Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: Expression of signaling molecules associated with cellular invasiveness including integrins and Rho GTPases under hypoxia. (a) Protein expression levels of integrins and phosphorylated FAK under normoxic or hypoxic conditions. (b) The expression levels of RhoA, ROCK1, Rac1/2/3, and phosphorylated Rac1/cdc42. GAPDH was used as the loading control. ∗ P < 0.05 (compared with normoxic group). ITGA4: integrin α 4 ; ITGA5: integrin α 5 ; ITGB7: integrin β 7 ; p-FAK: phosphorylated focal adhesion kinase; pRac1/cdc42: phosphorylated Rac1/cdc42.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Expressing

Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: Alteration of integrin α 4 -mediated signaling pathway in BM-MSCs under hypoxia. The mRNA expression levels of HIF-1 α (a) and integrin α 4 (b) in BM-MSCs were determined by real-time PCR. 18S rRNA was used as the loading control. Protein expression levels of integrin α 4 (c) and ROCK1 and Rac1/2/3 (d) in BM-MSCs were assessed by Western blotting. GAPDH was used as the loading control. ∗ P < 0.05 (compared with YC-1 nontreated group) and # P < 0.05 (compared with normoxic group). HIF-1 α : hypoxia-inducible factor-1 α ; ITGA4: integrin α 4 ; ROCK1: Rho-associated kinase 1.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: The effect of integrin α 4 inhibition on BM-MSC migration and activities of MMPs under hypoxia. (a) The mRNA expression of integrin α 4 in BM-MSCs was suppressed by transfection of integrin α 4 siRNA. 18S rRNA was used as the loading control. (b) BM-MSC migration was significantly increased after siITGA4 transfection. Invasiveness of BM-MSCs was assessed by invasion assay (left). BM-MSCs invaded through the inserts were counted for quantification (right). (c) Enzymatic activities of MMP-9 and MMP-2 in BM-MSCs after siITGA4 transfection were determined by zymography (left). Quantification of enzymatic activities of MMP-9 (middle) and MMP-2 (right). ∗ P < 0.05 (compared with siITGA4 nontransfected group) and # P < 0.05 (compared with normoxic group). MMP: matrix metalloproteinase; siITGA4: integrin α 4 siRNA.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Inhibition, Migration, Expressing, Transfection, Invasion Assay, Zymography

Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: Interaction between integrin α 4 and HIF-1 α and its effect on expression of Rho GTPases under hypoxia. (a) Protein expression levels of integrin α 4 , HIF-1 α , ROCK1, and Rac1/2/3 were assessed by Western blotting. GAPDH was used as the loading control. (b) HIF-1 α and integrin α 4 were localized with immunofluorescence in BM-MSCs after siITGA4 transfection under normoxic or hypoxic conditions. Blue: DAPI; green: HIF-1 α ; red: integrin α 4 . Scale bar = 80 μ m (400x original magnification). ∗ P < 0.05 (compared with siITGA4 nontransfected group) and # P < 0.05 (compared with normoxic group). DAPI: 4′,6-diamidino-2-phenylindole; HIF-1 α : hypoxia-inducible factor-1 α ; ITGA4: integrin α 4 ; ROCK1: Rho-associated kinase 1; siITGA4: integrin α 4 siRNA.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Rabbit anti-integrin α7 (ab182941), anti-integrin α2 (ab133557), anti-integrin α6 (ab97760) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR

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