MASTL inhibits cell spreading and attachment through kinase-independent functions. (A) Representative images of F-actin (Phalloidin-Atto) and DAPI staining in control (siControl) or MASTL-silenced (siMASTL; 48-h silencing) MDA-MB-231 cells plated on collagen for 2 h. Images were acquired on a Zeiss LSM 880 Airyscan confocal. (B) Quantification of cell area based on F-actin staining of 45 cells (three independent experiments) from A. (C) Real-time monitoring of siControl or siMASTL cell area (normalized cell index) during cell spreading measured with the xCELLigence RTCA system. Single-cell suspensions were plated on collagen. BSA coating was used as a negative control. Shown are representative curves from an individual experiment. (D) Representative F-actin (Phalloidin-Atto) and DAPI staining in EGFP-control and EGFP-MASTL WT–overexpressing (24 h) MDA-MB-231 cells plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (E) Quantification of cell area based on the F-actin staining of 45 cells (three independent experiments) from D. (F) Western blot analysis of MASTL levels in siControl, siMASTL, and siMASTL + EGFP-MASTL WT (24 h MASTL silencing + 24 h expression of siRNA-resistant EGFP-MASTL WT) MDA-MB-231 cells. Tubulin was used as a loading control. (G) Representative images of F-actin (Phalloidin-Atto) and DAPI staining in siControl, siMASTL, siMASTL + EGFP-MASTL WT, or siMASTL + EGFP-MASTL G44S MDA-MB-231 cells plated on collagen for 2 h (3i CSU-W1 spinning disk confocal). (H) Quantification of cell area based on the F-actin staining of 45 cells (three independent experiments) from G. (I) Representative cell spreading curves and cell area (normalized cell index; as in C) of siControl, siMASTL, or siMASTL + EGFP-MASTL WT MDA-MB-231 cells. Data in all graphs are from n = 3 biologically independent experiments (unpaired t test, mean ± SD). See also Fig. S1 and Table S1.
Figure 1.

MASTL inhibits cell spreading and attachment through kinase-independent functions. (A) Representative images of F-actin (Phalloidin-Atto) and DAPI staining in control (siControl) or MASTL-silenced (siMASTL; 48-h silencing) MDA-MB-231 cells plated on collagen for 2 h. Images were acquired on a Zeiss LSM 880 Airyscan confocal. (B) Quantification of cell area based on F-actin staining of 45 cells (three independent experiments) from A. (C) Real-time monitoring of siControl or siMASTL cell area (normalized cell index) during cell spreading measured with the xCELLigence RTCA system. Single-cell suspensions were plated on collagen. BSA coating was used as a negative control. Shown are representative curves from an individual experiment. (D) Representative F-actin (Phalloidin-Atto) and DAPI staining in EGFP-control and EGFP-MASTL WT–overexpressing (24 h) MDA-MB-231 cells plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (E) Quantification of cell area based on the F-actin staining of 45 cells (three independent experiments) from D. (F) Western blot analysis of MASTL levels in siControl, siMASTL, and siMASTL + EGFP-MASTL WT (24 h MASTL silencing + 24 h expression of siRNA-resistant EGFP-MASTL WT) MDA-MB-231 cells. Tubulin was used as a loading control. (G) Representative images of F-actin (Phalloidin-Atto) and DAPI staining in siControl, siMASTL, siMASTL + EGFP-MASTL WT, or siMASTL + EGFP-MASTL G44S MDA-MB-231 cells plated on collagen for 2 h (3i CSU-W1 spinning disk confocal). (H) Quantification of cell area based on the F-actin staining of 45 cells (three independent experiments) from G. (I) Representative cell spreading curves and cell area (normalized cell index; as in C) of siControl, siMASTL, or siMASTL + EGFP-MASTL WT MDA-MB-231 cells. Data in all graphs are from n = 3 biologically independent experiments (unpaired t test, mean ± SD). See also Fig. S1 and Table S1.

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