Figure 5.

SOX4/11 proteins are stabilized by inflammatory cytokines. A and B, Fold changes (FCs) in Sox4, Sox11, and Il6 mRNA levels (A) and SOXC protein levels (B). Mouse FLS at passage 4 were treated with 5 ng/ml tumor necrosis factor (TNF) for 8 hours. Messenger RNA levels were measured by quantitative reverse transcription–polymerase chain reaction, and protein levels were determined by Western blotting of whole‐cell extracts from triplicate cultures. C and D, Fold changes in RNA (C) and protein (D) expression in SW‐982 cells treated with 5 ng/ml TNF, 10 ng/ml interleukin‐1α (IL‐1α), 10 ng/ml IL‐6, or 10 ng/ml IL‐10 for 8 hours. Nuclear protein P84 and GAPDH cytoplasmic protein were used as loading controls. E and F, Fold changes in FLAG‐SOX mRNA (E) and protein (F) levels in whole‐cell extracts. SW‐982 cells were transfected with expression plasmids encoding FLAG‐SOX4 and FLAG‐SOX11 for 16 hours and treated with cytokines, as described in C and D. G, SOX4 and SOX11 protein levels in whole‐cell extracts, as determined by Western blotting. SW‐982 cells were transfected with expression plasmids encoding FLAG‐SOX4 and FLAG‐SOX11 for 16 hours, after which time the medium was left unsupplemented or supplemented with 5 μg/ml cycloheximide (CHX) for 15 minutes, with or without subsequent supplementation with 5 ng/ml TNF for 1, 2, or 4 hours. H, FLAG‐SOX4 and FLAG‐SOX11 protein levels in SW‐982 cells treated with or without MG‐132 and/or TNF, as determined by Western blotting. Fold‐change values are the mean ± SD. See Figure 1 for other definitions.