Fig 2: PHF8 and FOXA2 expression in adenocarcinoma and NEPC of patients' specimens. (A) Genetic changes of PHF8 in prostate cancer from the BioPortal database. (B) Immunostaining for PHF8 and FOXA2 in the same patient before and after ADT. (C, D) Statistical results of the IHC scores for (C) PHF8 and (D) FOXA2 in B (paired t-test). (E, F) Immunostaining for PHF8 (E) and FOXA2 (F) in adenocarcinoma, CRPC-Adeno, and NEPC or NED samples (non-parametric test).

Fig 3: PHF8 regulates FOXA2 expression and transcription. (A, B) The mRNA levels for several transcription factors involved in NEPC development were estimated by RT-PCR after PHF8 knockdown by siRNA with scrambled siRNA (siScr) as the control in (A) PC-3 and (B) NE1.3 cells (non-parametric test). (C, D) The expression of FOXA2 and NEPC markers (SYP and CD56) was examined by immunoblotting after siRNA-mediated PHF8 (siPHF8) knockdown in (C) PC-3 and (D) NE1.3 cells. (E) Double immunofluorescence staining analysis of PHF8 and FOXA2 was conducted after PHF8 knockdown by using siRNA. The knockdown cell is indicated by the red arrow. (F) Immunostaining for FOXA2 and NEPC markers (SYP and CD56) in xenografts of PC-3 cells with or without PHF8 knockdown (shRNA) (non-parametric test). (G, H) PC-3 cells with or without PHF8 knockdown (shRNA) were transfected with a FOXA2-Luc construct and Renilla luciferase vector, as well as indicated plasmids. Cell lysates were collected to investigate the expression of PHF8 (G) and measure luciferase activity after 48 h transfection (H) (non-parametric test). (I) PC-3 cells with or without PHF8 knockdown (shRNA) were subjected to CHIP assays with PHF8, H3K9me1, H3K9me2, H3K27me2, and H4K20me1 antibodies. The immunoprecipitated materials were used for qPCR analyses of the promoter regions of FOXA2 (t-test). (J, K) NEPC markers including SYP, CgA, and CD56 were estimated by western blot when FOXA2 was overexpressed in PHF8 KD (shRNA) PC-3 (J) and NE1.3 (K) cells. (L, M) CCK-8 assays were used to investigate the cell viabilities after 24 h of cell culture when FOXA2 was overexpressed in PHF8 KD (shRNA) PC-3 (L) and NE1.3 (M) cells (non-parametric test).

Fig 4: Comparative gene expression analyses of mDA progenitor induction as well as maturation into post-mitotic mDA neurons. (A) qRT-PCR analysis of FOXA2, LMX1A, TH, and GIRK2 at days 10, 20, and 35 of differentiation. The blue bars are illustrating an ameliorated phenotype of 3D cultures compared to the 2D counterpart. Statistical differences were calculated by multiple unpaired t-tests **p = 0.01, ***p = 0.001, ****p = 0.0001. (B) Bars represent expression fold change of FOXA2, LMX1A, TH and GIRK2 genes at each time point (day 10, 20, 35) with respect to differentiation start (hiPSCs) in the RNA-sequencing experiment. Values below 1 indicate down-regulation of expression. Red bars display gene expression change in 2D culture, whereas bars with red and white checker pattern visualize those from 3D culture. A single fold change value is derived from three replicates for each comparison presented here.

Fig 5: Phf8 knockout inhibits the NEPC signature. (A) Heatmap of global altered gene expression in Phf8-KO TRAMP mice compared with control TRAMP mice. (B) Heatmap of a selected gene cluster from an integrated 70-gene NEPC classifier revealed by Beltran et al [11] in both groups. (C) GSEA enrichment plot of the HALLMARK_ANDROGEN_RESPONSE gene set in the above-mentioned mice. (D) Heatmap of AR target gene expression in both groups. (E) Venn diagram revealing the commonly altered genes which were downregulated in Phf8-KO mice compared with control TRAMP mice but significantly upregulated in NEPC patients, by using Beltran et al's published dataset [11]. (F) Immunostaining for FOXA2 in the prostate samples from Phf8-KO TRAMP mice and control TRAMP mice (non-parametric test).