Fig. 2.

sPIF represses let-7 by destabilizing KSRP, which involves PI3K/AKT signaling. (A and F) Cells were incubated with sPIF (+) or PIFscr (−) (200 nM for RAW cells and 300 nM for N2a cells) for 24 h, followed by the addition of CHX. Proteins were harvested at the indicated time points and were analyzed. KSRP protein levels are presented after normalization against β-tubulin, with the KSRP level at the 0 time point arbitrarily set as 1. Results are representative of three independent experiments. (B, C, G, and H) Cells were treated with sPIF (+) or PIFscr (−) in the presence or absence of Akt Inh for 48 h. Proteins and RNAs were isolated, and levels were determined by Western blot and qRT-PCR, respectively. (D, E, I, and J) Cells were transfected with siCon or siRNA specific for KSRP (siKSRP). Proteins and RNAs were extracted 48 h later and were analyzed by Western blot and qRT-PCR, respectively. Except in A and F, all numbers are mean ± SD (n = 3), and are representative of three independent experiments. **P < 0.01. (K) Proposed model for sPIF-induced let-7 repression. PIF activates PI3K/AKT signaling through TLR4, leading to KSRP protein degradation, which in turn decreases the production of mature let-7.