Fig. 3

YBX1/G3BP1 complex up-regulates SPP1 to activate downstream NF-κB signaling pathway in RCC cells. (a) Analysis of the effect of YBX1 knockdown (upper panel) or G3BP1 knockdown (lower panel) on SPP1 mRNA levels using real-time PCR. (b) Western blot assays were performed to measure the YBX1, G3BP1, SPP1, p-p65 (Ser536), and total p65 in treated ACHN cells. GAPDH was used as an internal control. (c) Cells were co-transfected with NF-κB pathway firefly luciferase reporter together with internal control Renilla luciferase reporter (pRL-TK) vector. Then, luciferase activity was measured using a dual-Luciferase Reporter Assay System (Promega). (d) SPP1 knockdown by using three independent SPP1 siRNAs (si-SPP1–1, si-SPP1–2, si-SPP1–3) in ACHN cells were evidenced by western blot. β-actin was used as loading control. ACHN cells were transfected with pWPI+si-NC, pWPI-YBX1 + si-NC, pWPI-YBX1 + si-SPP1; pEGFP-C1 + si-NC, GFP-G3BP1 + si-NC, GFP-G3BP1 + si-SPP1, and then (e) the expression levels of YBX1, G3BP1, SPP1, p-p65 (Ser536), and total p65 were examined by western blot. β-actin was used as an internal control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001