Figure 6

Podoplanin-dependent VEGF-C downregulation is required for JNK activity. A) Following 24-hour cultivation of each clone under serum-free conditions, the medium was replaced with fresh serum-free medium containing JNK inhibitor, sp600125 (SP, 20 μM), or a negative control regent (NC, 20 μM) and incubated for 6 hours. Treated cells were harvested, and subjected to real-time RT-PCR for VEGF-C and GAPDH. The VEGF-C mRNA level normalized with each GAPDH level was expressed as a relative fold increase compared to that in EBC1-V1 (n = 3 each, *p < 0.05, **p < 0.005). B) After the same treatment as described in A, the cells were harvested and subjected to Western blot analysis for phospho-JNK and subsequently to re-probing for total JNK and then to β-actin. Two isoforms of JNK with different molecular weights, JNK1 (p46) and JNK2 (p54) were detected (arrows). C) SiRNA for human podoplanin and negative control oligonucleotides (siRNA podo. and siRNA cont., respectively) were transfected to EBC1-P4 cells as described in Methods. Twenty-four hours after transfection, the medium was replaced with serum-free fresh medium. Twenty-four hours later, cells were harvested and subjected to real-time RT-PCR for human podoplanin, VEGF-C, and GAPDH. Podoplanin and VEGF-C mRNA levels were normalized with the GAPDH level and expressed as relative fold increases compared to those of the control group (n = 3 each, *p < 0.05, **p < 0.0001). D) Following similar treatments of siRNA to those described in C, the medium was replaced with serum-free fresh medium. Six hours later, cells were harvested and subjected to Western blot analysis for phosphorylated JNK and subsequent re-probing for total JNK and β-actin.