Figure 1
TNFα activates the inflammatory IKK-β/NF-κB cascade in rHypoE-7 cells. A. Summary of gene transcripts detected in the hypothalamus and rHypoE-7 cell line by semi-quantitative reverse transcriptase-PCR (RT-PCR) (transcript absent (-), present (+), or abundant (++); neuron specific enolase (NSE), G-protein coupled receptor (GPR) 40 (GPR40), GPR120, Agouti-related neuropeptide (AgRP), pro-opiomelancortin (POMC), tumor necrosis factor alpha receptor (TNFαR), toll-like receptor 4 (TLR4), nuclear factor kappa B (NFκB), NFκB inhibitor alpha (IκBα), IkappaB kinase beta (IKK-β). B. Western blots showing activation and thus phosphorylation of TAK1 (pTAK1), IKK-β (pIKK-β), and JNK (pJNK) upon TNFα treatment (1 to 100 ng/mL, 10 min) or H20 control relative to β-actin. C. Bar graph showing the mRNA levels of IκBα (i) and TNFα (ii) after vehicle (H20) or TNFα treatment (1 to 100 ng/mL, 2 to 6 hr, n = 3 to 7). D (i). Western blot showing TNFα protein production after vehicle (H20) or TNFα exposure (10 ng/mL, 2 hr) in serum-free media followed by a 6-hr recovery incubation in media with FBS. (ii). Production of TNFα protein was significantly enhanced relative to vehicle (H20) control (1.39 ± 0.07 TNFα/βactin protein, n = 4). E. Pretreatment of rHypoE-7 cells with the IKK-β inhibitor, PS1145 (20 μM, 1 hr) prior to the addition of TNFα (10 ng/mL, 2 hr) significantly reduced the transcriptional inflammatory response as shown by a reduced mRNA level of IκBα (i) (DMSO; white bar: 2.95 ± 0.25 and PS1145; gray bar: 1.10 ± 0.08 IκBα/histone mRNA, n = 7) and TNFα (ii) (DMSO; white bar: 2.84 ± 0.32 and PS1145; gray bar: 0.56 ± 0.10 TNFα/histone mRNA, n = 7). Data are shown as mean ± SEM; *P <0.05; **P <0.01; ***P <0.01).