Journal: Scientific Reports

Article Title: Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells

doi: 10.1038/srep18430

Figure Lengend Snippet: ( A ) WT and KO cells were immunoblotted by anti-β1 antibody, α-tubulin was used as a loading control (left panel). WT and KO cells were collected and incubated with (bold line) or without (grey shadow) anti-β1, followed by incubation with Alexa Fluor 647 goat anti-mouse IgG subjected to flow cytometry analysis (right panel). ( B ) Cell lysates from WT and KO cells were immunoblotted with anti-α3, anti-α5, anti-α6, anti-αV and anti-β4 antibodies. α-Tubulin was used as a loading control (upper panel). Cells were collected and incubated with (bold line) or without (grey shadow) anti-α3, anti-α5, anti-α6 and anti-β4 antibodies, followed by incubation with Alexa Fluor 647 goat anti-mouse IgG subjected to flow cytometry analysis (lower panel). ( C ) Bright field pictures were taken to show the representative cell morphology. Scale bar, 50 μm (upper panel). WT and KO cells were stained with anti-β1 antibody, followed by incubation with fluorescent secondary antibody. Localization of F-actin was examined by staining with Alexa Fluor 546 phalloidin, the bar denotes 20 μm (middle and low panel). ( D ) The indicated cells were stained with anti-pFAK and anti-β4 antibody, followed by the incubation with fluorescent secondary antibody. Scale bar, 20 μm. The arrows indicate β4 integrin, the hemidesmosome maker, expressed in the cell-cell contact.

Article Snippet: The experiments were performed using the following antibodies: Antibody against human integrin β1 subunit (P5D2) was from Developmental Studies Hybridoma Bank, University of Iowa; Mouse mAb against Smad2, rabbit mAbs against EGFR, p-EGFR, ERK1/2, p-ERK1/2, AKT, p-AKT, p-Src, and p-Smad2 were from Cell Signaling Technology; rabbit pAb to β4 integrin and goat antibody against α3 integrin were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse mAbs against β1 integrin, α5 integrin, β4 integrin, αv integrin, FAK, p-FAK, and rat antibody against α6 integrin were from BD Biosciences; mAb against α-tubulin and α-SMA were from Sigma; mouse mAbs against Src was from upstate biotechnology.

Techniques: Control, Incubation, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells

doi: 10.1038/srep18430

Figure Lengend Snippet: ( A ) After attachment for 24 h, WT and KO cells treated with 3 μM of AG1478 for 48 h, the number of live cells were counted. Cell numbers were normalized to those at 0 h. Data are represented as the means ± s.e.m (*** p < 0.001 by two-tail unpaired t-test). ( B ) MDA-MB-231 cells were untreated, or treated with P5D2, AG1478, or P5D2 plus AG1478 for 48 h. Cell proliferation was evaluated by the number of live cells. Cell numbers were normalized to those at 0 h as 1. Data are represented as the means ± s.e.m (** p < 0.01 by two-tail unpaired t-test).

Article Snippet: The experiments were performed using the following antibodies: Antibody against human integrin β1 subunit (P5D2) was from Developmental Studies Hybridoma Bank, University of Iowa; Mouse mAb against Smad2, rabbit mAbs against EGFR, p-EGFR, ERK1/2, p-ERK1/2, AKT, p-AKT, p-Src, and p-Smad2 were from Cell Signaling Technology; rabbit pAb to β4 integrin and goat antibody against α3 integrin were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse mAbs against β1 integrin, α5 integrin, β4 integrin, αv integrin, FAK, p-FAK, and rat antibody against α6 integrin were from BD Biosciences; mAb against α-tubulin and α-SMA were from Sigma; mouse mAbs against Src was from upstate biotechnology.

Techniques:

Journal: PLoS ONE

Article Title: miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

doi: 10.1371/journal.pone.0070192

Figure Lengend Snippet: (A) Effects of miR-29c on endogenous integrin β1 protein. Changes in protein expression of Int β1 was determined by Western blotting analysis in 95D 29c , 95D MiNC, 95C 29ci and 95C IhNC. GAPDH was used as an internal loading control. (B) The gray levels of Western blotting are shown by bar gragh. miR-29c mimics inhibit endogenous integrin β1 in 95D cell lines (95D 29c and 95D MiNC), while miR-29c inhibitors promote integrin β1 protein expression. ** p <0.01 (Student's t -test, n = 3). (C) Effects of miR-29c on endogenous MMP2 protein levels by Western blotting. (D) The gray levels of Western blotting are shown by bar gragh. ** p <0.01 (Student's t -test, n = 3). (E) Effects of miR-29c on MMP2 and MMP9 enzyme activity in lung cancer by gelatin zymography. (F) The gray level of every band was measured to check the differences in MMP2 enzyme activity in 95C and 95D cells (The brightest band [95D MiNC] was set to 1 unit to compare with other gray levels). ** p <0.01 (Student's t -test, n = 3). (G) The gray level of every band was measured to check the difference in MMP9 enzyme activity between 95C and 95D cells (The brightest band [95D MiNC] was set to 1 unit to compare with other gray levels). ** p <0.01 (Student's t -test, n = 3).

Article Snippet: The membranes were blocked in 5% non-fat milk in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated overnight at 4°C by the appropriately diluted primary antibodies for rabbit monoclonal anti-human integrin β1 and MMP2 (diluted 1∶1000; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Control, Activity Assay, Zymography

Journal: Cell & Bioscience

Article Title: Extracellular domain of CD98hc is required for early murine development

doi: 10.1186/2045-3701-1-7

Figure Lengend Snippet: In vivo expression of ΔCD98hc-β geo fusion protein . A . ΔCD98hc-β geo fusion protein is expressed in CD98hc Δ/+ mouse. Protein was extracted from CD98hc Δ/+ ES cells (PST080) or various tissue samples harvested from wild type (CD98hc +/+ ) and heterozygous mutant (CD98hc Δ/+ ) mice for Western blotting. Anti-β-galactosidase (β gal) antibody yields ~220 kD bands specific to CD98hc Δ/+ samples, while anti-CD98hc antibody yields ~80 kD bands in all samples. Note that the different mobility of the proteins from different tissues is likely due to different posttranslational modifications. The expression of integrin β1, which can interact with CB98hc, was detected in ES cells, intestine, and testis samples, but not in brain or kidney sample. The β actin was analyzed as a loading control. *, nonspecific bands. B . The expression pattern of ΔCD98hc-β geo fusion protein mimics that of CD98hc protein in CD98hc Δ/+ mouse. Kidney sections from CD98hc +/+ and CD98hc Δ/+ mice were immunostained with anti-CD98hc antibody or stained with X-gal to localize β-galactosidase activity that represents ΔCD98hc-β geo fusion protein. Note that distribution of immunoreactive CD98hc is the same as that of β-galactosidase activity in CD98hc Δ/+ kidneys. Lower right panel shows the magnified image of boxed area indicated in lower middle panel.

Article Snippet: Rabbit anti-integrin β1 pAb was from Chemicon.

Techniques: In Vivo, Expressing, Mutagenesis, Western Blot, Staining, Activity Assay

Journal: Cell & Bioscience

Article Title: Extracellular domain of CD98hc is required for early murine development

doi: 10.1186/2045-3701-1-7

Figure Lengend Snippet: ΔCD98hc-β geo fusion protein interacts with integrin β1 similarly as wild type CD98hc . A . FLAG-tagged integrin β1 and wild type CD98hc or ΔCD98hc-β geo fusion protein were expressed in frog oocytes. Immunostaining of the sectioned oocytes with anti-integrin β1 antibody verified the cell surface expression (green) of FLAG-tagged integrin β1 protein. B . Co-immunoprecipitation analysis was performed with frog oocytes that expressed FLAG-tagged integrin β1 and wild type CD98hc or ΔCD98hc-β geo fusion protein. Anti-FLAG antibody immunoprecipitated FLAG-integrin β1 as expected and also co-immunoprecipitated CD98hc and ΔCD98hc-β geo fusion protein with similar efficiencies. C . Co-immunoprecipitation analysis was performed with PST080 ES cells. Anti-integrin β1 antibody pulled down ΔCD98hc-β geo fusion protein or CD98hc together with endogenous integrin β1.

Article Snippet: Rabbit anti-integrin β1 pAb was from Chemicon.

Techniques: Immunostaining, Expressing, Immunoprecipitation

Journal: Integrative Cancer Therapies

Article Title: Anti-Cancer Effects of the Ethyl Acetate Fraction From Opuntia humifusa on Human Triple-Negative Breast Cancer Cells

doi: 10.1177/15347354251401187

Figure Lengend Snippet: Effects of the ethyl acetate fraction of O. humifusa on metastasis- and hypoxia-associated proteins in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with control, DMSO, or the ethyl acetate fraction of O. humifusa (150 and 250 μg/mL) for 48 hours. Protein expression levels of MMP-7, MMP-9, HIF-1α, and integrin β1 were assessed. (B) Protein relative expression of MMP-7, MMP-9, HIF-1α, and integrin β1. Data are presented as mean ± SD (n = 3). *** P < .001 versus DMSO-treated group.

Article Snippet: Primary antibodies used were as follows (all 1:1000 dilution): GAPDH (Cell Signaling Technology Cat# 2118, RRID: AB_561053), cleaved caspase-3 (Cat# 9664, RRID: AB_2070042), cleaved caspase-8 (Cat# 9496, RRID:AB_561381), cleaved caspase-9 (Cat# 7237, RRID: AB_10895832), pro-caspase-3 (Cat# 9662, RRID: AB_331439), pro-caspase-8 (Cat# 4790, RRID: AB_10545768), pro-caspase-9 (Cat# 9502, RRID:AB_2068621), ERK1/2 (Cat# 9102, RRID: AB_330744), Akt (Cat# 9272, RRID: AB_329827), lamin A/C (Cat# 2032, RRID: AB_2136278), HIF-1α (Cat# 3716, RRID: AB_2116962), integrin β1 (Cat# 4706, RRID: AB_823544), MMP-7 (Cat# 71031, RRID: AB_2799796), MMP-9 (Cat# 3852, RRID: AB_2144868), Smac/DIABLO (Cat# 15108, RRID: AB_2798711), cyclin A2 (Cat# 67955, RRID: AB_2909603), CDK4 (Cat# 12790, RRID: AB_2631166), and CDK2 (Cat# 2546, RRID: AB_2276129), all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Expressing

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