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: Lung injury and fibrosis are attenuated in Vim-/- bone marrow chimeric mice.(a) Treatment protocol: CD45.1+ WT mice were lethally irradiated (6 Gy) and two sets of mice were transplanted either with 1 × 106 CD45.2+ WT or Vim-/- BM cells to generate chimeric mice. After 6.5 weeks (w), when >90% of AM were of donor (WT and Vim-/-) phenotype, chimeric mice were treated with saline or bleomycin and subjected to analyses. Bleomycin-induced lung injury was assessed by protein levels (b), and ELISA for IL-1ß (c), IL-6 (d) and TGF-ß (e) in BALF, 5 days (d5) after instillation. Bleomycin-induced fibrosis was assessed 21 days (d21) after instillation by measuring collagen content in lungs by Sircol assay (f) and by histological examination of lung tissue with H&E staining (g). Original magnification × 40; scale bar, 200 µm. Data in b are means±s.e., c–f are means±s.d. from two independent experiments, each with at least three animals per experiment. *P<0.05, **P<0.001, ***P<0.0001, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Fig 4: Activation of effector secretion requires S. flexneri docking(a) Images of docked WT bacteria at 30 min of infection. Bacteria activated to secrete effectors through T3SS express GFP (TSAR, green), all bacteria express mCherry (red), DNA labeled with DAPI (blue). Arrows, TSAR positive bacteria. Scale bar, 10 µm. Images are representative of three independent experiments. (b-c) Quantification of cell-associated bacteria that have activated TSAR at 30 min or 60 min of infection. *P<0.05; Student's t-test. Data are the mean ± SEM of at least three independent experiments. (d) Absence of TSAR expression among bacteria centrifuged onto cells yet non-adherent at 30 min of infection. Images are representative of three independent experiments. Scale bar, 10 µm. (e) Quantification of data presented in d. Data are the mean ± SEM of three independent experiments. (f) Rescue of docking of bacteria by genetic complementation of Vim-/- MEFs with human vimentin. Vim-/-:huVim, human vimentin expressed in Vim-/- MEFs. Data are the mean ± SEM of three independent experiments. *P<0.05, **P<0.01; one-way ANOVA with Tukey post hoc test.

Fig 5: Generation of LAMC2-EGFP knock-in human pancreatic cancer cells. a Design of LAMC2-EGFP donor and CRISPR/Cas9 sgRNA vectors. Blue circle represents the CRISPR/Cas9 protein complex and the yellow box underneath illustrates the guide RNA. b qPCR analysis for LAMC2 gene expression in EGFP+ and EGFP- cells. Data are normalized to GAPDH and are presented as fold change in gene expression relative to the EGFP- counterpart. c Sphere formation capacity for EGFP+ versus EGFP- cells. d Representative images of gelatin degradation for EGFP+ versus EGFP- cells. Nuclei were stained with Hoechst 33342 (blue), green represents actin (Alexa Fluor™ 488 Phalloidin) and red illustrates gelatin (Rodhamine). The white dashed line circles indicates the areas of degradation. e Invasive potential of sh empty versus LAMC2 knockdown cells. f qPCR analysis for EMT and MMP2 and MMP10 gene expression for EGFP+ versus EGFP- cells. Data are normalized to GAPDH and are presented as fold change in gene expression relative to the EGFP- cells. g Western blot analysis of VIM and MMP2 in EGFP+ and EGFP- cells. Parallel Tubulin immunoblotting was performed. *p < 0.05, **p < 0.005, ***p < 0.0005. n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge 3.$$\end{document}=3. Statistical significance was assessed by Student's t-test