Fig. 4.

NT-induced IL-6 secretion in 3T3-L1-NTR1 mouse preadipocytes involves PKCδ- and NF-κB–dependent pathways: (A) 3T3-L1-NTR1 preadipocytes were pretreated with dimethyl sulfoxide (DMSO; vehicle), CAPE (10 μM), Ca²⁺ dependent PKC inhibitor Go6976 (10 μM), Rottlerin (1–10 μM), PKCθ pseudosubstrate inhibitor (PKCθ PSI; 10 μM) or PKCε pseudosubstrate inhibitor (PKCε PSI; 10 μM) for 30 minutes, followed by NT (10 nM) or TFA (vehicle) for 8 hours. Conditioned media were then collected for mouse IL-6 ELISA. Results were representative of three independent experiments. (B) 3T3-L1-NTR1 preadipocytes were transfected with either full-length IL-6 promoter construct or IL-6 promoter constructs containing mutations in the NF-κB (m-NF-κB), AP1 (m3/m5-Ap1), or C/EBP (mC/EBP) transcription binding sites, followed by NT (10 nM) treatment for 8 hours. Cell lysates were then used to perform luciferase reporter assays. ***P < 0.001 vs. full-length group. (C) 3T3-L1-NTR1 preadipocytes were pretreated with DMSO or Rottlerin (3 μM) for 30 minutes, followed by NT exposure (10 nM) for 30 minutes. Cells were lysed, and equal amounts of protein were used to detect phospho-PKCδ, phospho-p65, and β-actin. (D, E) 3T3-L1-NTR1 preadipocytes were co-transfected with control siRNA or PKCδ siRNA together with a IL-6 promoter (D) and NF-κB luciferase construct (E), followed by exposure to NT (10 nM) for 8 hours. Cell lysates were used to perform IL-6/NF-κB luciferase reporter assay. ***P < 0.001 vs. control siRNA transfected NT-treated group. Results are representative of three independent experiments.