Fig 1.
Discovery approach to identify the common interactors in the SRF and AR pathways.
A. Schematic diagram of the experimental design. LNCaP Abl cells were transfected with either scramble siRNA, AR siRNA, SRF siRNA or with a plasmid overexpressing SRF, with or without DHT stimulation. LC-MS was performed following Co-IP pulldowns with antibodies specific for AR or SRF. B. Schematic diagram of data analysis following MS.
Fig 2.
WB validation of downregulation of AR and SRF and upregulation of SRF following siRNA/plasmid transfections.
Top panels show WB representative images of three independent experiments. Bottom panels show densitometry analysis of WB images. A. AR down-regulation; B. SRF down-regulation; C. SRF up-regulation. Bars represent average of three independent experiments ± standard deviation. Abbrev: Scr = scrambled, EV = empty vector, SRFV = SRF overexpressing vector, SRFV+DHT = SRF overexpressing vector + DHT. Two-tailed t-test was carried out followed by Welch’s correction. * = p<0.05.
Fig 3.
Summary of the identified proteins in different experimental groups.
A) Proteins from the SRF dataset before and after DHT. B) Proteins from the AR dataset before and after DHT.
Table 1.
List of AR/SRF common interactors, and their functions taken from Uniprot (Uniprot KB/Swiss-Prot).
Fig 4.
Pathway and network analysis of the proteins identified in the AR/SRF interactome.
STRING analysis of the common interactors between SRF and AR revealed other proteins involved in the AR/SRF interactome. A. Common interactors and their known interactions with AR and SRF. Common interactor nodes are shown in white. AR and SRF nodes are shown in red and green respectively. Yellow nodes represent other proteins following network expansion. B) Following network expansion, a list of highly significant KEGG pathways were highlighted. Blue nodes represent proteins involved in the PI3k-Akt pathways, red nodes proteins involved in prostate cancer signalling, and green nodes MAPK signalling pathway. The highlighted pathways all have a false discovery ratio below 0.05. The thickness of the lines between proteins indicates the strength of data support.
Table 2.
List of molecular targets identified through MS and STRING analysis.
Fig 5.
Cell viability graphs of inhibitors against HSP70, HSP90, PI3k and AKT.
Dose dependent curves of inhibitors Ver-155008, JG-98, Ipatasertib, Ganetespib and Alpelisib. A) LNCaP Parental. Concentrations used are as follows: Ver-155008 (μM): 0, 2.5, 5, 10, 20, 30, 40. JG-98 (μM): 0, 0.025, 0.050, 0.075, 0.1, 0.15, 0.25, 0.5. Ipatasertib (nM): 0, 5, 10, 20, 20, 40, 80, 100, 200. Ganetespib (nM): 0, 10, 20, 15, 20, 30, 40, 60. Alpelisib (μM): 0, 3, 6, 9, 12 B) LNCaP Abl. Concentrations used are as follows: Ver-155008 (μM): 0, 2.5, 5, 10, 20, 30, 40. JG-98 (μM): 0, 0.025, 0.05, 0.075, 0.1, 0.15, 0.25, 0.5. Ipatasertib (nM): 0, 5, 10, 20, 40, 80, 100, 200. Ganetespib (nM): 0, 8, 12, 15.8, 20, 30, 40. Alpelisib (μM): 0, 1, 5, 15, 20. C) 22Rv1. Concentrations used are as follows: Ver-155008 (μM): 0, 0.5, 1.5, 2, 3, 4, 5. JG-98 (μM): 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4. Ipatasertib (μM): 0, 0.4, 0.8, 1.6, 2, 3, 4. Ganestespib (nM): 0, 5, 10, 12, 15, 20, 25. Alpelisib (nM): 0, 60, 120, 240, 500, 1000, 1500, 3000. For each inhibitor, graphs represent the average of at least three biological replicates in triplicate. Error bars represent standard deviation.
Table 3.
IC50 values of inhibitors compared against enzalutamide in LNCaP parental cells.
IC50 values represent averages of at least three independent experiments in triplicate. One-Way-ANOVA was carried out on each drug, followed by Tukey’s test to compare the mean IC50 value against the mean IC50 value of enzalutamide.
Table 4.
IC50 values of inhibitors compared against enzalutamide in the LNCaP Abl cells.
IC50 values represent averages of at least three independent experiments in triplicate. One-Way-ANOVA was carried out on each drug, followed by Tukey’s test to compare the mean IC50 value against the mean IC50 value of enzalutamide.
Table 5.
IC50 values of inhibitors compared against EPI-7170 in the 22rv1 cells.
IC50 values represent averages of at least three independent experiments in triplicate. One-Way-ANOVA was carried out on each drug, followed by Tukey’s test to compare the mean IC50 value against the mean IC50 value of EPI-7170.
Fig 6.
Proposed common pathways of the SRF/AR interactome and the key proteins identified by APMS and bioinformatics.