C328S mutant vimentin affects interaction with actin in silico and F-actin formation in cells.

(A): Relative fold change of VIM mRNA in WT-VIM and C328S-VIM expressing MCF-7 cells normalised to POLR2A and YAP1. (B): Expression of WT-VIM and C328S-VIM in MCF-7 cells by western blotting. (C): Quantification of vimentin using ImageJ. (D): Immunostaining of MCF-7 cells expressing WT-VIM and C328S-VIM. Cells were immunostained with mouse anti-vimentin V9, AF-488 labelled goat anti-mouse showing green fluorescence. Nuclei were stained with DAPI in blue and the overlapping images are shown as Merge. Leica DM4000B Epi-fluorescence microscope was used for imaging (Scale bar = 20 µm; the scale bar in MCF-7C328S cells is much longer as these cells were reduced in size). (E): In silico modelling of actin binding at the interface where C328 and S325 residues are located. The solid rectangular area in the panel (i) is zoomed in the accompanying panel. The red colour indicates the C328 residue site whereas the green colour indicates the S325 residue site. (ii) In-silico modeling of C328 and S328 residue sites and rotamer conformations further zoomed in from the panel (i). The green colour indicates hydrogen bonds, the white colour indicates covalent bonds and the red colour indicates oxygen atom. (F): Immunostaining of MCF-7 cells expressing WT-and C328S-VIM. Cells were immunostained with AF568 Phalloidin (red colour) and anti-vimentin (green colour) antibody. Nuclei were in blue and the overlapping images are shown as Merge. Images were taken by Zeiss 880 laser scanning confocal microscope with Fast Airyscan and Multiphoton (inverted) system (scale bar = 20 µm). (G): Percentage of cells expressing F-actin in WT and C328 cells (see Figure S3 for clarification of high and low expression). Student’s t-test was used to calculate p values using Microsoft Excel and are given by asterisks (****p < 0.0001). Statistical analyses: n = 3, error bars= ±SEM, ns= not significant, number of cells counted=200. A) and western blotting (Figure 1B). The quantification of the relevant bands showed that the wildtype and mutant vimentin proteins were equally expressed in MCF-7 cells (Figure 1C). Immunostaining of the wildtype vimentin showed a fully extended vimentin network from perinuclear to peripheral cell boundaries whereas in cells transduced with the C328S mutant, the vimentin was more condensed around the perinuclear area (Figure 1D).

Effect of C328S-VIM on cell morphology, proliferation, adhesion and invasion.

(A): Morphology of MCF-7 expressing WT-VIM and C328S-VIM in brightfield (scale bar = 100 µm). (B): Morphology of WT and C328S cells stained with CellmaskTM deep red dye. Images were captured by INCA 2200 and analysed by INCarta software (scale bar = 50 µm). (C): Differences in nuclear area, nuclei form factor, nuclear major axis, nuclear minor axis, cell area, cell diameter, cell perimeter, cell minor axis, cell major axis and cell form factor, between the two cell lines. (D): Significant reduction in nuclear compactness and nuclei/cell area between the two cell lines. Proliferation rate was compared between WT and C328S cells by (E, F) colony, (G) MTT, and (H) CyQUANT assays. (I): CyQUANT cell adhesion assay was performed to compare the cell adhesion between WT and C328S cells without substrate, and (J): with the addition of laminin, fibronectin and collagen, separately. (K): Chemotactic migration of the WT and C328S cells through 8.0 µm culture inserts. The cells were fixed and stained with 0.1% (w/v) crystal violet before imaging. (L): The cells on the outer surface of the inserts were counted and compared between WT and C328S. (M): Chemotactic invasion in WT and C328S cells through 8.0 µm culture inserts coated with Matrigel. The cells were fixed and stained with 0.1% (w/v) crystal violet. (N): Total number of cells invaded on the outer surface of the inserts were counted. Statistical analyses: n = 3, Error bars= ± SEM, Student’s t-test was used to calculate p values using Microsoft Excel and are given by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Transcriptomic insight into tumorigenic potential induced by C328S-VIM both in vitro and in vivo.

(A): Volcano plot showing differentially expressed genes (DEGs) between WT and C328S cells. Gene Ontology (GO) showing overview of cellular functions, (B): downregulated and (C): upregulated by DEGs. (D): RNA seq analysis showing log2fold expression and validation by RT-qPCR for DEGs of interest. (E): XIST, the most upregulated gene, has been implicated in large number of solid tumours [34]. (F): Linear regression analysis of log2fold changes from RT-qPCR and RNA-Seq of DEGs. (G): Immunostaining of WT and C328S cells with V9, rabbit anti-K8 and rabbit anti-K18 using AF-488 (green) goat anti-mouse and AF-594 (red) goat anti-rabbit were used. Nuclei are in blue, overlapping images are shown as Merge. Leica DM4000B Epi-fluorescence microscope was used for imaging (scale bar = 20 µm). (H): VIM, K18, K19, K8, CDH2/N-cadherin and TWIST1 expression by western blotting in WT and C328S cells. Relevant bands were cropped from the original blots shown in Figure S1. (I): Quantification of the protein expression in panel H by ImageJ. (J): Relative log2fold changes in expression of EMT transcription factors, and (K): breast cancer stem cell markers in WT and C328S cells by RNA-Seq analysis. (L): Flow cytometry overlay dot plot of CD56-RY586 versus CD201-APC after gating on single and live cells for immunophenotype. (M): Comparison of mean fluorescence intensity (MFI) of CD56 in WT and C328S cells. (N): Comparison of MFI of CD201 in WT and C328S cells. (O): Transplantation of WT and C328 cells in nude mice without estrogen. (P): Average tumour burden after two weeks in nude mice injected with WT and C328S cells. (Q): H & E stained tumour sections scale bar= 50 µm. (R): Representative image from immunohistochemical staining of CD56 and CD201 in tumour sections as compared with IgG control, scale bar= 50 µm. (S): Quantification of CD56 and (T): CD201staining in tumour sections and control using ImageJ. Statistical analyses: n = 3, Error bars= ± SEM, Student’s t-test to calculate p values using Microsoft Excel, and are given as asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001).

Summary of cell lines used in this study.

shRNA mediated downregulation of XIST in C328S-VIM reverts cell phenotype.

(A): Downregulation of XIST in C328S cells by 4 different shRNAs (sh1-sh4) for XIST or NTC by RT-qPCR. shXIST2 was the most potent (p < 0.05) compared with NTC. (B): VIM expression in MCF-7C328S_shVIM and MCF-7C328S_shNTC as determined by RT-qPCR. (C): Vimentin expression in MCF-7C328S_shVIM and MCF-7C328S_shNTC by western blotting. (D): Quantification of the protein expression in panel C by ImageJ. (E): XIST RNA in MCF-7 cells expressing C328S_shVIM and C328S_shNTC by RT-qPCR. (F): Relative VIM mRNA fold change (%) in MCF-7 cells expressing C328S_shXIST2 and C328S_shNTC by RT-qPCR. Comparision of cell adhesion, (G): between MCF-7C328S_shVIM and MCF-7C328S_shNTC, and (H): between MCF-7C328S_shXIST2 and MCF-7C328S_shNTC cells without substrate by CyQUANT assay. Comparision of cell proliferation, (I): between MCF-7C328S_shVIM and MCF-7C328S_shNTC, and (J): between MCF-7C328S_shXIST2 and MCF-7C328S_shNTC by MTT assay. Comparision of chemotactic migration, (K): between MCF-7C328S_shVIM and MCF-7C328S_shNTC, and (L): between MCF-7C328S_shXIST2 and MCF-7C328S_shNTC cells through 8.0 µm culture inserts. Comparision of chemotactic invasion, (M): between MCF-7C328S_shVIM and MCF-7C328S_shNTC, and (N): between MCF-7C328S_shXIST2 and MCF-7C328S_shNTC cells through 8.0 µm Matrigel coated inserts. (O): Comparison of cell area, ratio of nuclei/cell area, cell diameter, cell perimeter, cell major axis, cell minor axis, between MCF-7C328S_shVIM and MCF-7C328S_shNTC. (P): Flow cytometry overlay dot plot of CD56-RY586 versus CD201-APC after gating on single and live cells for immunophenotyping of MCF-7C328S_shVIM and MCF-7C328S_shNTC cells. (Q): Flow cytometry overlay dot plot of CD56-RY586 versus CD201-APC after gating on single and live cells for immunophenotyping of MCF-7C328S_shXIST2 and MCF-7C328S_shNTC cells. Statistical analyses: n = 3, Error bars= ± SEM, Student’s t-test was used to calculate p values using Microsoft Excel when two groups were compared, one way ANOVA with Bonferroni test was applied using GraphPad Prism 10 when comparing more than two groups, (*p < 0.05, **p < 0.01 and ***P<0.001).