Anti brdu antibody

Primary mouse brain endothelial cells (BECs) derived from α5-EC-KO or littermate control mice primary BEC were cultured on fibronectin-coated (10 μg/ml fibronectin (Sigma) for two hours at 37 °C) glass coverslips in the presence or absence of 10 ng/ml TNF-α (R&D, Minneapolis, MN). One day after plating, BrdU (Invitrogen, Carlsbad, CA) was added to the culture medium, and the cells incubated overnight. The next morning cells were fixed in acid/alcohol and analyzed for BrdU incorporation by incubation with a rabbit polyclonal anti-BrdU antibody (Invitrogen) for one hour followed by anti-mouse-AlexaFluor 488 secondary (Invitrogen) for one hour, then labeled with the nuclear marker Hoechst (Sigma) for 5 mins before being washed and mounted on glass slides. BrdU-positive cells were expressed as the percentage of total cells (Hoechst staining and the results presented as the mean ± SEM of four experiments.

Cells were seeded in 10cm dishes, and treated with 10µM bromodeoxyuridine (BrdU; Abcam) for 30min at 37°C. 2x10⁶ cells were fixed in 70% (v/v) ethanol. These were digested to nuclei using 1mg/mL pepsin (Sigma-Aldrich), treated with 2M hydrochloric acid, and blocked using 0.5% BSA (Sigma-Aldrich) solution. Staining was with 1:100 anti-BrdU antibody (Invitrogen; 14-5071-82), followed by anti-mouse AF488 secondary antibody (A11029, Invitrogen). Nuclei were then treated with RNase A (Thermofisher) and 50µg/mL propidium iodide (PI; Thermofisher), and analysed using the LSRFortessa (Becton Dickinson, Franklin Lakes, USA). Cell cycle phase was assigned based on PI and BrdU staining gates in FlowJo v10.8.1.

Cells were labeled with 50 μM PKH26GL (Sigma-Aldrich) according to manufacturer's instructions. For in vitro assays, 3×10⁴ labeled HCCLM3 cells were seeded into 24-well plates and immunostaining for OPN was performed after 144 hrs in culture. For in vivo assays, 5×10⁵ labeled HCCLM3 cells were subcutaneously injected into nude mice. Six weeks later, the tumors were minced and digested with type IV collagenase (Sigma-Aldrich). Single-cell suspensions were obtained by filtration through a 70 μm filter (BD Biosciences) and immunostaining of OPN was performed. For BrdU-retaining assays, six weeks after intraperitoneal injections with BrdU (10 mg per kg body weight) three times a day for 2 days, the HCCLM3 cells-xenografted nude mice were sacrificed and tumors minced into sections and embedded in paraffin. BrdU-retaining cells were assayed by immunohistochemistry using anti-BrdU antibody (Invitrogen) and goat anti-OPN antibody (R&D Systems). Secondary antibodies were anti-mouse IgG Alexa Fluor 488 and anti-goat IgG Alexa Fluor 555 (Invitrogen).

The TNBC cell line (MDA-MB-231), and human mammary epithelial cells (HMEC) were purchased from ATCC and were maintained in recommended culture condition as per the ATCC instructions^{38 (link)}. The viral VEGF-E homologs (PCPV-VEGF, ORFV-VEGF, and BPSV-VEGF) were a kind gift from Dr. L.M. Wise, University of Otago, Dunedin New Zealand^{39 (link)}. Antibodies against GFP, PCNA, GAPDH, p-Erk, p-MEK, p-Raf, AKT, FoxO1, H2A were purchased from Santa Cruz Biotechnology Inc Dallas, TX, and ERK, PI3k110α, and p-AKT was purchased from Cell Signaling Technologies Inc. Danvers, MA and anti-BrdU antibody from Invitrogen, Thermo Fisher (SI Table 4). The secondary antibodies for western blot are IR conjugated and were from LI-COR Inc. Lincoln, NE, while for microscopy the secondary antibody is Alexa 594 conjugated purchased from Invitrogen Inc. Carlsbad, CA.
LY294002, an inhibitor for PI3K was purchased from Sigma Aldrich Inc. St. Louis, MO. MDA-MB-231 cells were treated with 50 µM and HMEC cells with 30 µM of LY294002 and incubated for 24 h before being used for assays.

To check the percentage of S-phase cells in the cell cycle, cells from EP, MP and LP were trypsinized from gels and TCP and were seeded on collagen-coated glass coverslips as described above. After 48 h of seeding, BrdU reagent (Invitrogen, 000103) was added in 1:100 (v/v) ratio in media and incubated for 4 h at 37°C in a humidified incubator with 5% CO₂. Thereafter cells were fixed (4% paraformaldehyde), permeabilized (0.5% Triton-X), denatured (2 M HCl), blocked (1.5% bovine serum albumin), and incubated with anti-BrdU antibody (Invitrogen, B35128, 1:100) and counterstained with AlexaFluor 568 (Invitrogen, A11061, 1:400). Immunofluorescence images were captured using EVOS-FL auto and BrdU positive and negative cells were counted manually using ImageJ.