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: Figure 1. 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: Figure 3. 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 (Figure 4 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: Figure 6. 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: Aging (Albany NY)

Article Title: ANGPTL2 promotes VEGF-A synthesis in human lung cancer and facilitates lymphangiogenesis

doi: 10.18632/aging.204581

Figure Lengend Snippet: ANGPTL2 increases VEGF-A synthesis in lung cancer cells via the integrin α5β1 receptor, p38 and NF-κB signaling. ( A – C ) Cells were transfected with ANGPTL2 cDNA, then stimulated with LILRB2 and integrin α5β1 siRNA or SB203580 and PDTC; VEGF-A expression was measured by Western blot (n=3). Quantitative data of the protein level were obtained using ImageJ software. Densitometric analysis of protein expression was normalized to β-actin. ( D , E ) Cells were transfected with ANGPTL2 cDNA, then stimulated with integrin α5β1 siRNA and SB203580; p38 and NF-κB activation was measured by Western blot (n=3) and NF-κB luciferase activity. Quantitative data of the protein level were obtained using ImageJ software. Densitometric analysis of protein expression was normalized to β-actin. * p < 0.05 compared with CL1-0; # p < 0.05 compared with Control.

Article Snippet: Small interfering RNAs (siRNAs) against integrin α5 (sc-29372) and β1(sc-35674), and immunoglobulin (Ig)-like transcript 4 (ILT4) (sc-45200) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Western Blot, Software, Activation Assay, Luciferase, Activity Assay, Control

Journal: Aging (Albany NY)

Article Title: ANGPTL2 promotes VEGF-A synthesis in human lung cancer and facilitates lymphangiogenesis

doi: 10.18632/aging.204581

Figure Lengend Snippet: Schematic diagram summarizes the mechanisms by which ANGPTL2 facilitates lymphangiogenesis in human lung cancer cells. ANGPTL2 increases VEGF-A production and subsequently facilitates LEC lymphangiogenesis in human lung cancer cells via the integrin α5β1 receptor, p38 and NF-κB signaling.

Article Snippet: Small interfering RNAs (siRNAs) against integrin α5 (sc-29372) and β1(sc-35674), and immunoglobulin (Ig)-like transcript 4 (ILT4) (sc-45200) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: PLoS Pathogens

Article Title: Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells

doi: 10.1371/journal.ppat.1010681

Figure Lengend Snippet: (A) Western blot showing that endothelial cell gC1qR binds to C . glabrata coated with fresh serum. Results are representative of 3 independent experiments. (B-E) Effects of inhibiting gC1qR function with siRNA knockdown (B and C) and specific monoclonal antibodies (D and E) on the endocytosis (B and D) and cell-association (C and E) of C . glabrata coated with fresh serum. Antibody 74.5.2 recognizes the high molecular weight kininogen binding site in the C-terminus of the gC1qR and antibody 60.11 is directed against the C1q binding site in the N-terminus of the gC1qR. Results shown in (B-E) are the mean ± SD of 3 experiments each performed in triplicate. Heat inact., heat inactivated serum; orgs/HPF, organisms per high-power field; ns, not significant, * P < 0.05, ** P < 0.01 by Student’s t-test (B and C) or ANOVA with the Dunnett’s test for multiple comparisons (D and E).

Article Snippet: The endothelial cells were transfected with gC1qR siRNA (Santa Cruz Biotechnology, Cat. # sc-42880), integrin α5 siRNA (Santa Cruz Biotechnology, Cat. # sc-29372), integrin αv siRNA (Santa Cruz Biotechnology, Cat. # sc-29373), or scrambled control siRNA (Qiagen, Cat. # 1027281) using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions.

Techniques: Western Blot, Knockdown, Bioprocessing, High Molecular Weight, Binding Assay

Journal: PLoS Pathogens

Article Title: Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells

doi: 10.1371/journal.ppat.1010681

Figure Lengend Snippet: (A-F) Effects of inhibiting αv integrin function with siRNA knockdown (A and B or)specific monoclonal antibodies (C-F) and on the endocytosis (A, C, D) and cell-association (B, E, F) of serum-coated C . glabrata . (G and H) Inhibition of gC1qR (with monoclonal antibody 74.5.2) and αv integrins has an additive effect on decreasing the endocytosis (G) but not cell-association of serum-coated C . glabrata (H). Results are the mean ± SD of 3 experiments, each performed in triplicate. Orgs/HPF, organisms per high power field; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by ANOVA with the Dunnett’s test for multiple comparisons (A, B, G, H) or the Student’s t-test (C-F).

Article Snippet: The endothelial cells were transfected with gC1qR siRNA (Santa Cruz Biotechnology, Cat. # sc-42880), integrin α5 siRNA (Santa Cruz Biotechnology, Cat. # sc-29372), integrin αv siRNA (Santa Cruz Biotechnology, Cat. # sc-29373), or scrambled control siRNA (Qiagen, Cat. # 1027281) using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions.

Techniques: Knockdown, Bioprocessing, Inhibition

Journal: PLoS Pathogens

Article Title: Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells

doi: 10.1371/journal.ppat.1010681

Figure Lengend Snippet: (A and B) Endocytosis of C . glabrata coated with either human or mouse serum by the indicated endothelial cells after 45 min (A) and 180 min (B). (C) Endocytosis of C . glabrata coated with fresh human serum by mouse liver endothelial cells expressing human gC1qR, integrin αv, or integrin β5. Data are the mean ± SD of 3 experiments each performed in triplicate. HUVEC, human umbilical vein endothelial cell; orgs/HPF, organisms per high power field; ns, not significant; ** P < 0.01, **** P < 0.0001. *** P < 0.001, **** P < 0.0001 by ANOVA with the Dunnett’s test for multiple comparisons.

Article Snippet: The endothelial cells were transfected with gC1qR siRNA (Santa Cruz Biotechnology, Cat. # sc-42880), integrin α5 siRNA (Santa Cruz Biotechnology, Cat. # sc-29372), integrin αv siRNA (Santa Cruz Biotechnology, Cat. # sc-29373), or scrambled control siRNA (Qiagen, Cat. # 1027281) using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions.

Techniques: Expressing