Figure 2. eIF4E antagonism attenuates TGF-β1-induced α-SMA expression in RLE-6TN cells.

(a) RLE-6TN cells were seeded at a density of 40,000 in 24-well-plates, and transduced with virus expressing eIF-4E shRNA (pLKO.1 eif4E shRNA; TRCN0000077474, Open Biosystems), or control non-silencing shRNA (SHC002, Sigma) on day 1 at MOI of 2.5, followed by TGF-β (2.5 ng/ml) treatment on day 2 (vehicle (V = 1 μl per 1 ml 4 mM HCl containing 1 mg/ml BSA) as control). Protein was harvested for Western analysis of eIF-4E, α-SMA, E-cadherin and Claudin 18 expression after 2 days of treatment; Lamin A/C is the loading control. Blots shown are representative of 3 independent experiments. Densitometric analysis demonstrated that knockdown of eIF-4E significantly reduced TGF-β-induced α-SMA expression as well as blocked TGF-β-induced reductions in E-cadherin and Claudin18 expression, suggesting eIF-4E plays an important role in regulation of genes involved in TGF-β-induced EMT in AEC. *P < 0.05, represents significant difference of eIF-4E expression from control shRNA, significant difference of α-SMA expression from other conditions, and significant difference of E-cadherin and Claudin 18 expression from vehicle control (1 μl per 1 ml 4 mM HCl containing 1 mg/ml BSA). (b) RLE-6TN cells were transduced with virus expressing a constitutively active form of the translational repressor HA-4EBP1 (pEF1α-3HA-4EBP1-TTAA-IRES-GFP) or control (pEF1α-eGFP). The next day, cells were treated with TGF-β1 (2.5 ng/ml) or vehicle (1 μl per 1 ml 4 mM HCl containing 1 mg/ml BSA). Cell lysates were harvested after 2 days of treatment. Shown is an immunoblot (left panel) probed for α-SMA expression (Lamin A/C served as the loading control). The immunoblot shown is representative of 3 independent experiments. The mean values for all 3 replicates are shown (right panel). *indicates P < 0.05 for the comparison of TGF-β1 treated cells expressing HA-4E-BP1 to all other conditions.