Fig 1: Blockade of the HIF‐1α/p53/miRNA‐34a axis mitigates subretinal fibrosis in a mouse CNV‐induced CNV model. The mice were divided into the following groups: normal, CNV 7 d, CNV 7 d + 0.1% DMSO, CNV 7 d + digoxin, CNV 7 d + AAV‐p53 mutant, CNV 7 d + miRNA 34a inhibitor and CNV 7 d + AAV‐Klotho full‐length plasmid. A, Western blot was performed to measure Fib, N‐cad and Vim protein levels in retina‐RPE‐choroid tissues. *** P < .001, CNV 7‐d group vs normal group. ## P < .01, compared with the CNV 7‐d group. B, Relative protein levels of Fib (B), N‐cad (C) and Vim (D) compared with GAPDH levels were analysed. E, IB4 (green) and type I collagen (colI; red) were stained on the choroidal flat mount. F, The volume of CNV was analysed. G, The subretinal fibrosis area was analysed. ** P < .01, *** P < .001, compared with the CNV 7‐d group in Figure 6F,G. H, A schematic diagram displaying the role of HIF‐1α/p53/miRNA‐34a in CNV is shown. Under hypoxic conditions, HIF‐1α up‐regulates p53 activation, increases miRNA‐34a, down‐regulates Klotho and promotes EMT in RPE cells, facilitating subretinal fibrosis and the progression of CNV. Moreover, blockade of the HIF‐1α/p53/miRNA‐34a axis alleviates subretinal fibrosis and the formation of CNV
Fig 2: The HIF-1a/p53/miRNA-34a/Klotho axis facilitates hypoxia-induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells. ARPE-19 cells were divided into the following groups: negative control (NC), hypoxia, hypoxia + 0.1% DMSO, hypoxia + digoxin, hypoxia + digoxin +nutlin-3a, hypoxia + digoxin + miRNA-34a mimics, hypoxia + AAV-p53 mutant infection, hypoxia + miRNA-34a inhibitor transfection (40 nM for 24 h) and hypoxia + AAV-Klotho full-length plasmid infection. A, Western blot was performed to measure the protein levels of the mesenchymal cell markers fibronectin (Fib), N-cadherin (N-cad) and vimentin (Vim). The relative protein levels of Fib (B), N-cad (C) and Vim (D) compared with GAPDH levels were analysed. *** P < .001, hypoxia group vs normal group. ## P < .01, compared with the hypoxia group. %% P < .01, % P < .05, compared with the hypoxia + digoxin group
Fig 3: MEG3 suppresses the PI3K/AKT pathway and the epithelial-to-mesenchymal transition process via miR21-5p. PC9 and H1299 cells were transfected with MEG3, empty vector, miR-21-5p inhibitor, NC-inhibitor, MEG3+ miR-21-5p mimic and MEG3+ NC-mimic. Representative western blots, and semi-quantitative analysis of relative changes in expression levels of E-cad, N-cad, Vim and MMP9 in (A) H1299 and (C) PC9 cells. The protein expression levels of p-PI3K, PI3K, p-AKT and AKT protein in (B) H1299 and (D) PC9 cells are also shown. **P<0.01. *P<0.05. MEG3, maternally expressed gene 3; miR, microRNA; NC, negative control; E-cad, E-cadherin; N-cad, N-cadherin; Vim, vimentin; MMP, matrix metalloprotein; p-, phosphorylated.
Fig 4: Effects of LKB1 on SIK1 expression and the EMT signaling pathway in ovarian tumor cells. (A) SIK1 expression was promoted by LKB1 upregulation in ovarian tumor cells, as determined by immunofluorescence. (B) LKB1 overexpression suppressed TGF-ß expression in ovarian tumor cells. (C) LKB1 upregulation promoted apoptotic sensitivity in ovarian tumor cells treated with paclitaxel. (D) LKB1 overexpression decreased E-cad, VIM, Snai2 and ?H2AX expression in ovarian tumor cells. (E) Upregulated SIK1 suppressed the growth of ovarian tumor cells compared with the control. (F) LKB1 overexpression inhibited the aggressiveness of ovarian tumor cells. **P<0.01. LKB1, liver kinase B1; SIK1, salt-inducible kinase 1; Snai2, zinc-finger protein SNAI2; VIM, vimentin; E-cad, E-cadherin; TGF-ß, transforming growth factor-ß; ?H2AX, ?-histone H2AX; LKB1OR, LKB1 overexpression; SIK1UE, SIK1 upregulation.
Fig 5: Vimentin interacts with SopB to facilitate the intracellular replication of Salmonella during infection.a Schematic diagram to detect vimentin interactome by precipitation and mass spectrometry. b List of the top Salmonella T3SS effectors interacting with vimentin from mass spectrometry. c Co-immunoprecipitation (Co-IP) assay followed by Western blotting to test for association of SopB-GFP with vimentin. SopB-GFP was enriched using anti-GFP agarose beads, and presence of vimentin or SopB was tested by Western blotting. Co-IP assays were conducted three times independently with similar results. d Immunofluorescence images of cells infected with ?sopB Salmonella (MOI = 10). Quantification of the relative vimentin area versus cell area is shown in the right panel. e Immunofluorescence images of cells infected with Salmonella (WT, ?sipB, ?phoP) (MOI = 10) at 24 hpi. Quantification of the relative vimentin area versus cell area is shown in the right panel. f Immunofluorescence images of vimentin, LAMP1, and bacteria in WT and VIM KO cells infected with Salmonella, ?sopB Salmonella, and full length sopB complemented ?sopB Salmonella (?sopB sopB-FL Salmonella) (MOI = 10) at 24 hpi, respectively. g Quantification of the percentage of SCV versus dispersive SCV in (f) was measured. h Quantification of relative MFI values by FACS in WT and VIM KO cells infected with mCherry-tagged Salmonella or ?sopB Salmonella (MOI = 50) from three independent experiments. i Schematic diagram of SopB from Salmonella induced vimentin remodeling to surround SCV. White dash lines in (d and e) indicate the outline of the cells. n = 20 views (60×/1.5 oil objective) from three independent experiments in (d, e and g). Scale bar, 10 µm (d, e and the cell images in f) and 5 µm (the magnified images in f). Data are represented as mean ± SD. Statistics (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001): one-way ANOVA with Dunnett’s analysis (right panel of d and e) or two-way ANOVA with Sidak’s analysis (g and h). Source data are provided as a Source Data file.
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