Additional file 1 of The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer
Creators: name: Chellini, Lidia
name: Pieraccioli, Marco
name: Sette, Claudio
name: Paronetto, Maria Paola
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary Fig. 1. DHX9 depletion affects tumorigenic phenotype in PC-3 cells. PC-3 cells were transfected with a control siRNA (siCTRL) or a siRNA specific for DHX9 (siDHX9) and DHX9 downregulation was confirmed by WB analysis (A) or by qRT-PCR (B). Histograms represent the relative DHX9 expression versus siCTRL. C PC-3 cells were transfected as in (A) and analyzed by MTS assay after 24, 48 or 72 hrs. Values are the mean ± SD of three independent experiments, each performed in triplicate, considering the siCTRL 24 hrs samples as 1. D PC-3 cells were transfected and as in (A) for 48 hrs and migration assay was performed. The crystal violet–stained migrating cells were photographed (left) and counted (right). Values are the mean ± SD of three independent experiments, considering the siCTRL samples as 100. Magnification, × 10. Statistical analysis in (A), (B), (C) and (D) were performed by Student’s t test, p values: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Supplementary Fig. 2. DHX9 depletion affects tumorigenic phenotype in LNCaP cells. A LNCaP cells were transfected with either control siRNA (siCTRL) or a siRNA specific for DHX9 (siDHX9) for 48 hrs. DHX9 downregulation was confirmed by qPCR. Histograms represent the relative DHX9 RNA expression versus siCTRL, normalized to GAPDH expression. B Heatmap representing differential gene expression in control (siCTRL) and DHX9 silencing (siDHX9) LNCaP cells. Each column represents a sample, and each row represents a gene. C LNCaP cells were transfected as in (A) and DHX9 downregulation was confirmed by WB analysis. Histograms represent the relative DHX9 protein expression versus siCTRL, normalized to GAPDH expression. D Gene Ontology (performed using DAVID) of terms regulated at gene expression levels by analyzing DEGs after DHX9 silencing. Histograms represent the TOP20 Biological Process categories, indicating the Fold Enrichment score and the -log10 (p-values). Statistical analysis in (A) and (C) were performed by Student’s t test, p values: *, p ≤ 0.05; **, p ≤ 0.01. E and (F) Gene Ontology (performed using DAVID) of terms regulated at gene expression levels by analyzing upregulated (E) and downregulated (F) transcripts after DHX9 silencing. Histograms represent the Fold Enrichment score (in black) and the -log10 (p-values; in red). Supplementary Fig. 3. Androgens modulate DHX9 expression. A The plot shows the Pearson correlation between AR and DHX9 expression in in 370 PCa patients (GSE21034) [25]. LNCaP cells were cultured in medium containing FBS or CSS and the expression of DHX9 was measured by qPCR (B) or WB analysis (C). Quantification of mRNA and protein level are shown in the bar graphs as the mean ± SD of three independent experiments, considering the control sample (FBS) as 1. Statistical analysis was performed by Student’s t test, p values: *, p ≤ 0.05; ***, p ≤ 0.001. Supplementary Fig. 4. DHX9 and AR act in a functional complex to modulate gene expression. A Total LNCaP extracts were immunoprecipitated with AR antibody (IP AR) and treated or not with RNAse. IP and input were subjected to WB to analyse the expression of DHX9 and AR. B Cytosolic, nuclear soluble and nuclear insoluble chromatin-associated fractions of LNCaP cells were analyzed upon DHT treatment by WB, using the indicated antibodies. Histone H3 and GAPDH were evaluated, respectively, as nuclear chromatin associated fraction and cytosolic markers. C Pearson correlation analysis on Prostate Cancer patients (GSE21034), performed between the expression of DHX9 and TMPRSS2, MAOA, NDRG1 or KLK3. Values are expressed as base 2-logarithm. In each panel, the correlation value (R) and the relative p-value are indicated. D LNCaP cells were transfected with a control siRNA (siCTRL) or a siRNA specific for DHX9 (siDHX9), cultured for 48 hrs in CSS, and treated or not with DHT (10 nM). qPCR was performed to analyze the DHX9 expression level. Histogram represents the mean ± SD of three independent experiments, considering the siCTRL CSS sample as 1. Statistical analysis was performed by Student’s t test, p values: ****, p ≤ 0.0001.