Figure 3. Effects of pharmacological inhibition of class I PI3Ks on invadopodia formation. (A) MDA-MB-231 cells were cultured on fluorescent gelatin-coated coverslips for 7 h in the presence or absence of various class I PI3K inhibitors, including PIK-75 for p110α, TGX-221 for p110β, and IC87114 for p110δ. The degraded areas on the gelatin matrix were quantified and are represented as the percentage of control DMSO-treated cells. (B) Dose–response curve of gelatin degradation obtained in the presence of increasing concentrations of PIK-75 is shown. (C) Representative images of MDA-MB-231 cells treated with various class I PI3K inhibitors are shown. Arrowheads denote the gelatin degradation sites. (D and E) The percentage of cells with invadopodia (D) and the relative number of invadopodia per cell (E) were determined in cells treated with control DMSO or 100 nM PIK-75. (F) MDA-MB-231 cells labeled with CellTracker green were analyzed for invasion through Matrigel-coated Transwell inserts in the presence or absence of 100 nM PIK-75 for 24 h. Invaded cells were then imaged by fluorescent microscopy and counted. Arrowheads denote invaded cells. (G) MDA-MB-231 cells were serum-starved overnight and treated with 300 nM of the indicated class I PI3K inhibitors for 1 h. The cells were subsequently stimulated with 8 nM EGF for 10 min and used for immunoblotting to determine the phosphorylation status of Akt (p-Akt) and ERK (p-ERK). Data are represented as means ± SEM of six (A, D, and E) and three (B and F) independent determinations. *, P < 0.01; and **, P < 0.0005 by Student’s t tests.
Figure 3.

Effects of pharmacological inhibition of class I PI3Ks on invadopodia formation. (A) MDA-MB-231 cells were cultured on fluorescent gelatin-coated coverslips for 7 h in the presence or absence of various class I PI3K inhibitors, including PIK-75 for p110α, TGX-221 for p110β, and IC87114 for p110δ. The degraded areas on the gelatin matrix were quantified and are represented as the percentage of control DMSO-treated cells. (B) Dose–response curve of gelatin degradation obtained in the presence of increasing concentrations of PIK-75 is shown. (C) Representative images of MDA-MB-231 cells treated with various class I PI3K inhibitors are shown. Arrowheads denote the gelatin degradation sites. (D and E) The percentage of cells with invadopodia (D) and the relative number of invadopodia per cell (E) were determined in cells treated with control DMSO or 100 nM PIK-75. (F) MDA-MB-231 cells labeled with CellTracker green were analyzed for invasion through Matrigel-coated Transwell inserts in the presence or absence of 100 nM PIK-75 for 24 h. Invaded cells were then imaged by fluorescent microscopy and counted. Arrowheads denote invaded cells. (G) MDA-MB-231 cells were serum-starved overnight and treated with 300 nM of the indicated class I PI3K inhibitors for 1 h. The cells were subsequently stimulated with 8 nM EGF for 10 min and used for immunoblotting to determine the phosphorylation status of Akt (p-Akt) and ERK (p-ERK). Data are represented as means ± SEM of six (A, D, and E) and three (B and F) independent determinations. *, P < 0.01; and **, P < 0.0005 by Student’s t tests.

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