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Fig. 2 | Arthritis Research & Therapy

Fig. 2

From: The compound LG283 inhibits bleomycin-induced skin fibrosis via antagonizing TGF-β signaling

Fig. 2

LG283 antagonizes EMT in vitro. The effect on EMT of human lung carcinoma epithelial cell line A549 cells in NanoCulture Plate (NCP). The cells were grown in either 2-D NCP conditions with or without 5 ng/ml rTGF-β2 only or TGF-β2-plus 0.5 μM of LG283. a Representative images of 2-D cultured A549 cells. The cells exhibited a round, confluent epithelioid appearance irrespective of the presence (right upper) or absence of LG283 (left upper). Treatment with rTGF-β2 for 72 h resulted in a morphological change to fibroblastic spindle shape (left lower), which was blocked by co-treatment with LG283 (right lower). Inset showed magnified images. Scale bars, 200 μm. b mRNA expression of epithelial (E-cadherin) and mesenchymal markers (FN-1, α-SMA, CTGF) and transcription factors associated with EMT (SNAIL1, 2 and ZEB1, 2) was quantified by real-time RT-PCR at 48h. Values were normalized to GAPDH levels and are shown as the mean of fold change compared to vehicle control ± SEM of three independent experiments; n=5 in each group; **p ≤ 0.01. c, d Protein expression of EMT-associated transcription factors was quantified by western blot analyses at 48–96h. Values were normalized to GAPDH or TUBULIN levels and are shown as the mean of fold change compared to vehicle control; n=3~4 in each group. Representative blot of α-SMA and E-cadherin is shown. All values represent mean ± SEM; *p ≤ 0.05; **p ≤ 0.01. Molecular weights of the paneled proteins; 98 kDa (Ecad), 48 kDa (α-SMA), and 50 kDa (TUBULIN). e After 48 h incubation of A549 cells with or without rTGF-β2 only or TGF-β2-plus LG283, protein expression of p-Smad3 was evaluated by western blotting. Values were normalized to TUBULIN levels and are shown as the mean of fold change compared to vehicle control ± SEM; n=4 in each group; **p ≤ 0.01. Molecular weights of the paneled proteins; 53 kDa (pSmad3) and 50 kDa (TUBULIN)

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