Figure 3: Elucidating the mechanism of GNPNAT1 KD driving an aggressive phenotype in CRPC-like cells.

(a) Heat map showing differential (P<0.05, FDR 30%) genes in LNCaP-ABL cells containing GNPNAT1 KD (L-KD1, n=4; and L-KD5, n=2) compared with NT controls (L-NT, n=3). (b) Immunoblot showing increased expression of phosphorylated and total PI3K (∼85 kDa, n=3 for PI3K-P85 and n=2 for PI3K-19H8), AKT (∼60 kDa, n=4 for AKT, n=3 for p-AKT (T308) and n=4 for p-AKT (S473)), m-TOR (∼281 kDa, n=5) and p-MAPK (∼44-42 kDa, n=3) in LNCaP-ABL KD cells compared with controls. In all cases, representative immunoblots are shown. (c) Treatment of LNCaP-ABL NT and KD cells with either 20 μM Perifosine (AKT/PI3K inhibitor, (n=3) or 50 μM of LY294002 (PI3K) inhibitor (n=3), significantly decreased KD1 cell proliferation and modestly reduced KD5, both compared with treated NT control. (d) Immunoblot showing increased expression of AR in GNPNAT1 KD LNCaP-ABL cells compared with control (∼110 kDa, n=3, representative immunoblot). (e) QPCR validating increased expression of AR-target gene KLK3 (PSA) in LNCaP-ABL KD cells (n=3). (f) QPCR validating increased expression of AR and its co-activator NCOA3 and downstream target KLK3 in xenograft tumours derived from LNCaP-ABL KD cells compared with NT control (n=4). (g) Heat map showing differential (P<0.05, FDR 20%) genes in 22Rv1 cells containing GNPNAT1 KD (22R-KD1 and 22R-KD5, n=4 each) compared with NT controls (22R-NT, n=4). (h) Promoter Scan on genes upregulated in KD cells revealed enrichment for SP1-binding sites. (i) Immunoblot showing significantly (P<0.05) increased protein levels for SP1 (∼81 kDa, n=5) in 22Rv1 cells containing GNPNAT1 KD (22R-KD1 and 22R-KD5, median band intensity of 0.66±0.23 and 0.51±0.08, respectively) compared with NT control (median band intensity of 0.35±0.06). Actin was used to control for protein loading. (j) Chromatin immunoprecipitation (ChIP) assay-coupled to QPCR to verify SP1 binding to the promoter of ChREBP. SP1 binding to the promoter of DihydroFolate Reductase (DHFR) was used as a positive control (n=3, median and standard deviation shown). (k) QPCR validation of ChREBP expression in 22Rv1 KD and NT cells (n=3). (l) Immunoblot showing elevated protein expression of ChREBP in 22Rv1 KD cells compared with NT control (∼65 kDa, n=3, representative immunoblot). P-values were computed using Unpaired Student’s t-test (for f) and ANOVA model (c,e,j and k). For e and f, the fold change was log 2 transformed to get normal distribution. In all cases, P-value: *P<0.05. For boxplots, the horizontal line represents median value, whereas Whiskers represent either <25 or >75 quartile ranges. All bar plots are represented in median±s.d.