Fig 2: Efficient T3SS translocation depends on intermediate filaments(a) Cell viability of MEFs following infection with S. flexneri for 24 h. Viability determined by trypan staining and counting of recovered cells. Vim-/-:huVim, human vimentin expressed in Vim-/- MEFs. Data are mean ± SEM of three independent experiments for each group. *, P<0.05; one-way ANOVA with Tukey post hoc test. (b) Levels of vimentin in cells used for experiments in panel a, by western blot. Images shown are from one experiment, representative of three independent experiments. (c-f) Assessment of translocation efficiency by conversion of CCF4-AM fluorescence emission from green to blue upon infection of MEFs with bacteria that translocate a bacterial type 3 effector-ß-lactamase reporter. (c) Images of fluorescence emission for S. flexneri expressing OspB-TEM. Images presented are representative of seven independent experiments. Scale bar, 100 µm. (d-f) Quantification of fluorescent images: (d) S. flexneri expressing OspB-TEM, mean ± SEM of seven independent experiments. (e) S. Typhimurium expressing SopE1-TEM, mean ± SEM of five independent experiments for media and WT and four independent experiments for ?invG. (f) Y. pseudotuberculosis expressing YopE-TEM, mean ± SEM of three independent experiments. Effector translocation was dependent on a functional T3SS apparatus, irrespective of the presence or absence of vimentin, as shown by ?spa15, ?invG, and ?yopD mutants, which are deficient in T3SS activity.. ***, P<0.001; two-way ANOVA followed by Sidak post hoc test.

Fig 3: Effect of the SMARCA4 complex depletion on the EMT program. (A) Q-RT-PCR for SMARCA4 knockdown. RNA was prepared from uninfected or 24 h infected scrambled or SMARCA4 knockdown cells. Data is normalized to internal control PP1A and expressed as fold change relative to uninfected WT cells. *p <0.01, ANOVA, with contrast using post-hoc Tukeys. (B) Hierarchical clustering of EMT signature genes. Expression patterns of signature epithelial or mesenchymal genes in the time course were z-score–normalized and subjected to hierarchical clustering using Euclidian distance. Abbreviations used are: MMP, matrix metalloproteinase; SNAI1, Snail Family Transcriptional Repressor 1; JunB, JunB Proto-Oncogene; CDH, Cadherin; VIM, vimentin; SPARC, Secreted Protein Acidic And Cysteine Rich; ITGB3, integrin Subunit Beta 3; TWIST, Twist Family BHLH Transcription Factor; S100A8, S100 Calcium Binding Protein A8; ZEB: Zinc finger E-box binding homeobox. (C) Q-RT-PCR for EMT pathway “signature genes”- MMP9, VIM, SNAI1, CDH2, and CDH1. RNA was prepared from uninfected or 24 h infected cells (MOI 1). Data is normalized to internal control PP1A and expressed as fold change relative to uninfected WT cells. **p <0.01, ANOVA, with contrast using post-hoc Tukeys. #p<0.01 compared to Scr. (D) Immunofluorescent staining of SMARCA4-depleted cells. Cells were stained with VIM (green) and CDH1 (red). Note the loss of cell surface CDH1 and the accumulation of VIM in the SMARCA4 complex-depleted cells, characteristic of pEMT.

Fig 4: Müller glial cells dedifferentiated into progenitor cells and generated rod-like cells in the presence of purmorphamine. (A) The percentage of cells expressing progenitor cell markers under different treatment conditions. Double staining revealed that purmorphamine treatment induced Müller glia (VIM- or GS-positive) to express the progenitor cell markers (B) Pax6, (C) Sox2 and (D) nestin. Following 2 days in fresh medium culture, Müller glia-derived progenitor cells expressed (E) the neuron marker Tuj1 and (F) the rod cell-specific marker rhodopsin. (G) mRNA transcripts of rod development genes, nrl and crx, were detected by semi-quantitative polymerase chain reaction following treatment with purmorphamine or SHH-N; however, no expression was detected in control cells or cells co-treated with cyclopamine. *P<0.05 vs. control. Scale bars, 30 µm. SHH+cyclo, data not shown. BrdU, 5-bromo-2'-deoxy uridine; cyclo, cyclopamine; crx, cone-rod homeobox; GS, glutamine synthetase; nrl, neural retina-specific leucine zipper; Pax6, paired box protein-6; Pur, purmorphamine; SHH, sonic hedgehog; Sox2, sex determining region Y-box 2; Tuj1, specific class III ß-tubulin; VIM, vimentin.

Fig 5: 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.