LPS reduces CXCL12-mediated migration by RGS1 in RPMI 8226 cells.

<p>RPMI 8226 cells (with or without LPS treatment) were assessed using the transwell migration assay. The upper chamber contained 1 × 10⁵ cells in 100 μL media, and the lower chamber contained 600 μL media. A) LPS reduced CXCL12-mediated transwell migration. Cells were cultured in the presence or absence of LPS for 18 hours before assessment using the migration assay. Cells were allowed to migrate for 5 hours in the absence or presence of 100 ng CXCL12 in the lower chamber of the transwell assay. After 5 hours of migration, 300 μL of the lower chamber media were removed, and cells were counted using FACS and PI. B) CXCR4 expression in LPS-treated RPMI 8226 cells. After 18 hours of incubation with LPS, 2 × 10⁵ cells were harvested, resuspended, and incubated with CXCR4-APC or mouse IgG-APC for 20 minutes on ice. The surface CXCR4 expression was then analyzed using FACS. C) In total, 2 × 10⁶ cells were transfected using a Neon electroporator with siRNA-targeting RGS1 (RGS1i) or nontargeting scrambled RNA (Scr). Three days after electroporation, cells were stimulated with LPS for 18 hours. The reduction in RGS1 RNA expression was assessed using quantitative PCR and RGS1 protein expression was accessed with western blot by anti-RGS1 antibody. D) Transwell migration of Scr-transfected cells and RGS1i-transfected cells, with or without LPS stimulation (*<i>p</i> < 0.05; ***<i>p</i> < 0.0005; N/S: nonsignificant).