Fig 1: (A) Exons differentially spliced between epithelial-like and mesenchymal-like cells (Mes-Epi exons) code for protein segments that are enriched in “NLS” term when compared to constitutive (CE) or alternative (ASE) exons. (*) P-value < 0.05. (B) RT-PCR performed with total RNAs obtained from four normal epithelial (1 = HEPic, 2 = HPAEPic, 3 = HMEC, 4 = AG01134) and four normal mesenchymal (5 = HMF, 6 = HCFaa, 7 = AG0449, 8 = AG0450) cell lines and from MCF-7 and MDA-MB-231 breast cancer cell lines. The selected genes correspond to genes bearing alternative exons coding for protein segments containing nuclear localization signal (NLS). Red and green rectangles correspond to alternative exons with higher and lower inclusion rate, respectively, in mesenchymal-like cells compared to epithelial-like cells. (C) SLK exon 15 that encodes for a NLS is more often included in MCF-7 than in MDA-MB-231 cells (see panel B). Immunofluorescence of SLK protein indicates that SLK is more restricted to the nucleus in MCF-7 (epithelial-like) than in MDA-MB-231 (mesenchymal-like) cells. (D) Depletion of SLK transcripts that contain exon 15 (TOSS E15 + siRNA E15) leads to a more diffuse SLK staining within transfected MCF-7 cells compared to control (CTRL) cells.
Fig 2: Role of ERM proteins and LOK/SLK kinases in shape regulation during cell detachment and in suspension(A) Time lapse of HeLa cells treated with Accutase over 15 min at RT following treatment with ERM inhibitor NSC668394 (10 μM overnight or 250 μM for 3 h). Orange – F-actin. Scale bar = 20 μm.(B–D) The shape changes of Control (n = 50) and NSC668394-treated (n = 50) HeLa cells following 20 min of Accutase treatment. Statistical difference, Kruskal-Wallis multiple comparison test, ∗p<0.05, ∗∗∗∗p<0.001.(B) Cell height (μm).(C) Cell surface area (μm2).(D) Aspect ratio for this experiment.(E) Immunofluorescence images of fixed HeLa cells treated with siRNA against GAPDH (Control) or combined LOK/SLK at 4 min after the Accutase treatment. Magenta – pERM, Gray - Tubulin, Blue – Nucleus. Scale bar = 20 μm.(F–H) Cell shape properties of HeLa cells treated with siRNA against GAPDH (as control) or combined LOK/SLK at 4 min after the Accutase treatment. Statistical difference, Mann-Whitney test, ∗∗∗∗p<0.001. F) Nucleus height (μm; GAPDH n = 147, LOK/SLK = 74), G) Cell surface area (μm2; GAPDH n = 100, LOK/SLK = 59), H) Aspect ratio (GAPDH n = 100, LOK/SLK = 59) for this experiment.(I) Western Blot showing Lok and Slk protein expression in HeLa cells immediately after detachment (0 min) and in suspension (15 min) following siRNA treatment against GAPDH (control), or following combined LOK and SLK RNAi.(J) Representative immunofluorescence images of GAPDH (Control) or combined LOK/SLK RNAi HeLa cells in suspension (15 min). Magenta – pERM, Gray - Tubulin, Blue – Nucleus. Scale bar = 5 μm.(K–M) Cell shape properties and pERM expression in HeLa cells previously treated with siRNAs against GAPDH (Control, n = 32) or both LOK/SLK (n = 24) after 15 min in suspension. Statistical difference, Mann-Whitney test, ∗∗p<0.01, ∗∗∗∗p<0.001. K) Cross-sectional area (μm2).(L) Ratio of the averaged cortical and cytoplasmic expression of pERM, M) Averaged intensity of cortical pERM for this experiment.
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