![MASTL regulates cell spreading independently of focal adhesion size, integrin activity or cell cycle (related to Fig. 2).(A) Western blot analysis of MASTL, tubulin and β-actin in MCF10A, MCF7 and MDA-MB-231 cells. (B) Representative images of F-actin (Phalloidin-Atto) and DAPI staining in control (siControl) or MASTL-silenced (siMASTL; 48 h silencing) MCF7 cells plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (C) Quantification of cell area based on F-actin staining of 45 cells (3 independent experiments) from B. (D and E) Quantification of the flow cytometry of total β1-integrin (P5D2; D) or active β1-integrin (12G10; E) in siControl and siMASTL (48 h) cells (n = 3 biologically independent experiments, mean ± SD, unpaired t test, MDA-MB-231). (F and G) Level of active β1 integrin (12G10) relative to total β1 integrin (K20) after MASTL#6 silencing (F; n = 6 independent experiments, mean ± SD, t test, MDA-MB-231) or after MASTL#7 silencing (G; n = 3 independent experiments, mean ± SD, unpaired t test). (H) Western blot analysis of MASTL and GAPDH in siControl and siMASTL (24, 48, or 72 h) MDA-MB-231 cells at different time points. (I) Quantification of the flow cytometry data (Fig. 2 I). Proportion of MDA-MB-231 cells in the G2 phase of the cell cycle (Watson model) after silencing of MASTL for 24, 48, and 72 h (n = 3 independent experiments [1 × #6, 2 × #7], mean ± SD, unpaired t test). See also Table S1.](https://cdn.rupress.org/rup/content_public/journal/jcb/219/6/10.1083_jcb.201906204/5/jcb_201906204_figs2.png?Expires=1767705168&Signature=I5ZSM43GtDThiKabDCDQiRSdWBeVGq6Oeo~wAwtAMq9N4wEQbMIryGsK1XRBHZAOnpyL3D5uwfUSpBWCb4n6~fb13EZc5l1JvnXEYUPopq9ug4ypG6sKMNL4Iw2TmaIya3YZIfmyoDBwR~JqlBZysw2M-4BS2HT1I18q1SSpxnqw-bjdpTRmT-Uk4fCJ~JgvuQjDR~WRgukQsWsGo1LZzW1wIuL9xlT~u2SeujBXkgMMWPXVhouj-3f3yp90ael6mqaOS2CccJB-xxxF~gAtIJU~AFlFmt45WPE-zJVA49Lk9hO8C7U9MqH-61XGMmyvr-G1jIPgVJhO8YSVCGYh-w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MASTL regulates cell spreading independently of focal adhesion size, integrin activity or cell cycle (related to Fig. 2 ). (A) Western blot analysis of MASTL, tubulin and β-actin in MCF10A, MCF7 and MDA-MB-231 cells. (B) Representative images of F-actin (Phalloidin-Atto) and DAPI staining in control (siControl) or MASTL-silenced (siMASTL; 48 h silencing) MCF7 cells plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (C) Quantification of cell area based on F-actin staining of 45 cells (3 independent experiments) from B. (D and E) Quantification of the flow cytometry of total β1-integrin (P5D2; D) or active β1-integrin (12G10; E) in siControl and siMASTL (48 h) cells (n = 3 biologically independent experiments, mean ± SD, unpaired t test, MDA-MB-231). (F and G) Level of active β1 integrin (12G10) relative to total β1 integrin (K20) after MASTL#6 silencing (F; n = 6 independent experiments, mean ± SD, t test, MDA-MB-231) or after MASTL#7 silencing (G; n = 3 independent experiments, mean ± SD, unpaired t test). (H) Western blot analysis of MASTL and GAPDH in siControl and siMASTL (24, 48, or 72 h) MDA-MB-231 cells at different time points. (I) Quantification of the flow cytometry data (Fig. 2 I). Proportion of MDA-MB-231 cells in the G2 phase of the cell cycle (Watson model) after silencing of MASTL for 24, 48, and 72 h (n = 3 independent experiments [1 × #6, 2 × #7], mean ± SD, unpaired t test). See also Table S1.