Luciferase Reporter Assays

To evaluate the activation of different signaling pathway, cells were transiently transfected with reporter plasmids p3TP-lux and SBE4-Luc (Addgene) for TGFβ signaling activation and pGL4.33 [luc2P/SRE/Hygro](Promega) for MAPK/ERK signaling activation. Moreover, to evaluate rat Nis and Tg promoter region activity, we used pNis 2.8 (18) and pGl-Tg (19) plasmids.

Briefly, 4 × 10⁴ cells seeded onto 24-well plate. After 72 h, cells were transfected with 300 ng of reporter plasmid plus 30 ng of pRL (Renilla luciferase) using Lipofectamine 2,000 (Invitrogen). Cell lysates were collected after 24 h of transfection, and luminescence was detected using Dual-Glo Luciferase Assay (Promega) in SpectraMax L2 luminometer (SpectraMax). Specifically for testing TGFβ signal responsiveness, PCCL3-ϕ and PCCL3-FAM83F cells were transfected with p3TP-lux and SBE4-Luc prior to the treatment with 1 ng/ml recombinant TGFβ1 (rTGFβ-Peprotech, Rocky Hill, NJ, USA) in culture medium for 24 h. For MAPK/ERK signaling analysis, cells were serum/hormones starved for 24 h before transfection of pGL4.33 [luc2P/SRE/Hygro], and after 2 h of serum addition, cell lysates were collected.