2.1 Cell culture and drugs

All CRPC cell lines, including DU145, PC3 and C4-2, were purchased from ATCC (USA) and cultured as ATCC’s suggestions. PC3 and DU145 monoclonal cells expressing GV492-β-arrestin2 (LV-ARRB2) or empty vector GV492 (LV-C) were generated and cultured in the presence of puromycin (2 μg/mL). The proteasome inhibitor MG132 and the ERK1/2 inhibitor U0126 were purchased from Selleck Chemicals.

2.2 Lentivirus packaging and cell transfection

Short hairpin RNA (shRNA) was synthesized by GenePharma (Suzhou, China). The shRNA sequences targeting human β-arrestin2, PKM2 and hnRNP A1 were 5′-GGACCGCAAAGTGTTTGTG-3′ (shRNA-ARRB2), 5′-GGAAAGAACAUCAAGAUUATT-3′ (shRNA-PKM2) and 5′-GUAUCCAUUAUCAUGUGUA (shRNA-hnRNP A1) respectively. The sequence of unrelated shRNA was 5-TTCTCCGAACGTGTCACGT-3′ (shRNA-NC). The lentivirus expression plasmids containing β-arrestin2 was constructed by Genechem (Shanghai, China) using GV492 vector. Cells were seeded in 60-mm dishes for transient transfections and transfected at 70% confluence. According to the manufacturer's instructions, the transfections were conducted with shRNAs using lentivirus soup in the presence of 1 µg/mL polybrene.

2.3 Cell viability assay

The cell viability was detected using MTT and colony formation methods. For MTT assay, equal amounts of cells contained 0.5% FBS, with or without DTX for indicated times, at the end of the experiment, 10 μL of MTT (5 mg/mL in PBS) were added and the cells were incubated for 3 h in the humidified incubator that contained 5% CO2 at 37 ˚C. Then remove the medium and add 100 µL of DMSO into each well, wrap the plate in foil and shake on an orbital shaker for 10 min. The absorbance was obtained via using a microplate reader at 490 nm. For the colony formation assay, cells were seeded in 60 mm cell culture dishes at a density of 1 × 103 per well and grown in complete medium with or without DTX (5 nM) for 14 days, then the cells were fixed with 4% paraformaldehyde, stained with crystal violet and the clone number was counted.

2.4 Western blot analysis

Western blot analysis was performed following the previously described protocol [7]. In brief, cells were lysed in RIPA buffer and equal amounts of protein were loaded onto a 10% SDS polyacrylamide gel. Subsequently, the proteins were transferred to a nitrocellulose membrane and immunoblotted with antibodies. The primary antibodies used included antibodies against β-arrestin2, phospho-PKM2, PKM2, phospho-ERK1/2, ERK1/2, hnRNP A1 (Cell Signaling Technology), Ubiquitin and GAPDH (Santa Cruz). The secondary antibodies were anti-rabbit IgG conjugated with IRDye680 and anti-mouse IgG conjugated with IRDye800 (Li-COR Biosciences). The Odyssey® Infrared Imaging System (LI-COR Biosciences) was used to detect proteins of interest. The band intensities were quantified with respect to GAPDH using ImageJ software.

2.5 Quantitative real-time polymerase chain reaction (RT-qPCR) analysis

RNA extraction from prostate cancer cells was performed using TRIzol reagent (Cat# 15596026, Invitrogen) following the manufacturer's instructions. Subsequently, 1 μg of RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Cat #RR047A, TaKaRa, Japan). The RT-PCR analysis was conducted on a LightCycler 480 System (Roche, Basel, Switzerland) using TB green (Cat #RR420A, TaKaRa) as the fluorescent dye. Specific primers for each target gene were utilized, and their sequences are shown in Table 1. The expression levels of the target markers were normalized to the expression of GAPDH.

Table 1 List of primers used in this study2.6 Microarray data

The matrix files of the gene expression profile of GSE33455 and GSE158494 datasets were extracted from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Both of them contain the expression data from docetaxel-resistant prostate cancer cell lines. Batch effects were removed through the “Rtsne” package (version: 0.15). The differential expression of β-arrestin2 is analyzed by t-tests.

2.7 GO enrichment analysis

GO enrichment analysis using the different expression genes were performed with R language with the aid of packages clusterProfiler, enrichplot and ggplot2. Only terms with both p- and q-value < 0.05 were considered significantly enriched.

2.8 Gene set enrichment analysis

The reference gene set “c5.bp.v7.1.symbols.gmt” were downloaded from Molecular Signatures Database as the target sets with which GSEA performed using the software GSEA_4.3.2 (http://software.broadinstitute.org/gsea/index.jsp). The threshold was set at |Normalized Enrichment Score (NES)|> 1 and P < 0.01.

2.9 Glycolysis analysis

Glycolysis analysis was carried out as previously described [19]. A Seahorse XF Cell Mitostress test kit and XFe24 extracellular flux analyzer (Seahorse Bioscience) were used to measure extracellular acidification rate (ECAR). In brief, 1 × 105 cells were seeded into 24-well plates and incubated for 24 h. After the cells were washed with Seahorse buffer, glucose, oligomycin, and 2-deoxy-glucose (2-DG) were added to measure the ECAR. The ECAR values were calculated after normalization to cell number and plotted as the mean ± SD.

2.10 Correlation test

Public data of β-arrestin2, PKM2, hnRNP A1, hnRNP A2B1 and PTP1B expressions in PCa patients were obtained from The Cancer Genome Atlas Project (TCGA). After excluding patients with incomplete information or normal types, 498 PCa patients’ expression data were analyzed.

2.11 Immunoprecipitation

Immunoprecipitation was conducted as we described previously [7]. In brief, cells were mechanistically broken in ice-cold RIPA buffer and incubated with indicated antibody or control IgG antibody (Santa Cruz) at 4 °C overnight. Then the lysate mixture was centrifuged at 4000 rpm/min for 5 min and washed. The precipitated proteins were eluted for western blot analyses.

2.12 Nude mice xenograft experiment

The nude mice xenograft experiment was carried out [20]. The male nude mice were purchased from the Experimental Animal Center of Guangdong province (Guangzhou, China). A total of 100 μL cells (1 × 107 cells/mL) were subcutaneously injected into the axillary region of mouse. Tumor size was measured twice a week with a vernier caliper. Tumor volume was calculated by the formula 0.524 × (length) × (width)2. Mice were injected docetaxel (5 mg/kg) intraperitoneally once a week when tumor volume was approximately 100 mm3. After 4 weeks observed, the mice were sacrificed and the tumors were dissected, weighted and immunohistochemical stained.

2.13 Immunohistochemical (IHC) staining

Immunohistochemistry was performed as we previous described [20]. The tissue samples were fixed, paraffin-embedded, sectioned at 4-μm thickness and then stained according to standard IHC protocol. Images were obtained via using a PathScope™ 4S scanner (DigiPath, USA) and quantified using Image Pro Plus software.

2.14 Statistical analysis

The data are reported as the means ± SD of at least three independent experiments. The mean differences were compared using ANOVA and the Student t-test. A P value of less than 0.05 was considered to be statistically significant.