Actin barbed-end formation at the invadopodia is regulated by pH. (A) Representative images of a barbed-end assay in WT cortactin cells in different pH conditions. A preserved barbed end increase in response to EGF was observed in these conditions. Insets show enlarged views of the boxed regions. (B) Quantification of barbed ends in response to EGF. Results are normalized to the Starved control barbed intensity (>150 invadopodia/group, n = 3; ***, P < 0.0001). (C) Quantification of barbed ends in response to EGF in NHE1KD cells. Results are normalized to the Starved control barbed intensity (> 180 invadopodia/group, n = 3; ***, P < 0.0001). Error bars indicate SEM.