Figure 2.

Septin2 expression is required for podosome-mediated matrix degradation and invasion in vitro. (A–D) Effect of Septin2 downregulation on the ability of ECs to form functional podosomes in response to Src activation. HPAECs were treated with 35 nM siRNA targeting Septin2 (SEPT2) or NT scramble control for 48 h before being seeded onto Alexa488-gelatin and transduced with adenoviral constructs expressing RapR-Src and FRB. After an additional 24 h, RapR-Src was activated for 30 min, 1 h, and 2 h with rapamycin (500 nm), and cells were fixed and stained for F-actin. (A) Representative epifluorescence images of HPAECs 30 min following Src activation. White arrows indicate functional podosomes defined as in Fig.1 A (scale bar = 50 µm). (B) Percentage of HPAECs displaying the matrix-degrading podosome phenotype before (0 h) and after activation of Src (time points are indicated). For each condition, a minimum of 10 fields of view were analyzed per experiment. Data show results from three independent experiments. KD, knockdown. (C) Western blot analysis displays downregulation of endogenous Septin2 with respect to GAPDH loading control used in A. (D) Average relative Septin2 expression normalized to scramble control (n = 3). Error bars represent standard deviation. (E) Role of Septin2 in VEGF-stimulated matrix degradation. Septin2 expression was downregulated as in A. Cells were deprived of VEGF for 48 h, serum starved in for 2 h, and then seeded onto Alexa405-gelatin. Total matrix degradation was measured after 5 h of VEGF stimulation. At least 10 fields of view per condition were analyzed from three independent experiments. Individual data points are shown around mean value ± SE (whiskers). (F and G) Septin2 localizes to invading sprouts in vitro. (F) HPAECs were seeded atop a 3D collagen matrix and stimulated using growth factor/S1P mix for 20 h. Following stimulation, cells were fixed and stained for endogenous Septin2 and MT1-MMP (confocal images, scale bar = 10 µm). (G) Representative 3D surface rendering of an invading sprout shown in F created with Imaris (indicated by white box; DAPI-stained nuclei represented in blue). (H and I) Effect of Septin2 downregulation on cell invasion. Septin2 expression was downregulated as in A and invasion was stimulated as in F. Following stimulation, cells were fixed and stained with toluidine blue. (H) Representative images showing cell invasion (lateral view of invading sprouts; scale bar = 0.15 mm). (I) Quantification of invading sprouts. Each data point represents an average of two to four fields per well imaged over three independent experiments. Data are represented as mean ± SE. Significance was determined using a two-sample t test (comparisons indicated by lines; B, E, and I).