Figure 2. Class I PI3K catalytic subunit p110α is an essential regulator of invadopodia formation. (A) Real-time quantitative PCR analysis of the expression of PI3K isoforms in MDA-MB-231 cells. The relative mRNA levels of PI3K isoforms normalized with the mRNA levels of cyclophilin B are shown. (B and C) MDA-MB-231 cells were transfected with siRNAs targeting individual PI3K isoforms for 48 h, and the expression profiles of PI3K isoforms were determined by RT-PCR (B) and immunoblot analyses (C). Cyclophilin B (Cycl) and β-actin were used as internal controls. (D) MDA-MB-231 cells transfected with the indicated siRNAs were cultured on fluorescent gelatin-coated coverslips for 7 h, and the degraded areas on the gelatin matrix were quantified. (E) Representative images of cells transfected with siRNAs targeting p110 isoforms and stained for F-actin. Arrowheads denote the gelatin degradation sites. (F and G) The percentage of cells with invadopodia (F) and the relative number of invadopodia per cell (G) were determined in cells transfected with control or p110α siRNA. (H) MDA-MB-231 cells plated onto fluorescent gelatin-coated coverslips for 4 h were stained with anti-p110α antibody and phalloidin. Insets are magnified images of the boxed regions. Arrowheads denote accumulation of p110α signals at invadopodia. (I) MDA-MB-231 cells transfected with control or p110α siRNA were labeled with CellTracker green and analyzed for invasion through Matrigel-coated Transwell inserts for 24 h. Invaded cells were then imaged by fluorescent microscopy and counted. Arrowheads denote invaded cells. Smaller dots represent pores of the membrane of Transwell inserts. (J) MDA-MB-231 cells transfected with the indicated siRNAs were serum-starved overnight and stimulated with 8 nM EGF for 10 min. The cells were then analyzed by immunoblotting to determine the phosphorylation status of Akt (p-Akt) and ERK (p-ERK). Data are represented as means + SEM of three (A and I), eight (D), and five (F and G) independent determinations. *, P < 0.01; and **, P < 0.0002 by Student’s t test.
Figure 2.

Class I PI3K catalytic subunit p110α is an essential regulator of invadopodia formation. (A) Real-time quantitative PCR analysis of the expression of PI3K isoforms in MDA-MB-231 cells. The relative mRNA levels of PI3K isoforms normalized with the mRNA levels of cyclophilin B are shown. (B and C) MDA-MB-231 cells were transfected with siRNAs targeting individual PI3K isoforms for 48 h, and the expression profiles of PI3K isoforms were determined by RT-PCR (B) and immunoblot analyses (C). Cyclophilin B (Cycl) and β-actin were used as internal controls. (D) MDA-MB-231 cells transfected with the indicated siRNAs were cultured on fluorescent gelatin-coated coverslips for 7 h, and the degraded areas on the gelatin matrix were quantified. (E) Representative images of cells transfected with siRNAs targeting p110 isoforms and stained for F-actin. Arrowheads denote the gelatin degradation sites. (F and G) The percentage of cells with invadopodia (F) and the relative number of invadopodia per cell (G) were determined in cells transfected with control or p110α siRNA. (H) MDA-MB-231 cells plated onto fluorescent gelatin-coated coverslips for 4 h were stained with anti-p110α antibody and phalloidin. Insets are magnified images of the boxed regions. Arrowheads denote accumulation of p110α signals at invadopodia. (I) MDA-MB-231 cells transfected with control or p110α siRNA were labeled with CellTracker green and analyzed for invasion through Matrigel-coated Transwell inserts for 24 h. Invaded cells were then imaged by fluorescent microscopy and counted. Arrowheads denote invaded cells. Smaller dots represent pores of the membrane of Transwell inserts. (J) MDA-MB-231 cells transfected with the indicated siRNAs were serum-starved overnight and stimulated with 8 nM EGF for 10 min. The cells were then analyzed by immunoblotting to determine the phosphorylation status of Akt (p-Akt) and ERK (p-ERK). Data are represented as means + SEM of three (A and I), eight (D), and five (F and G) independent determinations. *, P < 0.01; and **, P < 0.0002 by Student’s t test.

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