Effect of TD-derived peptides on CME, Tfn recycling, and AP1-mediated Golgi trafficking. (A) Effect of the inhibitory activities of the six peptides on the surface-bound TfnR. (B and C) Single-round Biotin-Tfn uptake revealed that Wbox2 strongly enhanced surface bound Biotin-Tfn (B) and reduced Biotin-Tfn uptake efficiency (C). (D–F) The Wbox2 peptide inhibited TfnR uptake in Fibroblast, HCC4017, and HeLa cells. (G and H) Effects of Wbox2 on Biotin-Tfn recycling after either 10- or 30-min pulse. (I–K) Immunostaining of AP1 γ-adaptin and M6PR in control HeLa cells as well as cells treated with Wbox2 (10 µM, preincubation for 30 min at 37°C) or siRNA knockdown CLCa+b (siCLCs). The AP1 and M6RP distribution in cells was quantified by grading them on the basis of degree of concentration at the perinuclear region: lowest score for completely dispersed phenotype, highest score for majority of signal being concentrated at perinuclear region, as representative images of M6PR shown in I. Scale bars = 10 µm. (J and K) Wbox2 treatment did not alter AP1 or M6PR distribution, whereas siCLCa+b as a positive control showed a strong effect by accumulating AP1 and M6PR in the perinuclear region. Number of cells quantified in J: 94 for control, 106 for Wbox2, and 33 for siCLCs. Number of cells quantified in K: 95 for control, 62 for Wbox2, and 37 for siCLCs. Error bars are SD. ***, P ≤ 0.001. N.S., not significant.