Nb100 304

ECs and VSMCs were fixed with 4% paraformaldehyde and washed three times. The cells were blocked with a blocking buffer including 2% bovine serum albumin (BSA; Fisher Scientific #BP9706-100) and 0.2% Tween 20 (Sigma-Aldrich) for 1 h at room temperature, and primary antibodies were added to the cells overnight at 4°C. The following day, the cells were washed three times. The cells were incubated with secondary antibodies (anti-rabbit Alexa-488 and anti-rat Alexa-555, Thermofisher) for 1 h at room temperature and then washed with PBS. Slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI). Pictures were taken by using the FluoView FV3000 confocal microscope. Primary antibodies included the following: Smoothelin (Abcam #ab204305), α-smooth muscle actin (Abcam), CD144 (Biosciences #555661), VWF (citeab #A0082), Lamin A (Invitrogen #MA1-06101), γH2A (Millipore #05-636), 53BP1 (Novus #NB100-304), histone H1 (Abcam #ab61177), and HP1α (Cell Signalling #2616s). γH2A.X and 53BP1 were reported for colocalization. Fluorescence intensity for histone H1 and HP1α were normalized with nuclei area.

Prostate tissue immunohistochemistry staining for androgen receptor (AR) and prostate-specific antigen (PSA) has been described previously [12 (link)]. Tissue fragments were fixed overnight in 10% formalin and placed in embedding cassettes. After, dehydration in 70% ethanol, formalin-fixed tumors were processed using automated standard procedures and subsequently embedded in paraffin. Four-micrometer tissues sections obtained with Leica microtome were mounted on coated microscope slides. For immunofluorescent staining, sections were deparaffinized using xylene and hydrated with declining concentrations of ethanol. Target antigen retrieval was performed using Vector Antigen Retrieval buffer (pH 6.0), which was heated to 100 °C for 20 min. Cells were permeabilized using phosphate buffered saline (PBS) with 0.2% Triton X-100 for 20 min. Blocking was achieved using PBS with 2.5% horse and goat serum. Primary antibodies [anti-p-ATM (abcam ab36801) 1/300, anti-γ-H2AX (Millipore 3292608) 1/300, anti-53BP1 (Novus Biologicals NB100-304) 1/300, anti-p-DNA-PKcs (abcam ab18192) 1/300] were diluted in blocking buffer and incubated over night at 4 °C cold room. Secondary Fluorescein or Texas Red (1/500) antibodies were used to visualize the primary antibody. Sections were mounted using Prolong Gold antifade reagent with DAPi.