Figure 8.
Figure 8. Genetic and functional interference with αVβ3, but not α5β1, integrin inhibits RAB5A- and HGF-induced invadosome formation and matrix degradation. (A) Confocal analysis of RAB5A-HeLa cells stained with phalloidin (red) or the indicated Ab (green). (B) Still images from TIRF microscopy time-lapse of HeLa cells transfected with RAB5, GFP-β3, and RFP-LifeAct (see also Video 8). (C) HeLa cells cotransfected with RAB5A and MT1-MMP were subjected to PLA (red dots) with abs against MT1-MMP and integrin β3 or stained with phalloidin (green). Negative controls (Ctr) were cells stained with oligonucleotide-labeled PLA probes alone. Confocal images in A, B, and C are representative of >100 analyzed cells. (D) HeLa cells were transfected with scrambled (Ctr), anti-β1, or anti-β3 siRNA, or incubated with the inhibitory antibodies, 4B4 and LM609, against β1 and αVβ3, respectively. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) for 3 h, or left in serum free conditions (SF), and stained with phalloidin (green). Insets are magnifications of boxed regions. Quantification of gelatin degradation was expressed as a degradation index (relative to the area of degradation of control, HGF-stimulated cells normalized for cell number). Data are the mean ± SEM (error bars; n = 60 cells/experiment in five independent experiments). Two independent siRNAs were used for each integrin with similar results. Silencing of β1 and β3 genes was verified by QRT-PCR (right). **, P < 0.005. Arrows in A–D indicate examples of invadosomes. (E) MDA-MB-231 and MCF10.DCIS.com cells were transfected with scrambled (Ctr) or anti-β3 siRNA. Serum-starved cells were then plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) for 3 h, or left in serum free conditions (SF), and stained with phalloidin (green). Gelatin degradation is expressed as a degradation index. Data are the mean ± SEM (error bars; n = 40 cells/experiment in three independent ones). Two independent siRNAs were used for each integrin with similar results. Silencing β3 genes was verified by QRT-PCR (right). **, P < 0.005. Bars: (A) 15 µm; (B–E) 20 µm.

Genetic and functional interference with αVβ3, but not α5β1, integrin inhibits RAB5A- and HGF-induced invadosome formation and matrix degradation. (A) Confocal analysis of RAB5A-HeLa cells stained with phalloidin (red) or the indicated Ab (green). (B) Still images from TIRF microscopy time-lapse of HeLa cells transfected with RAB5, GFP-β3, and RFP-LifeAct (see also Video 8). (C) HeLa cells cotransfected with RAB5A and MT1-MMP were subjected to PLA (red dots) with abs against MT1-MMP and integrin β3 or stained with phalloidin (green). Negative controls (Ctr) were cells stained with oligonucleotide-labeled PLA probes alone. Confocal images in A, B, and C are representative of >100 analyzed cells. (D) HeLa cells were transfected with scrambled (Ctr), anti-β1, or anti-β3 siRNA, or incubated with the inhibitory antibodies, 4B4 and LM609, against β1 and αVβ3, respectively. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) for 3 h, or left in serum free conditions (SF), and stained with phalloidin (green). Insets are magnifications of boxed regions. Quantification of gelatin degradation was expressed as a degradation index (relative to the area of degradation of control, HGF-stimulated cells normalized for cell number). Data are the mean ± SEM (error bars; n = 60 cells/experiment in five independent experiments). Two independent siRNAs were used for each integrin with similar results. Silencing of β1 and β3 genes was verified by QRT-PCR (right). **, P < 0.005. Arrows in A–D indicate examples of invadosomes. (E) MDA-MB-231 and MCF10.DCIS.com cells were transfected with scrambled (Ctr) or anti-β3 siRNA. Serum-starved cells were then plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) for 3 h, or left in serum free conditions (SF), and stained with phalloidin (green). Gelatin degradation is expressed as a degradation index. Data are the mean ± SEM (error bars; n = 40 cells/experiment in three independent ones). Two independent siRNAs were used for each integrin with similar results. Silencing β3 genes was verified by QRT-PCR (right). **, P < 0.005. Bars: (A) 15 µm; (B–E) 20 µm.

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