data ( d , e ), and one-way ANOVA followed by Tukey’s ( f ). Cell model: MDA-MB-231.

Article Snippet: The following primary antibodies were used: anti-TRPV4 antibody (raised in mouse; 1B2.6; Millipore Sigma; MABS466; 1:1,000), anti-ARP3 (mouse; FMS338; Abcam; ab49671; 1:5,000), anti-ARPC4 (rabbit; Abcam; ab217065; 1:2,000), anti-integrin β1 antibody (rabbit; Cell Signaling; 4706S; 1:1,000) and anti-NHE1 (raised in mouse; 54; Santa Cruz Biotechnology; sc-136239; 1:200).

Techniques: Western Blot, Migration, Control, Expressing, Transformation Assay

Journal: Oncology Reports

Article Title: Adipocytes induce the resistance of ovarian cancer to carboplatin through ANGPTL4

doi: 10.3892/or.2020.7647

Figure Lengend Snippet: Downstream effect of ANGPTL4 following binding to integrin α5β1 on surface of the tumor cell membrane. (A) Co-IP was performed and the immunoprecipitates were analyzed using western blot analysis with antibodies against integrin α5β1, claudin-5, ANGPTL4 and GAPDH. (B) Statistical results of the co-IP assay. The data are presented as the mean ± SD. **P<0.01 vs. SKOV3. (C and D) The regulatory effect following silencing of integrin α5β1 on the ANGPTL4-induced resistance of SKOV3 cells in vitro . The data are presented as the mean ± SD. **P<0.01 vs. SKOV3 shMock /CM/carboplatin or SKOV3 shMock /adipocyte/carboplatin). (E and F) The regulatory effect following silencing of claudin 5 on the ANGPTL4-induced resistance of SKOV3 cells in vitro . The data are presented as the mean ± SD. **P<0.01 vs. SKOV3 shMock /CM/carboplatin or SKOV3 shMock /adipocyte/carboplatin). ANGPTL4, angiopoietin-like 4; Co-IP, co-iummunoprecipitation; sh, short hairpin; CM, conditioned medium.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk, prepared with the TBS-Tween-20 buffer, and incubated with primary antibody against ANGPTL4 (1:1,000; cat. no. ab2920; Abcam), integrin α5β1 (1:1,000; cat. no. orb13515; Biorbyt Ltd.), claudin 5 (1:2,000; cat. no. ab131259; Abcam), VE-cadherin (1:1,000; cat. no. 2158; Cell Signaling Technology, Inc.), Bcl-xl (1:1,000; cat. no. ab32370; Abcam), ABCB1 (1:1,000; cat. no. 13342; Cell Signaling Technology, Inc.), ABCC1 (1:1,000; cat. no. 72202; Cell Signaling Technology, Inc.), ABCG2 (1:1,000; cat. no. ab207732; Abcam), β-actin (1:10,000; cat. no. BS6007M; Bioworld Technology, Inc.), PI3K (1:2,000; cat. no. 4257; Cell Signaling Technology, Inc.), phospho-PI3K (1:1,000; cat. no. 4228; Cell Signaling Technology, Inc.), c-Myc (1:2,000; cat. no. 18583; Cell Signaling Technology, Inc.), phospho-c-Myc (1:1,000; cat. no. 13748; Cell Signaling Technology, Inc.), p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), phospho-p65 (1:1,000; cat. no. ab194726; Abcam), Akt (1:2,000; cat. no. 4691; Cell Signaling Technology, Inc.), phospho-Akt (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.) overnight at 4°C, whilst avoiding non-specific background.

Techniques: Binding Assay, Membrane, Co-Immunoprecipitation Assay, Western Blot, In Vitro

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 5. Expression changes of key GBM proteins and their response to FN1 Variants in TBMN. Immunofluorescence experiments assessed the expression of Laminin α5β2, COL4A3/4/5 and Integrin β1 in the GBM. The findings revealed non-homogeneous linear structure expression of these key GBM proteins in the presence of FN1 variants, indicating their potential involvement in the pathogenesis of TBMN. HC = healthy control.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Expressing, Immunofluorescence, Control

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 6. Abnormal co-localization of FN1 variants with Integrin β1 and its impact on other GBM components. Immunofluorescence studies investigated the rela tionship between FN1 variants and the expression patterns of GBM components. The findings reveal abnormal co-localization of FN1 variants with Integrin β1, accompanied by reduced co-localization of other GBM components. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Immunofluorescence, Expressing

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 7. Competitive Binding of FN1 Variants to Integrin β1. (A and B) Duolink proximity ligation assay and Co-IP were employed to analyze the protein interaction between FN1 variants and Integrin β1. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Binding Assay, Proximity Ligation Assay, Co-Immunoprecipitation Assay