Journal: International Journal of Molecular Sciences
Article Title: PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-κB Signaling Pathway
doi: 10.3390/ijms231911414
Figure Lengend Snippet: PAUF activates the MyD88-dependent, not the TRIF-dependent, TLR4 downstream pathway. ( A ) Panc-1_Mock and Panc-1_TLR4 OE cells were treated with rPAUF (0.1 μg/mL) for 0, 10, 30, and 60 min. Collected proteins were immunoprecipitated with MyD88 antibody (α-MyD88) using protein A agarose beads. Immunocomplexes were determined by Western blot against MyD88 and TLR4. ( B ) Panc-1_Mock and Panc-1_TLR4 OE cells were treated with rPAUF (0.1 μg/mL) for 0, 60, and 120 min. Collected proteins were immunoprecipitated with TRIF antibody (α-TRIF) using protein A agarose beads. Immunocomplexes were determined by Western blot against TRIF and TLR4. ( C ) Western blot showed that Panc-1_trans TLR4 OE cells were successfully engineered to transiently overexpress TLR4, compared to the control, Panc-1_trans Ctrl cells. ( D ) Panc-1_trans Ctrl and Panc-1_trans TLR4 OE cells were pretreated with or without IKK inhibitor XII (5 μM) for 4 h before rPAUF (0.1 μg/mL) or PBS treatment. After overnight culture, cells were collected for dual-luciferase activity assay. Data are represented as a mean ± SD from triplicates (**** p < 0.0001 were obtained from two-way ANOVA and post-hoc multiple comparisons with Bonferroni correction). ( E ) Panc-1_Mock and Panc-1_TLR4 OE cells were treated with rPAUF (0.1 μg/mL) for 0, 10, 30, 60, 120, and 240 min, and activation of TRIF-dependent TLR4 downstream molecules (p-TBK1 and p-IRF3) was estimated using Western blot.
Article Snippet: For luciferase reporter assays, the pGL4.32[luc2P/NF-κB-RE/Hygro] and pRL-TK vectors, Lipofectamine 2000 transfection reagent (cat# 11668019) was purchased from Invitrogen, and Dual-Luciferase Reporter Assay System (cat# E1960) were purchased from Promega (Madison, WI, USA), the pDUO-mcs vector (cat# pduo-mcs) was purchased from InvivoGen (San Diego, CA, USA), and the IKK inhibitor XII (cat# 401491) were purchased from Sigma-Aldrich.
Techniques: Immunoprecipitation, Western Blot, Luciferase, Activity Assay, Activation Assay
