Silencing of MASTL inhibits cancer cell invasion and regulates cell morphology in 3D. (A) Representative images of F-actin (Phalloidin-Atto) staining in siControl and siMRTF-A–silenced (48 h) MCF10A cells plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (B) Quantification of cell area based on the F-actin staining of 45 cells (three independent experiments, unpaired t test). (C) Western blot analysis of MRTF-A, GEF-H1, vinculin, paxillin, and GAPDH protein levels in siControl and siMRTF-A–silenced (48 h) MCF10A cells. (D–G) Inverted invasion assessment of siControl, siMASTL, and siMRTF-A MDA-MB-231 cells. Schematic illustration of the invasion assay used and representative images for each condition after 96 h invasion and staining with phalloidin-488; n = 3 biological replicates, with nine stacks/condition/replicate, mean ± SD, unpaired t test with Welch’s correction (D), along with quantification (E). Representative high-magnification images (F) and quantification (G) of invading MDA-MB-231 cells assessed for their roundness (n = 3 biological replicates, with at least seven cells/condition/replicate, mean ± SD, unpaired t test with Welch’s correction;). (H and I) Representative high-magnification images of MRTF-A staining and Hoechst (H) and MRTF-A nuclear/cytoplasmic ratio quantification (I) of invading MDA-MB-231 cells. (J) Schematic illustrating how MASTL regulates cell morphology, migration, and transcription of actin regulators by controlling the nuclear localization of MRTF-A and SRF activity. Data in all graphs are from n = 3 biologically independent experiments (mean ± SD). See also Table S1.
Figure 10.

Silencing of MASTL inhibits cancer cell invasion and regulates cell morphology in 3D. (A) Representative images of F-actin (Phalloidin-Atto) staining in siControl and siMRTF-A–silenced (48 h) MCF10A cells plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (B) Quantification of cell area based on the F-actin staining of 45 cells (three independent experiments, unpaired t test). (C) Western blot analysis of MRTF-A, GEF-H1, vinculin, paxillin, and GAPDH protein levels in siControl and siMRTF-A–silenced (48 h) MCF10A cells. (D–G) Inverted invasion assessment of siControl, siMASTL, and siMRTF-A MDA-MB-231 cells. Schematic illustration of the invasion assay used and representative images for each condition after 96 h invasion and staining with phalloidin-488; n = 3 biological replicates, with nine stacks/condition/replicate, mean ± SD, unpaired t test with Welch’s correction (D), along with quantification (E). Representative high-magnification images (F) and quantification (G) of invading MDA-MB-231 cells assessed for their roundness (n = 3 biological replicates, with at least seven cells/condition/replicate, mean ± SD, unpaired t test with Welch’s correction;). (H and I) Representative high-magnification images of MRTF-A staining and Hoechst (H) and MRTF-A nuclear/cytoplasmic ratio quantification (I) of invading MDA-MB-231 cells. (J) Schematic illustrating how MASTL regulates cell morphology, migration, and transcription of actin regulators by controlling the nuclear localization of MRTF-A and SRF activity. Data in all graphs are from n = 3 biologically independent experiments (mean ± SD). See also Table S1.

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