Journal: International Journal of Molecular Sciences

Article Title: Interaction of Serratia proteamaculans with Integrins Activates Invasion-Promoting Signaling Pathways

doi: 10.3390/ijms26093955

Figure Lengend Snippet: Involvement of integrins in S. proteamaculans invasion. ( A ) Comparison of the effect of inhibitors on the sensitivity of A549 and M-HeLa cells to bacteria. The sensitivity to bacteria of cells pre-incubated with inhibitor of EGFR (30 μM AG1478), c-Src kinase (5 μM Src I1), FAK (5 μM Y15) for 1 h, and ILK (2.5 μM cpd22) for 24 h was assessed quantitatively. Control—intensity of invasion into untreated cells. ( B ) Comparison of the effect of MAP kinase signaling pathway inhibitors on the sensitivity of M-HeLa cells to bacteria. The sensitivity to bacteria of cells pre-incubated with inhibitor of Raf/MEK/ERK (10 μM U0126), JNK (10 μM SP600125), p38 (5 μM SB203580) for 1 h was assessed quantitatively. Control—intensity of invasion into untreated cells. ( C ) Comparison of the effect of treating M-HeLa cells with siRNA targeting β1 and α5 integrins on cell sensitivity to S. proteamaculans . Pretreating cells with an siRNA reduced the β1 and α5 integrins expression by 46% and 25%, respectively. Control—M-HeLa cells transfected with siRNA containing scrambled nucleotide sequence. The insert shows the total amount of integrins and internal control GAPDH in untreated M-HeLa cells and pretreating cells with small interfering RNA targeting β1 and α5 integrins. The number of intracellular bacteria was estimated as a percentage, taking the number of intracellular bacteria in control samples as 100%. Values are expressed as mean S.D. (error bars) of a representative experiment. The difference in the control was considered significant at the * p < 0.05 level.

Article Snippet: The expression of host-cell proteins was inhibited using siRNA targeting α5 integrin (sc-29372) and β1 integrin (sc-35674) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Comparison, Bacteria, Incubation, Control, Expressing, Transfection, Sequencing, Small Interfering RNA

Journal: International Journal of Molecular Sciences

Article Title: Interaction of Serratia proteamaculans with Integrins Activates Invasion-Promoting Signaling Pathways

doi: 10.3390/ijms26093955

Figure Lengend Snippet: Effect of RGD peptide on S. proteamaculans invasion. ( A ) Effect of incubation with RGD peptide (5 μg/mL and 50 μg/mL) on receptor expression in M-HeLa cells. ( B ) Effect of pre-incubation of M-HeLa cells with RGD peptide (5 μg/mL and 50 μg/mL) for 30 min on the intensity of bacterial invasion. ( C ) Effect of pre-incubation of M-HeLa cells with 50 μg/mL RGD peptide for 30 min on the intensity of bacterial adhesion. ( D ) Effect of bacterial infection on amount of α5 and β1 integrin in the host cell. The mean fluorescence intensity of integrin normalized to the mean fluorescence intensity of DAPI estimated with ImageJ2 Fiji on 6–9 images obtained using confocal microscopy ( shows an example). Values are expressed as mean S.D. (error bars). The difference to the control was considered significant at the * p < 0.05. ( E , F ) Effect of incubation with 50 μg/mL RGD peptide, S. proteamaculans , or co-incubation with RGD peptide and bacteria on expression ( E ) and total amount ( F ) of α5 and β1 integrins in M-HeLa cells.

Article Snippet: The expression of host-cell proteins was inhibited using siRNA targeting α5 integrin (sc-29372) and β1 integrin (sc-35674) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Incubation, Expressing, Infection, Fluorescence, Confocal Microscopy, Control, Bacteria

Journal: International Journal of Molecular Sciences

Article Title: Interaction of Serratia proteamaculans with Integrins Activates Invasion-Promoting Signaling Pathways

doi: 10.3390/ijms26093955

Figure Lengend Snippet: Schemes of interaction between S. proteamaculans and the host cell. The OmpX surface protein of S. proteamaculans binds to α5β1 integrin, which is anchored in a lipid raft with EGFR. Integrin binding activates FAK and ILK kinases, which mediate actin and tubulin rearrangements, and Src kinase, which phosphorylates EGFR at Tyr845. This leads to autophosphorylation of EGFR at Tyr1086. Phosphorylation is a signal for the transport of EGFR to the cell nucleus and the initiation of ERK and JNK signaling cascades. In this way, bacteria induce an increase in the expression of α5, β1 integrin, and EGFR involved in S. proteamaculans invasion.

Article Snippet: The expression of host-cell proteins was inhibited using siRNA targeting α5 integrin (sc-29372) and β1 integrin (sc-35674) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Binding Assay, Phospho-proteomics, Bacteria, Expressing

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Diarrheagenic E. coli bind to integrin α 5 β 1. (a) EAEC, EHEC, and ETEC were added to integrin α 5 β 1- and BSA-coated wells and bacterial binding was detected by ELISA, using anti-O44, anti-O157, and anti-O78 serum, respectively. (b) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells. EAEC binding was detected by ELISA, using anti-O44 serum. *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Fibronectin/integrin α 5 β 1 complex increases the adhesion of EAEC strain 042. (a) Integrin α 5 β 1- and BSA-coated wells were incubated with increasing concentrations of fibronectin (25, 50, and 100 ng) or (b) with 100 ng of fibronectin (Fn) for 15, 30, or 60 min. Bound fibronectin was detected by ELISA, using anti-fibronectin antibodies. (c) EAEC 042 was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin. (d) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells incubated with fibronectin. EAEC binding was detected by ELISA using anti-O44 serum. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: AafA binding to fibronectin/integrin α 5 β 1 complex. (a) AafA- dsc protein was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin (Fn). AafA- dsc binding was detected by ELISA using anti-AafA antibodies. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05). (b) AafA- dsc and fibronectin or (c) AafA- dsc , fibronectin, and integrin α 5 β 1 were mixed and the complex formed was added to control column or a column with anti-AafA antibodies for coimmunoprecipitation analysis. The eluted fraction obtained from control column (E1 and E3) or a column with anti-AafA antibodies (E2 and E4) was analyzed by Dot blot, using anti-AafA, anti-integrin α 5, and anti-fibronectin antibodies. The resulting autoradiography was scanned and signal intensity was quantified by UN-SCAN-IT 6.1 software. One representative experiment of three independent experiments with similar result is shown.

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control, Dot Blot, Autoradiography, Software

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Adhesion of EAEC strain 042 to T84 cells. T84 cells preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn), Fn and anti-integrin α 5 β 1 (RGD), or anti-integrin α 5 β 1 ( α 5) were infected with EAEC strain 042. The number of adherent bacteria was determined by colony forming unit counts (CFU). *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Control, Infection, Bacteria

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Integrin α 5 expression knockdown reduces EAEC strain 042 fibronectin-mediated binding to epithelial cells. HEp-2 cells nontransfected and transfected with scrambled or integrin α 5 shRNA were preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn) and then infected with EAEC strain 042. Numbers of adherent bacteria were determined by colony forming unit counts (CFU). *Significantly different compared to control treatment ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Knockdown, Binding Assay, Transfection, shRNA, Control, Infection, Bacteria

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: The expression level of integrin α5 subunit in Ph+ leukemia cell line SUP-B15 is significantly increased after serum starvation . (A) The percentages of cells expressing integrin α4, α5, and β1 subunits before and after serum starvation for 7 h detected by flow cytometry. (B) The mean fluorescent intensity (MFI) of integrin α4, α5, and β1 subunits from whole alive cell population before and after serum starvation for 7 h detected by flow cytometry.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Integrin α5 subunit antibody inhibits the adhesion of Ph+ leukemia cells to human fibronectin and enhances the killing of imatinib . (A) Cell adhesion assay showed that α5 subunit inhibitory antibody (CD49e, clone IIA1 BD Biosciences) was the only tested integrin antibody that significantly inhibited the adhesion of Ph+ leukemia cells to fibronectin with adhesion percentage of 6.6 ± 3.8% compared with control IgG of 44.8 ± 7.9%, p < 0.01. (B) . Annexin-V plus PI by flow cytometry. (i) P2 gate identifies the CD38 positive cell population representing SUP-B15 Ph+ leukemia cells. (ii) Ph+ leukemia cells grown on HS-5 stromal cells. (iii) Ph+ leukemia cells treated with 10 μM imatinib (IM). (iv) SUP-B15 cultured with stromal cells treated with 10 μM imatinib. (v) SUP-B15 cultured with stromal cells treated with 10 μM imatinib and 10 μg/ml anti-α5 Ab. (C) Quantitative analysis for apoptosis rate of different conditions. *After culture on HS-5 cells for 24 h, the apoptosis rate of leukemia cells SUP-B15 decreased from 29.4 ± 2.3 to 16.7 ± 3%, p < 0.05. # When inhibitory antibody to integrin α5 was combined with imatinib to treat Ph+ leukemia cells cultured on stromal cells, the apoptosis rate 38.0 ± 8.0% was significantly increased compared with imatinib by itself 25.7 ± 3.3%, p < 0.05.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Cell Adhesion Assay, Control, Flow Cytometry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Blocking integrin α5 affects the engraftment of Ph+ leukemia cells in immunodeficient mice . (A) The incubation for 1 h of Ph+ leukemia cells with disintegrin, a peptide inhibitor of integrins, impaired the engraftment of leukemia in NSG mice. Representative figures of n = 2 showed bioluminescence imaging from day 7 to 28 after inoculation of leukemia cells. (B) Animal total body bioluminescence was measured using the Xenogen IVIS Imaging System 200 Series with total imaging time of 2 min and compared with control animals that received cells that were not treated with Echistatin. (C) Anti-integrin α5 inhibitory antibodies clone IIA1 (BD Biosciences) and clone P1D6 (Millipore) decreased the engraftment of Ph+ leukemia cells in the bone marrow of NOD/SCID mice ( n = 2).

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Blocking Assay, Incubation, Imaging, Control

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Knocking down integrin α5 delayed the engraftment of Ph+ leukemia cells in immunodeficient NOD/SCID mice . (A) Real-time QPCR assay showed that we had successfully knocked down the expression level of the integrin α5 knocking in clone 10. The cycle threshold (Ct) of the target gene (integrin α5) was normalized to the chosen reference gene GAPHD. Relative quantification, R = 2 −(ΔCt sample − ΔCt control) . Result shows the fold change normalized to SUP-LUC2 cells. (B) Western blot showed a reduced protein expression level of integrin α5 in clone 10. (C) Flow cytometry showed reduced mean fluorescent intensity of integrin α5 in clone 10. (D) Representative figures showed bioluminescent imaging at 2 weeks, and 5–8 weeks post leukemia cells inoculation. (E) Animal total body bioluminescence was quantified to compare with control. **The levels of bioluminescence became most significantly different 2 months post inoculation between the α5 knock-down group and control group [mean (SD) radiance vs. control, 5.3 (0.1) vs. 2.1 (0.2) × 10 6 p/s/cm 2 /sr, p < 0.01 at 2 months, n = 3.].

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Quantitative Proteomics, Control, Western Blot, Flow Cytometry, Imaging, Knockdown