Figure 1.
The correlation between endogenous expression of Snail and E-cadherin in ARPE-19 cells.
(A) Compared to HGF cells, ARPE-19 cells showed the typical epithelial morphology. Original magnifications 100×. RT-PCR (B) and Western blot (C) analysis of Snail and E-cadherin in ARPE-19, MCF-7 and HGF cells at the mRNA and protein levels respectively. MCF-7 cells were used as a positive control for E-cadherin expression and HGF cells were used as a positive control for Snail expression.
Figure 2.
EMT of ARPE-19 cells induced by TGF-β1.
ARPE-19 cells were grown on glass coverslips for 24 hr, starved for 24 hr, and then incubated for 24 hr, 48 hr, 72 hr with 10 ng/ml TGF-β1. Compared with control cells (A, E, I, M, Q), stimulated cells displayed an altered mesenchymal morphology by phase-contrast microscopy (B, C, D), with decreasing expression of E-cadherin (F, G, H) and ZO-1 (J, K, L) and an increasing expression of fibronectin (N, O, P) and α-SMA (R, S, T) by immunofluorescence microscopy. The green signal represents the staining of corresponding protein, and the red signal represents the nuclei staining by DAPI. Original magnifications 100× (A–D); 400× (E–T).
Figure 3.
TGF-β1 increased Snail expression in cultured ARPE-19 cells.
Total RNA was extracted from ARPE-19 cells at the time points as indicated after TGF-β1 (10 ng/ml) treatment. (A) The increasing expression of Snail mRNA was time-dependent. The increasing expression of mesenchymal markers fibronectin (B) and α-SMA (C) and the decreasing expression of epithelial markers E-cadherin (D) and ZO-1 (E). Values were normalized to GAPDH mRNA levels and represent fold change as compared to untreated cells. Data shown as mean ± SD of two independent experiments each performed in triplicate. (F) Western blot analysis of Snail, fibronectin, α-SMA, E-cadherin and ZO-1 with proteins extracted from ARPE-19 cells treated with 10 ng/ml TGF-β1 at differential time points. (G) Imunofluorescence analysis and subcellular localization of Snail (green) in ARPE-19 cells treated with 10 ng/ml of TGF-β1 for 48 hr. Nuclei were stained with DAPI (red). Original magnifications 400×.
Figure 4.
Snail silencing attenuated TGF-β1 induced EMT.
(A) Immunofluorescence staining of Snail in Snail-shRNA or control-shRNA transfected cells which were starved for 24 hr in serum-free media and treated with 10 ng/ml of TGF-β1 for 48 hr. The green signal represents the staining of Snail protein, the red signal represents the nuclei staining by DAPI. (B) Photomicrographs of Snail-shRNA or control-shRNA transfected ARPE-19 cells are showing different cell morphologies. Cells transfected with Snail-shRNA or control-shRNA plasmid were starved for 24 hr in serum-free media and treated with 10 ng/ml of TGF-β1 for 48 hr. The expression of Snail, fibronectin, a-SMA, E-cadherin and ZO-1 were analyzed by Western blot (C) and real-time PCR (D). Values represent fold change as compared to control cells. Data shown as mean ± SD of two independent experiments each performed in triplicate. Original magnifications 400× (A); 100× (B).
Figure 5.
Migration after Snail knockdown in ARPE19 cells with or without TGF-β1 treatment.
(A) Cells transfected with control-shRNA or Snail-shRNA were allowed to migrate transwell chambers for 18 hr in the presence or absence of TGF-β1. After 18 hr, the migrated cells were fixed, stained, and photographed. (B) The number of migrated cells. Data shown represent the average of three independent experiments. *P<0.05, compared with control-shRNA, TGF-β1(−) samples and control-shRNA, TGF-β1(+) samples.
Figure 6.
Snail distribution and protein levels in PVR membranes.
(A) Immunofluorescent staining of Snail (green) in epiretinal membranes. Nuclei were stained with DAPI (red). Arrowheads indicated colocalization of Snail expression and nuclei. Original magnifications 50× (a); 200× (b–e). (B) 50 µg and 80 µg total protein extracted from PVR epiretinal membranes were detected by Western blot. GAPDH was used as internal control.