Additional file 8 of ERG inhibition selectively sensitizes ERG-positive prostate cancer to radiation through DNA repair vulnerabilities and enhances response to androgen receptor inhibitors
Year: 2026
Type: article
Abstract: Supplementary Material 8: Supplementary Fig. S1. Effect of ERGi on ERG expression and cell proliferation in prostate cancer cells. (A) Western Blot analysis showing ERG protein levels in panel of PCa cell lines VCaP, DU145 and PC3 and in the benign prostatic hyperplasia cell line BPH1. HSC70 was used as a loading control. (B) Proliferation assay demonstrating the inhibitory effects of increasing concentrations of ERGi on cell viability in the indicated cell lines. IC50 values are indicated. Data presented as mean±SEM of at least 3 independent experiments. (C) Upper panel: Representative western blot showing ERG protein levels in VCaP cells treated with different concentrations of ERGi ranging from 0.1 to 0.8 µM for 48 h. Lower panel: Quantification of the ERG expression from the upper panel. The intensities were measured and quantified using Licor Odyssey CLx imaging system (D) Upper panel: Schematic representation of the experiment, in which VCaP cells were treated with 0.25 µM for 48 h and subsequently released into ERGi-free medium. Middle panel: Western blot analysis of ERG protein levels following the indicated treatment scheme. Lower panel: Bar graph showing the quantification of the experiment in middle panel. Supplementary Fig. S2. (A) Quantification of gH2AX/53BP1 foci in VCaP, DU145 and BPH1 cells under the indicated treatment conditions. The number of foci after DMSO treatment was used to calculate the relative foci numbers in the indicated treatments. Shown are the means ± SEM of three independent experiments. Statistical significance was determined using unpaired Mann-Whitney test; ns, not significant. (B) Kinetic analysis of DSB foci resolution after irradiation. Quantification of γH2AX, 53BP1, and co-localized foci per cell in control and ERGi-treated VCaP cells following 2 Gy irradiation at the indicated time points. Data are shown as mean± SD of foci measured in at least 50 cells. Statistical significance was determined using unpaired Mann-Whitney test between individual treatments (*P< 0. 05, ****P < 0. 0001); ns, not significant. (C) Upper panel: Representative EdU-based cell cycle anaylsis of VCaP cells cultured in 2D setting at 12h, 24h and 48h after the indicated treatments. Middle Panel: Bar graphs showing the distribution of VCaP cells across cell cycle phases, including G1 (black), S (light grey), and G2 (white), following treatment with or without 250 nM ERGi for 48 h. Cells were subsequently exposed to 0 Gy or 2 Gy ionizing radiation and EdU was added 30 min prior to indicated time points pre fixation. Lower Panel: Table showing mean percentages for each cell cycle phase. (D) Same as in C, but using a 3D culture setting. EdU was added for 6 h prior to indicated time points. Data presented in C&D are shown as means ±SD of at least 3 biological replicates. (E) Upper panel: Representative metaphase preparation showing lethal chromosomal aberrations (arrows) in VCaP cells pretreated with 250 nM ERGi or mock treated (CTR) prior to irradiation with 2Gy. Metaphases were analyzed 24h after irradiation. Lower panel: Quantification of lethal chromosomal aberrations per metaphase for the experiment depicted in the upper panel. A total of 246 metaphases were analysed. (F) Upper panel: Representative immunofluorescence images of VCaP cells following the indicated treatments and stained with DAPI to visualize DNA. Micronuclei are indicated by arrows. Lower panel: Quantification of the percentage of micronucleated cells in the experiment shown in the upper panel. At least 500 cells were analyzed per treatment. Data presented in C-F are shown as means ±SD of at least 2 biological replicates. Statistical significance was determined using unpaired Mann-Whitney test comparing unirradiated cells to the indicated time point post-treatment (*P < 0. 05, (**P < 0. 01, (***P < 0. 001, ****P < 0. 0001); ns, not significant. Supplementary Fig. S3. ERG knockdown or blocking of the ERG-ETS domain inhibits the repair of IR-induced DSBs in VCaP cells. (A) Representative immunofluorescence images showing gH2AX (red), 53BP1 (green) in VCaP cells treated with 50 µM VPC-18005. Scale: 20µm. (B) Quantification of the data shown in A. Data shown are means ±SEM of 2 independent experiments. Statistical significance was determined using unpaired Mann-Whitney test (****P < 0. 0001); ns, not significant. (C) Western blot demonstrating ERG protein levels in VCaP cells after treatment with either 25 µM or 50 µM VPC-18005 for 48 h. (D) Western blot showing ERG protein levels in VCaP cells after the indicated time points post-transfection with ERG siRNA. (E) Western blot showing ERG expression in VCaP cells (i) untreated (DMSO), (ii) treated with ERG siRNA for 72 h, (iii) scrambled (iv) dual treatment with ERGi and ERG siRNA and (vi) treatment with 0.25 µM ERGi. (F) Representative immunofluorescence images for gH2AX (red) and 53BP1 (green) in VCaP cells under the indicated treatment conditions. Scale: 20µm. (G) Quantification of the experiment indicated in F. D stands for DMSO, sc for scrambeled RNA siE for siERG, and Ei for ERGi. Data are presented as means ±SEM of 2 independent experiments. Statistical significance was determined using unpaired Mann-Whitney test (**P < 0. 01); ns, not significant. Supplementary Fig. S4. (A) Representative immunofluorescence images showing gH2AX (red), 53BP1 (green) in DU145 (upper panel) and BPH1 (lower panel) cells following the indicated treatments. Scale: 20µm. (B&C) Quantification of the data shown in A. Shown are the means ±SEM of at least 3 independent experiments. Statistical significance was determined using unpaired Mann-Whitney test (****P < 0. 0001); ns, not significant. (D) Immunoblot analysis of PAR, PARP1, ERG and PSA in VCaP cells under the indicated treatments. Supplementary Fig. S5. (A & B) Representative 3D colonies generated from VCaP or DU145 cells following the indicated treatments. (C) Representative 2D colonies formed in BPH1 cells treated with ERGi (0.25 µM), ATMi (10 µM), either alone or combined prior irradiation with the indicated x-ray doses. (D) Survival fractions measured from the experiment presented in C. Data shown are means ± SEM from three independent experiments. (E) Cell viability of normal human fibroblasts (HF184) measured by CellTiter-Glo after irradiation in cells treated with DMSO or ERGi. (F) Quantification of gH2AX foci in TSCs from the indicated PCa patients at 24h post-3Gy before (DMSO) and after ERG inhibition (ERGi). Data shown are means ± SD of three biological replicates. Supplementary Fig. S6. Differential quantitative proteomics of ERGi-treated cells. (A) Heatmap visualization of significantly differentially abundant proteins (ANOVA-test, FDR, p < 0.05) across the cell lines before and after ERG inhibition. Z-scores (row-wise) are shown for each protein. Red indicates higher relative abundance, while blue represents lower relative abundance. Samples (in biological replicates, n=6) are grouped by cell line (VCaP, DU145, BPH1) and their ERG-inhibited counterparts (VCaP+ERGi, DU145+ERGi, BPH1+ERGi). (B&C) Heatmaps showing two-sample tests (FDR, p < 0.05) of changes in protein abundances in (B) DU145 ±ERGi and (C) BPH1±ERGi. (D-G) Bubble plots showing the top ten significantly enriched pathways from the MSigDB Hallmark gene set library in treated (C) DU145 (protein cluster size 1549) and (E) BPH1 (protein cluster size 1478), and from KEGG-enriched pathways in (F) DU145 and (G) BPH1. (H) Heatmap showing the expression of the indicated proteins in the studied cell lines before and after ERG inhibition. (I) Volcano plot showing log2 fold change in protein abundance (x-axis) against -log10 p-value (y-axis) of VCaP against VCaP+ERGi. Points above the horizontal dashed line represent proteins with significant changes in abundance (p < 0.05), while points outside the vertical dashed lines represent proteins with a fold change of at least 1.5 in abundance. EZH2 protein is depicted in gold. Supplementary Fig. S7. (A) Schematic illustration of the NIRL ablation setup. Snap-frozen patient tissue sections were placed on a cooling stage at -10°C inside a closed ablation chamber. The generated tissue aerosol was condensed and collected on a glass slide positioned opposite the ablation site. The collected material was solubilized in 1% SDC/TEAB buffer and processed for LC-MS/MS-based proteomic analysis. The ablated area measured 1 mm2, and each replicate yielded approximately 250 nL of collected material. (B) Bubble plot showing the top significantly enriched KEGG pathways identified from proteins with higher abundance in the ERG-positive patient sample (Pat #1) relative to the ERG-negative patient sample (Pat #2) (n=1,581 proteins). (C) Heatmap showing the relative abundance of the indicated proteins in the analyzed fresh-frozen patient tissue samples. Supplementary Fig. S8. (A&B) Representative images of PDOs derived from (A) ERG-positive and (B) ERG-negative PCa patients following the indicated treatment (upper panels; with 10X objective). Lower panels show the harvested MTT-stained PDOs: with 4X objective. (C&D) Quantification of gH2AX (red) and 53BP1 (green) foci in PDOs from (C) an ERG-positive (Pat. #1) and (D) an ERG-negative (Pat. #2) PCa patient, analyzed 1h (left panels) and 24 h (right panels) after 3 Gy irradiation following a 48-h pretreatment with DMSO or ERGi. Data are presented as the mean ± SD of at least 50 cells for each treatment. Supplementary Fig. S9: (A&B, left panels) Representative images of MTT-stained DU145 spheroids treated with the indicated concentrations of (A) abiraterone (Abi) or (B) apalutamide (Apa), either alone or in combination with 50nM ERGi. Right panels: Quantification of survival fractions determined by CFA based on the corresponding treatments shown in the left panels. (C&D, left panels) Representative images of MTT-stained LNCaP spheroids treated wi
Source: Figshare
Authors: Ayham Moustafa, Mohamed E. Elsesy, Bente Siebels, Jan Hahn, Su-Jung Oh-Hohenhorst, Carlotta Schudy, Thorsten Rieckmann, Susanne Burdak-Rothkamm, Derya Tilki, Tobias Maurer, Cordula Petersen, Kai Rothkamm, Gunhild von Amsberg, Carsten Bokemeyer, Hartmut Schlüter, Sabrina Köcher, Wael Y. Mansour
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7223561147