The mouse AC1, AC3, C1, and C2 cell lines were isolated from PbCre4
+;
Pten
fl/fl
;TP53
fl/fl
Luc + mouse prostate tumors and were established as previously described [
24,
26]. AC1 and AC3 cells were cultured in PrEGM medium (Lonza, Walkersville, MD, USA); C1 cells were cultured in PrEGM/DHT with 5% serum and 5% 3 T3-conditioned medium; C2 cells were cultured in PrEGM/DHT with 5% 3 T3-conditioned medium. The mouse wild-type (WT) prostatic basal cell line was provided by Dr. Lei Fang (NCI/NIH, Bethesda, MD, USA) and was cultured in WIT-P medium (Stemgent, San Diego, CA, USA) as previously described. DU145, PC3, LNCaP, and 22RV1 human prostate cancer cell lines were obtained from ATCC (Rockville, MD, USA). The metastatic RasB1 cell line was previously characterized and used to study molecular mechanisms of prostate cancer metastasis in multiple peer-reviewed articles [
27‐
33]. All human prostate cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). RasB1 and PC3 cells with stable expression of ETV6 were established by transfection with an ETV6 complementary (c)DNA-encoding or empty pCDH-CMV-MCS-EF1-Puro vector (System Biosciences, Palo Alto, CA, USA); 2 × 10
5 cells were seeded and transfected with 5 μg DNA and selected with puromycin for 1 month. Mouse and human ON-TARGETplus SMARTpool siRNAs (scrambled and ETV6) and a human shRNA vector (LacZ and ETV6) were from Dharmacon (Thermo Scientific, Waltham, MA, USA) and the RNAi Core Lab (Academia Sinica, Taipei, Taiwan), respectively. Transient transfections of plasmids and siRNAs were carried out using the X-tremeGENE HP DNA transfection reagent (Roche, CA, USA) or Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). Cells were treated with EGFR inhibitors, CI1033 (10 ng/ml) and AG1478 (10 μM) for 24 h in medium containing 10% serum. For EGF treatment, cells were subjected to serum-starvation for 24 h, followed by the addition of 100 ng/ml EGF for 24 h also in serum-free medium. The EGF was from R&D Systems (Minneapolis, MN, USA), and the EGFR inhibitors (CI1033 and AG1478) were from Selleck (Houston, TX, USA). The mouse Etv6-binding site was located upstream of mouse
Twist1 on chromosome 12: 33957354 at GRCm38. The Twist1-red fluorescent protein (RFP) reporter containing the mouse
Twist1 promoter with the Etv6 response element was constructed using a Clone-it Enzyme free Lentivectors Kit (System Biosciences). ETV6 response element mutations were made using a Site-Directed Mutagenesis System kit (Invitrogen). All primers used for these constructs are listed in Additional file
1; Table S1. All constructs were verified by a DNA sequence analysis.