Figure 4.

PODXL1 accelerates PDAC cellular motility by activating C5aR with triggering EMT. (A) IP assay demonstrated PODXL1 specifically bound to C5aR. Endogenous expression of PODXL1 in the PODXL1-WT and the KO MiaPaCa-2 cells (left). Precipitates by anti-PODXL1 Ab probed with anti-C5aR Ab showed PODXL1-C5aR binding not in the KO cells but in the WT cells (right). (B) Visualizing the alteration of C5aR intracellular localization with or without PODXL1 by Fluoppi assay. Cellular localization of C5aR was examined on HeLa cotransfected with PODXL1-Ash and C5aR-red compared with the cells transfected with C5aR-red alone (lower panel). Correct system behavior was confirmed by the PODXL1-Ash + CTTN-red cotransfected cells (CTTN is a known binding partner of PODXL1) as a positive control (upper panel). Arrows indicated membrane localization of fluorescent dots. (C) Loss of the cellular motility in the PODXL1-KO cells treated with C5a by invasion assay (Boyden chamber assay). Accelerated motility of the PODXL1-WT MiaPaCa-2 in response to C5a (left), loss of the response in the PODXL1-KO cells (right). (D) Endogenous expression of C5aR in the PODXL1-WT and the KO cells of MiaPaCa-2 or AsPC-1 by IF using confocal-laser scanning microscope. (E) Decreased expression of C5aR in the PODXL1-KO PDAC cells in comparison with that in the WT cells (left). Marked shift of intracellular localization of C5aR demonstrated by IB for the extracts between membrane fraction and cytosolic fraction from the PODXL1-WT and PODXL1-KO PDAC cells (right). Transferrin receptor and CXADR were shown as positive control for membrane extract. (F) EMT triggered with C5a stimulation in the PODXL1-WT PDAC cells. Decreased expression of E-Cadherin, upregulation of Snail and Vimentin in the WT cells only when stimulated with C5a by IB. Tubulin was shown as a control.