Figure 1

Knockdown of RNF11 exaggerates inflammatory responses following TNF-α treatment. (A) and (B) Stably transduced SH-SY5Y short hairpin RNA (shRNA)-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 24 hours. The relative amount of mRNA expression in stimulated and unstimulated cells was calculated using quantitative RT-PCR (qRT-PCR) and glucuronidase β (GUSB) as an internal control. (C) Stably transduced SH-SY5Y shRNA-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 4 hours before cells were rinsed and fresh media were replaced in the plate. Media were collected after 20 hours and analyzed for TNF-α protein levels using a human TNF-α ELISA. ND, not detectable. (D) Murine primary cultures were harvested, and qRT-PCR was used to measure relative RNF11 mRNA levels. (E) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and mRNA levels were examined by qRT-PCR. (F) Murine primary cortical neurons were transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and protein levels of monocyte chemoattractant protein 1 (MCP-1) were measured using a mouse MCP-1 ELISA. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM with GUSB (for SH-SY5Y cells) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for primary cultures) levels as an internal control for three independent experiments. ELISA values are expressed as averages of protein levels extrapolated from standard curve analysis from three independent experiments. ^*** P < 0.001.