Figure 4.

GnP triggers GPRC5A-mediated CXCL8 secretion by PDAC cells and leads to NET formation and chemoresistance. (A) mRNA expression of genes in GnP-sensitive or -resistant MIA PaCa-2 cells, detected by qPCR. (B) HIF1A, GPRC5A, and YAP1 protein expression in GnP-sensitive or -resistant MIA PaCa-2 cells, detected by western blot. (C) GPRC5A protein expression in GnP-sensitive or -resistant MIA PaCa-2 and PANC-1 cells treated by si-NC or si-GPRC5A, detected by western blot. NC: GnP-sensitive PANC-1 cells transfected with si-NC; GP^knock: GnP-sensitive PANC-1 cells transfected with si-GPRC5A; GnP: GnP-resistant PANC-1 cells transfected with si-NC; GnP+GP^knock: GnP-resistant cells transfected with si-GPRC5A. (D) CXCL8 concentration in the supernatant of GnP-sensitive or -resistant MIA PaCa-2 and PANC-1 cells treated by si-NC or si-GPRC5A, detected by ELISA. (E) MPO-DNA and (F) the fluorescence intensity of SYTOX Green in HL-60-induced neutrophils treated by the supernatant of GnP-sensitive or -resistant MIA PaCa-2 and PANC-1 cells treated by si-NC or si-GPRC5A. (G) Detection of co-localized staining of NETs for DAPI, citrullinated histone H3 (citH3), and myeloperoxidase (MPO) using a confocal microscope in the bone marrow-derived neutrophils treated by the CM of GnP-sensitive or -resistant PANC-1 cells. Scale bar, 50 μm. All experiments were performed at least three times. For statistical analyses, a one-way ANOVA was used for (D), (E), and (F). ***, P < 0.001; ns, not significant. n = 6, Data are the mean ± SD.