Fig. 4: DPYSL5 is a key factor in the regulation of NEPC phenotype and cell proliferation and promotes G1 arrest.

a GSEA reveals that DPYSL5 silencing decreases the expression of genes commonly upregulated in NEPC and epithelial mesenchymal transition in LNCaP EnzR cells. In addition, E2F and EZH2 targets are downregulated. b DPYSL5 silencing leads to the downregulation of EZH2 and truncated JARID2 in LNCaP EnzR cells, and to the downregulation of EZH2, truncated JARID2, EZH1, SUZ12, AEBP2, and H3K27me3 in C42B EnzR cells based on western blot analysis. c Upregulation of luminal markers in DPYSL5 silenced LNCaP EnzR cells. d DPYSL5 silencing decreases neurite branch points and (e) neurite length in LNCaP-EnzR cells. f 122 common genes involved in cell cycle and cell division were identified when genes downregulated by DPYSL5 siRNA in LNCaP EnzR cells (EnzR LNCaP siDPYSL5 down) were compared with genes downregulated by EZH2 (NYUTTEN_EZH2_TARGETS_DN). Left: Venn-diagram of the comparison. Right: Bar graph showing the gene ontology (GO) of the 122 common genes. g DPYSL5 regulates the expression of E2F and CCND1 targets, and cell cycle related genes (p-value > 0.001 was used as a cut off). h Proliferation of LNCaP siRNA control cells (siCTRL, in blue), ENZ treated LNCaP siRNA control cells (siCTRL ENZ, in light blue), DPYSL5 silenced LNCaP cells (siDPYSL5, in red) and ENZ treated, DPYSL5 silenced LNCaP cells (siDPYSL5 ENZ, in light pink) was analyzed using IncuCyte. A significant decrease in proliferation with DPYSL5 siRNA was observed, and the effect was further enhanced when combined with ENZ. Left: Proliferation curves based on confluency analysis with IncuCyte software. Right: Bar graph of the relative confluencies at 96 h timepoint. Bars represent mean ± SD with n = 3. p-values shown as asterisks (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001). i IncuCyte cell cycle analysis and (j) PI staining-based FACS analysis of LNCaP control siRNA cells (siCTRL +DMSO), ENZ exposed LNCaP siRNA control cells (siCTRL +ENZ), DPYSL5 silenced LNCaP cells (siDPYSL5 +DMSO) and ENZ exposed, DPYSL5 silenced LNCaP cells (siDPYSL5 +ENZ) showing an increase of cells in G1 phase in response to DPYSL5 silencing. Red: mKate-labeled Cdt1 (G1/S transition and G1 phase), Green: GFP-labeled Geminin (S/G2 transition and at G2 phase). The effect was more drastic when DPYSL5 silencing was combined with ENZ supplementation when compared to the control cells.