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: Cell Host & Microbe

Article Title: β1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections

doi: 10.1016/j.chom.2017.05.001

Figure Lengend Snippet:

Article Snippet: The samples were stained with a rat anti-mouse β1-integrin (1:100 dilution, clone MB1.2, Merck Millipore), anti-acid ceramidase (1:100 dilution), anti-ceramide (1:100 dilution), anti-sphingosine (1:1000 dilution), anti-mouse β2-integrin antibodies (1:100, clone M1812, 1:100, #557437, BD) or FITC-Annexin (1:200, #11 828 681 001, Roche) in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl 2 , 0.7 mM MgCl 2 , 0.8 mM MgSO 4 ) plus 1% FCS at room temperature for 45 min.

Techniques: Virus, Isolation, Recombinant, Software

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Domain architecture of an active (open headpiece/extended) integrin α5β1 heterodimer. α5 Subunit is grey; β1 subunit headpiece and leg are, respectively, in shades of blue and green. The localization of three different mAb epitopes, exposed only in the conformationally active β1 subunit, is represented. Epitopes of mAb 12G10 and mAb HUTS4, respectively, lie in the βI domain and hybrid domain of the headpiece, whereas mAb 9EG7 epitope is in the I-EGF2 domain. (B) Confocal immunofluorescence microscopy analysis of the subcellular localization of the three different anti-active β1 integrin mAbs employed to stain fixed ECs. All three mAbs bind to active β1 integrins mainly located within typical elongated fibrillar adhesions. Scale bar 20 µ m; magnification scale bar 10 µ m. (C) Confocal immunofluorescence microscopy analysis of anti-active β1 integrin mAbs localization after 10 min of incubation on living ECs. Anti-I-EGF2 domain mAb 9EG7 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, whereas anti-βI domain mAb 12G10 recognizes active β1 integrins located both outside and inside highly fragmented and tiny adhesions. Similar to mAb 9EG7, the anti-hybrid domain mAb HUTS4 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, hinting that anti-active headpiece mAb-elicited fragmentation specifically depends on βI domain binding. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Selected frames from (top row) and (bottom rows), respectively, illustrating dynamic mAb 9EG7-Alexa Fluor 488 binding to active β1 integrins over time upon live incubation on ECs either in the absence (top row) or in the presence (bottom rows) of mAb 12G10–Alexa Fluor 647. When incubated alone (top row), mAb 9EG7–Alexa Fluor 488 preferentially binds active β1 integrins located within fibrillar adhesions and remains stable over time. When mAb 12G10–Alexa Fluor 647 is pre-incubated on ECs, mAb 9EG7–Alexa Fluor 488 does no longer localize in fibrillar adhesions. Scale bar 20 µ m. (E) Representative g -STED confocal microscopy pictures of anti-active β1 integrin 9EG7 mAb localization after 10-min incubation on living ECs either in the absence (top left panel) or the presence (middle left panel) of 12G10 or TS2/16 (bottom left panel). To thoroughly analyze the morphology of ECM adhesion sites, g -STED confocal images were acquired close to the basal EC surface. 9EG7-labeled adhesions were then analyzed with ImageJ software (right panels) and classified, according to their shape factor (SF), into elongated (red) and round (yellow) structures. 9EG7-labeled adhesions were classified as elongated, if their SF was < 0.5, and round, if the SF was ≥ 0.5. Scale bar 20 µ m. The maximum Feret’s diameter was measured to quantify the morphological features of 9EG7 + elongated structures. Compared with control ECs incubated live with 9EG7 alone, 9EG7 + elongated structures were significantly shortened in the presence of 12G10 or TS2/16. Data are mean ± SD, n ≥ 20 cells per condition pooled from two independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy, Staining, Incubation, Binding Assay, Confocal Microscopy, Labeling, Software, Control

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Low magnification confocal immunofluorescence microscopy analysis of ECs that were treated live for 10 min with either mAb 9EG7 mAb ( green ) alone or in combination with mAb 12G10 ( red ). Scale bar 100 µ m.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Representative confocal microscopy analysis of SNAKA51 + active α5 integrin (A), β3 integrin (B), and soluble rhodamine-FN (C) localization in ECs that were incubated (bottom panels) or not (top panels) for 10 min with the anti-βI domain mAb 12G10. Scale bar 20 µ m; magnification scale bar 10 µ m. (A, C) When compared with untreated control (CTL) ECs, the mFD of SNAKA51 + fibrillar adhesions or rhodamine-FN + fibrils was significantly reduced in ECs treated with mAb 12G10. Data are mean ± S.D, n ≥ 34 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (B) The incubation of cultured ECs with mAb 12G10 did not influence number, mean area, or mFD of β3 integrin + focal adhesions. Data are mean ± S.D, n ≥ 32 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control, Cell Culture

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Representative g -STED confocal microscopy analysis of tensin 1 localization in ECs that were incubated or not for 10 min with the anti-active β1 integrin mAb 9EG7 or mAb 12G10 or the Fab fragment of mAb 12G10 (12G10-Fab) or mAb TS2/16. Scale bar 20 µ m; magnification scale bar 10 µ m. When compared with untreated control (CTL) ECs or those treated with mAb 9EG7, the mFD of tensin 1 + fibrillar adhesions was significantly reduced in ECs treated with either mAb 12G10, 12G10-Fab or mAb TS2/16. Data are mean ± SD, n ≥ 22 cells per condition pooled from three independent experiments. The number of structures was normalized on cell area and on those in control cells. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (B) MAb 9EG7 affects the lifetime of FN–α5β1 bonds. AFM measurement of mAb 9EG7 effect on force-dependent lifetime of single bonds between a FNIII 7–10 fragment and an integrin α5β1-Fc fusion protein. Lifetime versus force plots of α5β1-Fc–functionalized Petri dish dissociating from FNIII 7–10 -coated cantilever tips in Mn 2+ either in the absence (grey) or the presence (green) of 10 µ g/ml mAb 9EG7 mAb. Data are mean ± SEM of several tens to several hundreds of measurements per point. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Selected frames from (A), (B), and (C), monitoring mAb 12G10 ( red ) binding to active β1 integrins on living: control ECs (A); ECs previously oligofected with tensin-EGFP (B); ECs pre-incubated for 10 min with mAb 9EG7 (C). (A, B, C) As expected, mAb 12G10 mAb binds active β1 integrins located within and outside fragmented adhesion sites (A), but, either when Tensin-EGFP is overexpressed (B) or upon pre-incubation with mAb 9EG7 (C), mAb 12G10 mAb localizes instead within fibrillar adhesions that remain stable over time. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Representative confocal images showing pre-bleaching, post-bleaching, and recovery on the region of interest (indicated by arrows) of tensin-EGFP–positive fibrillar adhesions in ECs treated with DMSO (as control) or with the FAK inhibitor PF-562271. Scale bar 5 µ m. Recovery rate was measured, and data were normalized by employing as reference the fluorescence intensity acquired on the same ROI before bleaching. Data were then normalized on control DMSO-treated samples. Data are mean ± SD, n ≥ 31 adhesions per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.001 ***. The effectiveness of inhibition of FAK autophosphorylation by PF-562271 was verified by Western blot analysis of EC lysates. (E) Representative confocal microscopy images of anti-active β1 mAb 12G10 ( green ) localization in ECs treated or not with the FAK inhibitor PF-562271; ECs were also stained for auto-phosphorylated FAK on tyrosine 397 (pFAK-Y397, red ). Scale bar 20 µ m; magnification scale bar 10 µ m. Measurement of mFD of 12G10 + adhesions revealed that, compared with control EC incubated with DMSO, 12G10 + adhesive structures are significantly longer in presence of FAK inhibitor PF-562271. Data are mean ± SD, n ≥ 19 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (F) Western blot analysis of FAK autophosphorylation (Y397) levels in ECs treated with or without mAb 9EG7 or mAb 12G10 for 2 or 15 min. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Binding Assay, Control, Incubation, Fluorescence, Inhibition, Western Blot, Confocal Microscopy, Staining, Adhesive

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Representative confocal microscopy analysis of the impact of mAb 12G10 on living ECs previously incubated for 10 min either with intact mAb 9EG7–Alexa Fluor 488 (9EG7) or its Fab (9EG7-Fab). Scale bar 10 µ m; magnification scale bar 10 µ m. Upon pre-incubation with intact dimeric 9EG7, but not monomeric 9EG7-Fab, mAb 12G10 localizes within fibrillar adhesions. Quantitative analysis showed that the incubation of cultured ECs with 12G10 shortens the mFD of elongated active β1 integrin + clusters only when cells are pre-incubated with monomeric 9EG7-Fab, but not intact dimeric 9EG7. Data are mean ± S.D, n ≥ 22 cells per condition pooled from three independent experiments. Statistical analysis: one-way ANOVA, P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Cell Culture

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Confocal immunofluorescence microscopy analysis of ECs live treated with mAb 9EG7 ( green ) alone or in combination with mAb 12G10 ( blue ). The internalization of mAb 9EG7–bound active β1 integrins in EEA1 + early endosomes ( red ) was quantified by Pearson correlation coefficient (PCC). Treatment with the anti-βI domain mAb 12G10 promotes mAb 9EG7–bound active β1 integrin endocytosis. Data are mean ± SD, n ≥ 23 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. (B) Time-course analysis of the relative amounts of endocytosed 9EG7 + active β1 integrins in control (CTL; green ) versus mAb 12G10–treated ( light blue ) ECs, evaluated by internalization and capture ELISA assays. Treating living ECs with the anti-βI domain mAb 12G10 elicits a strong increase in 9EG7 + active β1 integrin endocytosis. Data are mean ± SD, of eight technical replicates per condition pooled from three independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (C, D) Real-time analysis of EC haptotactic migration towards FN (xCELLigence RTCA DP system) either in the absence (CTL) or the presence of anti-active β1 integrin mAb 9EG7 alone (C, D) or mAb12G10 alone (C) or combined mAb 9EG7 and mAb 12G10 (D). Data are mean ± SD, n ≥ 14 technical replicates per condition pooled from four independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.05 * , # ; P ≤ 0.01 ** , ## ; P ≤ 0.001 *** , ### ; P ≤ 0.0001 **** , #### . (C, D) *CTL versus mAb 9EG7; # CTL versus mAb 12G10 (C); or CTL versus mAb 9EG7 + mAb 12G10 (D). Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy, Control, Enzyme-linked Immunosorbent Assay, Migration

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Confocal immunofluorescence microscopy analysis of ECs live treated with mAb SNAKA51 ( red ) alone or in combination with mAb 12G10 ( green ). The internalization of mAb SNAKA51–bound active α5 integrins in EEA1 + early endosomes ( blue ) was quantified by Pearson correlation coefficient. Treatment with the anti-βI domain mAb 12G10 promotes mAb SNAKA51–bound active α5 integrin endocytosis. Data are mean ± SD, n ≥ 26 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy