FX-9 (3-(p-Tolyl)isoquinolin-1-amine) was synthesized by Feng and Wu . The 10 mM stock solution was dissolved in dimethylsulfoxide (DMSO; Merck KGaA, Darmstadt, Germany) and stored at − 20 °C. For the experiments a final concentration of 5 μM was used in accordance with our previous results  and prepared immediately prior to each experiment.
Cell lines and cell culture
PC-3 is an androgen-insensitive human cell line of a bone metastasis from a prostate carcinoma of a 62-year-old man . The cell line was cultivated in 25 cm2 cell culture flasks in medium 199 (Gibco™, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum superior (Biochrom GmbH, Berlin, Germany) and 2% penicillin-streptomycin (Biochrom GmbH). The cells were cultivated at 37 °C and 5% CO2 in a humidified atmosphere.
RNA-isolation and –integrity
The cell lines were seeded at a density of 600,000 cells in 5 ml culture medium in 25 cm2 cell culture flasks and incubated for 12 h at 37 °C and 5% CO2 in a humidified atmosphere. The cells were exposed to 5 μM FX-9 for 6 and 12 h, cultured in an incubator at 37 °C and 5% CO2 in a humidified atmosphere. The concentration was selected as it had shown significant anti-proliferative effects on PC-3 in our previous study . Time points were chosen in accordance with our previous cell biological results characterizing early transcriptome alteration before induction of cell death. Untreated cells served as control. The cells were detached with a cell scraper and collected in phosphate-buffered saline. The cell suspension was transferred into tubes and centrifuged at 20 °C and 150 x g for 10 min. The supernatant was discarded, the cell pellets were lyzed using 600 μl chaotropic buffer (RLT plus buffer, Qiagen GmbH, Hilden, Germany). The lyzates were transferred into cryotubes and stored at − 80 °C. RNA extraction was performed using the RNeasy Plus Kit (Qiagen GmbH) including a DNA removal step in accordance with the manufacturer’s protocol. The RNA samples were quantified spectrophotometrically (Nanodrop 1000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA)) and diluted to a concentration of 70 ng per microliter for RNA integrity analysis (Agilent RNA 6000 Nano Chip using Bioanalyzer 2100 instrument (Agilent Technologies, Inc., Santa Clara, Ca, USA)). Samples were prepared as independent biological replicates. All samples showed an RNA integrity number of 10.0.
Gene expression level profiling
200 ng RNA was used as starting material in the GeneChipR Whole Transcript Sense Target Labeling protocol (Affymetrix, Thermo Fisher Scientific, Inc.). The microarray hybridization was performed using the Affymetrix Clariom™ S Array Kit in accordance with the manufacturer’s instructions (Affymetrix, Thermo Fisher Scientific, Inc.): in detail, the so called WT (Whole Transcriptome) protocol started with first strand synthesis by introducing T7 promoter tags to all RNA molecules using N6 3` ends. After strand replacement in accordance with Eberwine, non-labeled aRNA (antisense RNA) was produced by in vitro transcription in agreement with the linear amplification of all RNA molecules without a 3’bias. After an aRNA cleanup (magnet bead based), a new strand identical single-strand DNA was produced using the aRNA as template by adding random primers and deoxyribonucleoside 5′-triphosphates (dNTPs). In the meantime, a certain amount of dTTP was replaced by dUTP. After removing the aRNA by RNaseH digestion and clean-up (magnet bead based), this enabled an enzymatic endpoint fragmentation. Therefore, uracyldeglycosidase removed the uracils in combination with APE1 (apurinic apyrimidinic endonuclease 1), which is cleaved the deuracylized phosphodiester backbone of the single strand DNA molecules. Desoxynucleotidyltransferase was added to the DNA labeling reagent (Biotin-11-dXTP) to the 3` ends of the single strand DNA fragments. The hybridization was carried out overnight (16 h) at 45 °C in the GeneChipR Hybridization Oven 645 (Affymetrix, Thermo Fisher Scientific, Inc.). Washing and staining protocols, including an antibody amplification, were applied by the GeneCip Fluidis Station 450. The microarrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix, Thermo Fisher Scientific, Inc.) at 0.7 μm resolution.
Analysis of the microarray data
Affymetrix Clariom™ S Arrays interrogate more than 20,800 genes using about 800,000 probes. The data was analyzed by the Transcriptome Analysis Console (TAC) version 188.8.131.52 (Applied Biosystems, Thermo Fisher Scientific, Inc.) with the implemented SST-RMA normalization algorithm. Replicate groups were summarized by LIMMA statistics. Differentially expressed genes (DEGs) were identified using filter parameters fold change (FC) < 2 or < − 2, LIMMA p-value < 0.05, and FDR-value < 0.05.
Analysis of signaling pathways and biological processes
To analyze the cellular signaling pathways effected by FX-9, the total number of DEGs were examined in the known pathway network Reactome . Analyzing DEGs by gene ontology functional processes was performed via the gene network Database for Annotation, Visualization and Integrated Discovery (DAVID, DAVID Bioinformatics Resources 6.8) . DEGs enriched signaling pathways with an FDR-value < 0.05 were considered for further analysis.
Immunocytochemical (ICC) staining
PC-3 cells were seeded at a density of 5 × 106 cells per 75 cm2 culture flask and were incubated over night to adhere. After exposure to 5 μM FX-9 or cell culture medium for control for 12 h or 24 h, the cells were harvested by TrypLE™ Express Enzyme (Gibco™, Thermo Fisher Scientific, Inc.) and pelleted in 1.5 ml tubes by centrifugation (20 °C, 150 x g, 10 min). The cell pellets were fixed in 1 ml cold 4% paraformaldehyde and stored at 4–8 °C for a maximum of 7 days until embedding in paraffin. After slicing the paraffin-embedded cell pellets, they were mounted on slides. Thermal antigen demasking in citrate buffer was performed for 20 min in a microwave. To exclude non-specific binding, the slides were blocked with 20% goat serum for 20 min. This was followed by incubation with mouse monoclonal primary antibodies p21 Waf1/Cip1 (diluted 1:100, sc-6246, Santa Cruz Biotechnology, Inc., Dallas, TX, USA,), E2F-1 (diluted 1:200, sc-251, Santa Cruz Biotechnology, Inc.,), PAI-1 (diluted 1:50, sc-5297, Santa Cruz Biotechnologys, Inc.), and rabbit monoclonal primary antibody NFkB2/NFkB p100 (diluted 1:200, JM82–03, Novus Biologicals, LLC, Littleton, CO, USA) at 4 °C over night. After a 45-min incubation period with biotinylated secondary antibodies goat anti-mouse (diluted 1:200, BA-9200, Vector Laboratories, Inc., Burlingame, CA, USA) or goat anti-rabbit (diluted 1:200, BA-1000, Vector Laboratories, Inc.,) indirect avidin-biotin-peroxidase staining with VECTASTAIN®Elite® ABC-kit (Vector Laboratories, Inc.) was performed in accordance with manufacturer’s protocol. Reaction was visualized by incubation in 0.1 g 3,3′-diaminobenzidine tetrahydrochloride (DAB, Acros Organics, BVBA, Fair Lawn, NJ, USA) in 200 ml PBS and counterstaining was performed with hematoxylin. The experiment was performed in triplicates. Staining intensities and the fraction of positively stained cells were scored as proposed by Sorenmo et al.  and Pagliarone et al.  by an experienced and blinded pathologist.