Silencing of Rab7b expression promotes LPS-induced production of proinflammatory mediators in macrophages. Rab7b expression was regulated by LPS. (A) Peritoneal macrophages and (B) Raw 264.7 cells were treated with 100 ng/mL LPS as indicated. Expression level of Rab7b was examined by both RT-PCR (upper panels) and quantitative PCR (lower panels). For quantitative PCR, the results were presented as folds expression of Rab7b mRNA to that of β-actin. (C) Efficiency of transient Rab7b silencing. 1.5 × 105 mouse peritoneal macrophages were transfected with 0.1 nM control small RNAs (Ctrl siRNA1 and Ctrl siRNA2) or Rab7b siRNA (Rab7b siRNA1 and Rab7b siRNA2); 48 hours later, the efficiency of silencing was examined by either RT-PCR (upper panel) or quantitative PCR (lower panel). Results were presented as described for panels A and B. (D) Production of proinflammatory mediators by Rab7b-silenced peritoneal macrophages and (E) Raw 264.7 cells after LPS treatments. Cells were silenced with small RNAs as described for panel C, and the proinflammatory mediators were measured either by enzyme-linked immunoassay (for TNF-α, IL-6, and IFN-β), or Griess reaction assay (for NO). Similar results were obtained in 3 independent experiments. Data are presented as means (± SE). ▴P < .05; *P < .05; **P < .01; ***P < .001.
