Fig. 6

TAEKDEL peptide transactivates EGFR-STAT3 signaling pathway. A, B Endocytosis of KDELR induced by KDEL ligand was inhibited by a EGFR inhibitor. HeLa cells expressing Halo-KDELR were treated with non-permeable HaloTag Alexa Fluor 488 ligand and DMSO, 250 nM PD153035 or 20 nM Crizotinib at 37 °C for 2 hous, prior to incubation with 50 μM TAEKDEL peptide at 4 °C for 30 minitures. Cells were then incubated at 37 °C for 0 or 30 min and stained for endogenous EGFR (A). Co-colization of EGFR and KDELR in the peri-nuclear region was represented by Pearson’s coefficient and quantified using one-way ANOVA with a Dunnett’s multiple comparisons test (B). n = 20 cells pooled from 3 independent experiments. ****: P < 0.0001. Scale bar = 10 μm. C, D Phosphorylation of EGFR and STAT3 was increased by TAEKDEL incubation. HT1080 cells stably overexpressing KDELR-mCherry were incubated with DMSO, 1.5 nM EGF, or 50 μM TAEKDEL at 37 °C for 30 min. Cell lysates were prepared and subjected to immunoblotting. (C). Fold change of phosphorylated proteins was statistically analyzed using one-way ANOVA with Dunnett’s multiple comparisons (D). n = 3 independent experiments. *: P < 0.05. **: P < 0.01. ****: P < 0.0001. ns: not significant (D). E, F Cyclin-D1, Bcl-2, Vimentin, ICAM-1 mRNA in HT1080 KDELR-mCherry cells treated with GRP78 (D) or TAEKDEL (E) at 37 °C was quantified using RT-PCR and analyzed by the ΔΔCT method. Statistical significance was calculated using two-way ANOVA with a Dunnett’s post-hoc test for multiple comparisons. n = 3 independent experiments. *: P < 0.05. ***: P < 0.001. ****: P < 0.0001. ns: not significant