Fig 2: Silencing of vimentin induced cell detachment. (A,B) Images from the representative transwell assay; silencing of vimentin increased the leading cells detachment. A549 and PANC-1 cells were silenced with shLacZ or shVIM and seeded in the upper chamber overnight, and the transwell assay was performed for 24 and 48 h. The membranes were stained by crystal violet and the cell number from membranes or the lower chamber was counted. Scale bars: 100 µm. *** p < 0.001. Data are means ± s.d. (two-way ANOVA with Tukey’s multiple comparisons test) from experiments with three replicates (n = 3). (C) Relative expressions of vimentin mRNA in BFTC909-shLacZ, -shVIM#1, and shVIM#2 were determined by the real-time qPCR assay. (D) Relative expressions of vimentin mRNA in PANC-1-shLacZ, -shVIM#1, and shVIM#2 were determined by the real-time qPCR assay. *** p < 0.001. Data are means ± s.d. (one-way ANOVA with Dunnett’s multiple comparisons test) from experiments with three replicates (n = 3). VIM, vimentin.

Fig 3: Cav-1 was negatively regulated by vimentin filaments. (A) Representative immunofluorescence images showed the subcellular distribution of endogenous vimentin and Cav-1. The white dotted outline showed the perinuclear vimentin-rich region. Bars, 10 µm. (B) The correlation analysis indicated by Pearson’s coefficient of the fluorescence signals of endogenous vimentin and Cav-1. The correlation between exogenous expressed Cav-1-mCherry and endogenous antibody stained Cav-1 was used as a positive control. (C) Representative images from time-lapse videos (Supplementary Materials) of U2OS cells expressing vimentin-GFP and Cav-1-mCherry. Magnified panels showed representative images at different time points in the yellow box of the left panel, and white arrows indicated the positions of individual Cav-1 vesicle observed in the video (Supplementary Materials). Bars, 10 µm in the left panels and 2 µm in the right magnified panels, respectively. (D) The extracts of U2OS cells with wild-type and vimentin-knockout (VIM KO) were subjected to Western blot analysis. (E) The quantified relative levels of phosphorylated Cav-1 (P-Cav-1) protein normalized to the internal control GAPDH in wild-type and vimentin-knockout cells from Western blots. The data was from five independent experiments (n = 5). (F,G) The intensity of P-Cav-1 was increased in vimentin-knockout U2OS cells and was able to recover in vimentin full-length (FL) reintroducing cells. In (F), it was showed the representative images of wild-type (arrowheads in WT and VIM KO Mix), vimentin-knockout and vimentin-knockout/full-length (arrowheads in VIM KO and FL Mix) cells. In (G), it was showed the quantification of normalized relative fluorescence intensities in wild-type (n = 21), vimentin-knockout cells (n = 13) and full-length rescue cells (n = 22). Bars, 10 µm. All the quantified data was presented as mean ± s.e.m. ** p ≤ 0.01, *** p ≤ 0.001 (t-test).

Fig 4: Renal-directed differentiation of hESC on renal ECM or PSS. (A-D). H&E staining of cell-scaffold constructs after 20 days in culture with two growth factor protocols (A or B). (E-H). Tubule-like structures were present and positive for CK but not VIM. (I-L). Regions positive for PAX2 (intermediate mesoderm and induced metanephric mesenchyme) were typically distinct from regions positive for WT1 (induced mesenchyme) under Protocol A. These markers were expressed in a more diffuse pattern throughout the construct under Protocol B. Tubule-like structures in Protocol B with renal ECM were WT1-positive and surrounded by a ring of PAX2-positive cells. (M-P). The proximal tubule marker AQP1 was expressed with greater frequency under Protocol A and on PSS while the Loop of Henle marker UMOD was rarely expressed in any construct. (Q-T). The distal tubule/collecting duct marker ECAD was widely expressed on cells with epithelial morphology in all constructs while the ascending Loop of Henle and collecting duct marker CALB was only expressed in constructs under Protocol A with PSS. (U-X). The proximal tubule marker EMA was expressed on some tubule structures in all constructs with small vessel-like structures positive for the endothelial marker CD31. Nuclei were visualized with DAPI (blue). Representative images shown; N=3 experiments. Scale bar = 100 µm.

Fig 5: Widespread changes in protein abundance occur in rat liver following repeated acetaminophen exposure.(a–d) Volcano plots of all common proteins quantified by iTRAQ analysis, at each time-point (a) 24 h, (b) 48 h, (c) 72 h, (d) 96 h, relative to vehicle control. A complete list is provided in Supplementary Tables 1a–d. Coloured circles represent differential expression (blue - raw P value, p < 0.05; red – FDR, p = 0.05). (e) Principal Components Analysis identified the greatest differences between single and repeat dose samples. (f ) Heat map representing the 1169 proteins common to all samples and all time-points identified distinct changes in protein abundance in repeat-dosed animals (red indicates increased abundance, blue indicates decreased abundance). (g) Western blots for GSTP1, NQO1, PCNA and VIM, performed in order to validate proteomic findings. Representative blots of two rats at each time-point are shown. Actin was used as a loading control.