Elucidating THP-1 and LX2 Cell Crosstalk

On the one hand, THP1 cells were seeded 1x10⁶ cells per well in six-well plate and treated with palmitate (PA,1 mM, Sigma-Aldrich, USA). Meanwhile, different siRNA-lipofectamine™ 3000 (Cat# L3000015, Invitogen) mixture was added into cell culture medium. After 48h, total RNA of THP1 cells were extracted to detect the levels of Jun, spp1, Socs3 and Rac1. On the other hand, THP1 cells were seeded 1x10⁶ cells per well on six-well upper trans-well insert (0.4µM), LX2 cells were seeded 1x10⁵ cells in the lower wells of trans-well plates and attached overnight. After 24h, THP1 cells were washed with phosphate-buffered saline solution (PBS), and treated with PA (1 mM) and different siRNA-lipofectamine™ 3000 mixture, each upper insert was transferred to a lower plate containing the LX2 cells. Thereafter, THP1 cells and LX2 cells were co-cultured in serum-free medium for 48 hours. Total RNA of LX2 cells were extracted to detect the levels of α-smooth muscle actin (SMA), collagen type I alpha 1 (Col1a1) and Fibronectin (Fn). The sequences of different siRNA were listed in Supplementary Table 1.