Fig. 2. KIF1A overexpression induces NEPC phenotype.

A The morphology of cells was imaged by Zeiss light microscope, Scale bar = 20 μm. B Quantitative result of protrusions length in LNCaP or LNCaP-AI cell. C Heatmap of AR signaling and NE associated genes in LNCaP cells or LNCaP-AI cells. D Gene sets significantly enriched in LNCaP cells compared to LNCaP-AI cells. NES and p value were reported. E Cell viability measured in the indicated cell lines by CCK-8 assay. LNCaP and LNCaP-AI cells were treated with titrated doses of ENZ for 3 days. F NE markers and KIF1A protein levels in LNCaP or LNCaP-AI cell. G Representative images of immunofluorescence of KIF1A in LNCaP or LNCaP-AI cells. Cells were imaged by confocal microscopy. Scar bar = 5 μm. H The mRNA levels of KIF1A in LNCaP/LNCaP-AI cells. The mRNA was first measured by RNA-seq (lower panel) and then validated through qPCR (upper panel). I Western blot of NE markers (NCAM1, ENO2, SYP) and KIF1A in indicated PCa cells with KIF1A overexpression or knockdown. J Heatmap showing NE associated genes expression in indicated LNCaP-AI cells. siKIF1A-2 were utilized as siKIF1A. K Gene sets significantly enriched in high- KIF1A group (NC) compared to siKIF1A in LNCaP-AI cells. NES and pvalue were reported. L GSEA of the EMT hallmark genes in NC and siKIF1A LNCaP-AI cells. M GSEA of the stem cell up-regulated genes in NC and siKIF1A LNCaP-AI cells. N Western blot of KIF1A and EMT markers in indicated PCa cells with KIF1A overexpression or knockdown. O Western blot of KIF1A and stemness related genes in indicated PCa cells with KIF1A overexpression or knockdown. All results were presented as the mean ± SD. ****p <0.0001, based on Student’s t test. ADT androgen deprivation therapy, ENZ enzalutamide, NSE normalized enrichment score.