Fig 2: Population effect of PA on EMT marker expression.(A) Density plots showing distribution of cells co-stained with zombie violet (Y-axis) and either CDH, DSP or VIM (X-axis) in HepG2 cells treated with BSA or PA (0.3 mM). The bars represent staining thresholds for zombie violet (horizontal bar) or the protein markers (vertical bar). Lower left quadrant represents background staining, upper left quadrant represents cells stained with only zombie violet, upper right quadrant represent cells co-stained with zombie violet and protein marker and lower right quadrant represent cells stained with protein marker only. The numbers in the plot represent the percentage of total cells within that quadrant. (B) Confocal images showing specific regions with altered expression levels of CDH and VIM in HepG2 and Hep3B cells. Blue indicates nuclear staining and green indicates CDH or VIM staining. Images were recorded at 40× with identical exposure and PMT settings maintained between BSA and PA treated cells for a given marker (scale bar = 100 µm). C Bar graph showing fold-change in mRNA expression levels of various EMT transcription factors determined by qRT-PCR in PA (0.3 mM) treated HepG2 cells compared to corresponding expression levels in BSA treated cells (GAPDH normalized). P-values indicate significance levels compared to BSA determined by Student’s t-test (two tailed), n = 3.

Fig 3: Vimentin Forms a Sub-cortical Layer in Mitosis and Controls Actin Cortex Organization and Mechanics(A) Representative time lapse of HeLa cells expressing mCherry-vimentin through the cell cycle (images from Video S1). Time in minutes. Scale bar, 20 µm.(B) Top: representative two-color dSTORM image of vimentin (magenta) and F-actin (cyan) in a mitotic cell. Scale bars, 5 and 1 µm. Bottom: distance between the peaks of the actin and vimentin fluorescence profiles across the cortex (actin-VIM; see STAR Methods for details). To rule out any systematic shift, we also measured peak-to-peak distance for actin profiles where actin was labeled with two differentially labeled phalloidin probes (actin-actin; Figure S4B). Graph: mean ± standard deviation (actin-actin, n = 10 and actin-VIM, n = 14; at least 3 independent experiments). Mann-Whitney test.(C) Left: representative images of endogenous vimentin localization in mitotic HeLa cells transfected with non-targeting control siRNA (siNTC), siRNA against plectin (siPLEC), or vimentin (siVIM). Scale bars, 5 µm. Right: quantification of vimentin enrichment at the cell cortex. Graph: mean ± standard deviation; 3 independent experiments. siNTC, n = 30 and siPLEC, n = 29. Mann-Whitney test.(D) Cortex tension in mitotic (prometaphase) cells measured by atomic force microscopy (top schematic, see STAR Methods for details on tension measurement) for cells treated with non-targeting control siRNA (siNTC, n = 47), siRNA against vimentin (siVIM, n = 28), or siRNA against plectin (siPLEC, n = 18). Graph: median and interquartile range; at least 3 independent experiments. Kruskal-Wallis test.See also Figure S4.

Fig 4: Sub-cortical Vimentin Directly Controls Cortical Actin Organization in Mitosis(A) Left: representative images of total vimentin (immunostaining) in mitotic Hela cells stably expressing GFP-vimentin mutants. Right: corresponding quantification of vimentin enrichment at the cell cortex. Scale bar, 5 µm. Right: graph, mean ± standard deviation; 3 independent experiments. WT, n = 31; 56A, n = 31; 56E, n = 33; and 83E, n = 29. Kruskal-Wallis test; ns, non-significant.(B) Representative maximum z projections of confocal stacks across mitotic HeLa cells stably expressing vimentin mutants and stained for total vimentin. Scale bar, 10 µm. See Video S3 for 3D visualizations.(C) Left: examples of STORM images of the actin cortex in cells expressing the different vimentin mutants. Scale bars, 0.5 and 5 µm. Right: values of full width at half maximum (FWHM) of the cortical actin signal in cells expressing the different vimentin mutants. NT, n = 16; WT, n = 26; 56E, n = 20; 56A, n = 19; 83E, and n = 21. 3 independent experiments. Graph: mean ± standard deviation. ANOVA Tukey’s multiple comparison.(D) Cortex tension in mitotic (prometaphase) cells expressing VIM-WT, VIM-56E, or VIM-83E. Graph: median with interquartile range; 3 independent experiments. WT, n = 60; 56E, n = 29; and 83E, n = 26. Kruskal-Wallis test.(E) Left: representative time lapses of curvature maps along the cell contour (see STAR Methods for details) during ablation experiments in HeLa cells stably expressing GFP-VIM-WT or GFP-VIM-56E (displayed in insets; yellow circles, ablation region). Right: quantification of the effects of laser ablation in HeLa cells expressing GFP-VIM-WT or GFP-VIM-56E. N = 4 independent experiments for VIM-WT (n = 52 cells) and N = 3 for VIM-56E (n = 57 cells). In some cells (15 cells out of 52 for VIM-WT and 26 cells out of 57 for VIM-56E), ablation elicited bleb formation at the site of ablation (Video S7, right panel); these cells were excluded from the quantifications, as bleb formation indicated ablation disrupted the actin cortex (Tinevez et al., 2009). Scale bars, 5 µm.See also Figure S5.