Journal: bioRxiv

Article Title: Transcriptional network involving ERG and AR orchestrates Distal-Less Homeobox 1 mediated prostate cancer progression

doi: 10.1101/2020.08.28.271916

Figure Lengend Snippet: (A) Schematic showing the androgen response elements (AREs) at DLX1 putative enhancer (top). ChIP-qPCR data depicting AR recruitment at DLX1 putative enhancer in R1881 (10nM) stimulated VCaP cells treated with or without Enzalutamide (10μM). (B) Q-PCR and immunoblot showing relative expression of target genes in VCaP cells under culture conditions as mentioned. (C) Schematic representation showing the possible interaction between ERG and AR on DLX1 promoter (Top). Re-ChIP data showing co-enrichment of AR and ERG on EBM at the DLX1 promoter (Bottom). (D) Integrated genome view of 3D chromatin structure and binding of transcription factors at DLX1 genomic and nearby region. (E) Quantitative PCR showing relative expression of DLX1 in siRNA mediated AR, ERG and/or FOXA1 silenced VCaP cells. (F) Same as (E) except for immunoblot data. (G) ChIP-qPCR data depicting AR (top) and FOXA1 (bottom) enrichment at DLX1 putative enhancer in 22RV1 cells stimulated with R1881 (10nM) for 16 hours. KLK3 is used as a positive control. (H) ChIP-seq data (GSE94013) using AR-V7 and AR C-terminal (AR-C19) antibody in DHT stimulated 22RV1 cells. (I) Relative expression of KLK3 and DLX1 in dox inducible LNCaP AR-V7 overexpressing cells stimulated with R1881 (10nM) and treated with Dox (40ng) or vehicle control for 24 and 48 hours. (J) Immunoblot showing expression of AR-FL, AR-V7, DLX1 and PSA using same cells as in (I). β-actin used as loading control. (K) Q-PCR showing DLX1 and KLK3 expression in LNCaP AR-V7 cells under culture conditions as mentioned for 48 hours. Data shown from three biologically independent samples. In the panels (A-C), (E), (G), (I) and (K), data represent mean ± SEM. *P≤ 0.05 and **P≤ 0.005 using two-tailed unpaired Student’s t-test.

Article Snippet: PVDF membrane was incubated overnight at 4°C with the following primary antibodies at 1:1000 dilution: DLX1 (Thermo, PA5-28899), E-Cadherin (CST, 3195), Vimentin (Abcam, ab92547), phospho-Akt (CST, 13038), total-Akt (CST, 9272), Caspase-3 (CST, 9662), Cleaved PARP (CST, 9541), Bcl-xL (CST, 2764), ERG (Abcam, ab92513), FOXA1 (CST, 58613), 1:2000 diluted AR (CST, 5153), 1:2000 diluted PSA (CST, 5877), and 1:5000 diluted β-actin (Abcam, ab6276).

Techniques: ChIP-qPCR, Western Blot, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Positive Control, ChIP-sequencing, Control, Two Tailed Test