Fig. 1

miR-346 Induces Potent DNA Damage and Transcription-Dependent Replication Stress in Prostate Cancer. A Immunofluorescent microscopy analysis of i,iii) phospho-Ser¹³⁹-γH2AX and ii,iv) 53BP1 protein levels in C42 cells treated with Carboplatin (2.5 μM) or transfected with 20 nM miR-346 for 72 h. Representative images of three independent experiments are shown. Foci were quantified using ImageJ. B, C Western blot analysis of phospho-Ser¹³⁹-γH2AX protein levels in B) C42 cells and C) PNT1A non-cancerous prostate cells transfected with miR-346 (20 nM) for 96 h (B) or indicated durations (C). D Immunofluorescent microscopy analysis of DNA:RNA hybrids (R-loops) in C42 cells treated with Carboplatin (2.5 μM) or transfected with 20 nM miR-346 for 72 h. Representative images of three independent experiments are shown. E RNA-seq analysis of RNASEH1 transcript levels in C42/miR-346 cells treated ± Dox (100 ng/ml). F Western blot analysis of RNASEH1 protein levels in 22RV1 cells treated with 10 nM NC mimic or 2.5, 7.5 or 20 nM miR-346 for 72 h. G, H Western blot analysis of: G) phospho-Ser³³-RPA32 and phospho-Ser³⁴⁵-CHK1 protein levels in C42 cells transfected with miR-346 (20 nM) for the indicated durations, H) phospho-Ser³³-RPA32 protein levels in C42 cells transfected with 10 nM miR-346 and treated with 10 μM α-Am for 8 h. J DNA fibre assay analysis of replication fork speed and stalled/terminated forks (per quantifiable fibre) in C42 cells transfected with miR-346 or negative control miR (20 nM) for 24 h. A minimum of 100 fibres were quantified for each measurement and different replication events quantified in ImageJ as described [32]. Fields were selected using one fluorescence channel only for the avoidance of bias. Scale bars = 20 μm. K Flow cytometric analysis of cell cycle distributions of LNCaP (i) and 22RV1 (ii) cells transfected with NC or miR-346 mimic (10 nM) for 72 h. L Western blot analysis of phospho-Thr¹⁵-CDK2, phospho-Ser¹⁰-Histone H3, phospho-Thr¹⁵-CDK1 and total CDK1 in LNCaP cells transfected with10nM miR-346 or NC miR for 48 and 72 h. B, C, F, G, H, L Representative images of three independent experiments are shown, β-actin, GAPDH and VCL were used as a loading controls. CBP = carboplatin, α-Am = alpha-amanitin. Etoposide and CBP were used as positive controls for DNA damage induction. Columns: mean ± SEM for a minimum of three independent experiments. * P ≤ 0.05, # P ≤ 0.005, α P ≤ 0.0001. See also Fig. S1–4