Fig. 3

Effect of ZPT on PKD–NF-κB Signaling and Proinflammatory Gene Expression. (A) Caco2 and SW480 cells were serum-starved for 3 h in the presence of the indicated concentrations of ZPT or a vehicle (DMSO) and subsequently stimulated by 20 µM LPA for 30 min. PKD phosphorylated at Ser916 and total PKD in the cell lysates were quantified by immunoblotting with anti-phospho-PKD (Ser916) and anti-PKD Abs, respectively, as described in Methods. Numbers below the immunoblots indicate fold increase of the phospho-PKD signals divided by the total PKD signals over that at 0 min after LPA stimulation of the control cells. Each of the experiments was performed three times yielding equivalent results. A representative result is shown. The original blots are presented in Supplementary Figures S4 and S5. (B) Caco2 cells treated with 5 µM ZPT or the vehicle as described in (A) were subjected to subcellular fractionation and the resulting nuclear and cytoplasmic fractions were subjected to immunoblotting with the anti-NF-κB p65 Ab. TATA-binding protein (TBP) and α-tubulin were used as markers for the nuclear and the cytoplasmic fractions, respectively. A fold change of the nuclear NF-κB caused by 5 µM ZPT treatment is shown in numbers. Each of the experiments was performed three times yielding equivalent results. A representative result is shown. The original blots are presented in Supplementary Figure S6. (C) Total cellular RNAs isolated from Caco2 cells treated as described in (B) were subjected to qRT-PCR for quantification of the mRNA levels of the proinflammatory molecules: CXCL1, CXCL8, CCL2, CCL20, TNF-α and COX-2, by using the b-actin mRNA as an internal control. The obtained values are expressed as relative fold changes of nuclear NF-κB normalized to TATA-binding protein (TATA), with the value from the − LPA/−ZPT condition set to 1. The experiments were performed three times in duplicates.