Observer 2

NT and SMC1A shRNA expressing DU145 and PC3 cells were subjected to immunocytochemistry (ICC) to quantify γH2AX foci (DNA DSB marker) as described previously.^{19 (link),32} Briefly, 20 000 cells/well were grown in 1.5 polymer tissue culture treated μ-slides with 8-well (Ibidi) and after 24 h, exposed to 2 Gy radiation. At specific time points (pre-radiation, 0.5, 2, 12, and 24 h after 2 Gy), medium was aspirated and cells were fixed with ice-cold methanol, acetic acid (7:1), permeabilized with 0.25% Triton X-100, and incubated with 2% BSA in phosphate-buffered saline, 0.05% Tween 20 (PBST) for 1 h at room temperature. Cells were incubated with rabbit γH2AX (phosphor-Ser139) antibody (from Cell Signaling 1:1000 dilution in 1% BSA in PBST) overnight at 4°C, followed by incubation with goat anti-rabbit DyLight®550-conjugate (from Bethyl Labs, 1:500 dilution) for 1 h at 37°C in a humidified chamber. Cells were washed with PBS and cell nuclei were stained with DAPI and visualized by fluorescence microscopy (Zeiss observer II). For each data point, γH2AX foci were counted in at least 20 cells to yield average foci/cell and plotted.

Prostate cancer cells (DU145 and PC3, NT) and their corresponding SMC1A knockdown (SMC1A shRNA) cells were irradiated (5 Gy), seeded on 24-well ultra-low attachment plates (Thermo Scientific) at a density of ~1000 cells/well and grown in stem cell culture medium (StemCell, Vancouver, Canada). Sphere formation was evaluated every 24 h for up to 5 days and quantitated at day 5 by counting spherical cell clusters (>60 μm) in 10 random fields. Images were captured by fluorescence microscope (Zeiss observer II) at 10× magnification.

Prior to incubation with bHs-ST in vitro, sections of PANC-1 tumors were de-paraffinized and rehydrated. Uninduced and induced χ8768-bHs (10⁸ CFU), PBS or bovine hyaluronidase (Sigma) were incubated on tissue sections overnight in a humidified 37°C incubator. Following treatment, specimens were incubated with a biotinylated HA binding protein (HABP, Sigma) at 5 μg/mL final concentration for 2 hours at 37°C. Slides were then washed, incubated with streptavidin-HRP at RT for 1 hr and visualized with a DAB kit (Vectastain). H&E and Masson’s Tricrhome were used for staining. PANC-1 tumor sections, skin and decalcified joints from NSG mice treated intravenously with χ8768-bHs (uninduced and induced). Sections were de-paraffinized and rehydrated and stained overnight with 2.5 μg/mL HABP, 1:100 anti-ST antibody (Santa Cruz, sc-52223), 1:100 anti-pan-cytokeratin (AE1/AE3) or according to H&E and Masson’s Trichrome protocols used by the Pathology Research Services Core (City of Hope). Streptavidin-PE (Vector), anti-mouse-Cy5 (Invitrogen), or anti-mouse-HRP 1:250 were then used to visualize HA, ST or pan-cytokeratin by brightfield or fluorescence microscopy (Zeiss Observer II), in addition to DAPI for visualizing nuclei during fluorescence imaging. Tiling was performed at 5X or 10X, while higher resolution images for ST, HA and DAPI were done at 100X (oil).