Nb100 449

Treated cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer for whole-cell lysates, containing protease and phosphatase inhibitors. Protein concentrations were determined using the Pierce Bicinchoninic (BCA) Protein Assay Kit, and total protein concentration between 10 and 20 μg of protein was loaded for Western blot analysis. Membranes were probed with antibodies for BMP2 (NBP119751, Novus Biologicals, Centennial, CO, USA, 1:1000), total HIF-1α (NB100–449, Novus Biologicals, 1:500), VEGFA (ab461154, Abcam, Trumpington, Cambridge, UK, 1:2000), total VEGFR2 (2479; Cell Signalling Technology, Danvers, MA, USA, 1:1000), phosphorylated (y¹¹⁷⁵) VEGFR2 (2478, Cell Signalling Technology, 1:1000), total eNOS (610297, BD Biosciences, San Jose, CA, USA, 1:1000), and phosphorylated (S¹¹⁷⁷) eNOS (61293, BD Biosciences, 1:1000). Even loading was confirmed by α-tubulin (ab729, Abcam, 1:5000) for total lysate. Protein levels were quantified and analysed using Bio-Rad ImageLab software (v.5.0, 170-9692, Bio-Rad Laboratories, Gladesville, CA, USA).

Adherent cells were fixed for 10 min in 4% paraformaldehyde in PBS and then permeabilized in 0.1% Triton X-100 in PBS as previously described^{29 (link)}. Cells were then incubated with the primary antibody in blocking buffer for 1 h at room temperature and then washed and incubated with an Alexa-conjugated secondary antibody (1/1000, Molecular Probes) and with 2 Units of AlexaFluor 594 phalloidin (Thermo Fisher scientific) per coverslip for one hour. Cells were counterstained with DAPI (1 µg/ml, Sigma) and mounted with Fluoromount (Southern Biotech). The primary antibodies used were rabbit anti-HIF1α (NB100-449, Novus Biological), mouse anti-HIF1α (NB100-105, Novus Biological) and MA1-516, Thermo Fisher Scientific), goat anti-JMY (L16, Santa Cruz), mouse anti-JMY (G11, Santa Cruz) and rabbit anti-JMY (M300, Santa Cruz). Images were captured using a BX51 (Olympus) coupled with a Retiga200R camera or using a Leica TCS SPE confocal microscope (Leica Microsystems). Nuclear HIF1α, cytoplasmic JMY and F-actin mean fluorescence intensities were measured using DAPI or phalloidin staining for object segmentation with ImageJ software.

HIF-1α expression was evaluated by flow cytometry using a specific antibody anti- HIF-1α. 10⁵ cells were seeded in a 48-well-plate and let them attach for 24 h at 37 °C and 5% CO₂. Next day, cell culture media was removed, cells were trypsinized, transferred into a flow cytometry tube and washed with PBS three times by centrifugation. Then, cells were fixed using 4% PFA for 10 min at 4 °C. After performing three washes with PBS, cells were permeabilized with 0.1% saponin and incubated for 20 min at RT. Subsequently, saponin was removed by washing three more times and primary antibody anti- HIF-1α (1:700, Novus Bio #NB100-449) was incubated for 1h at RT. Secondary anti-rabbit antibody Alexa Fluor^® 488 (1:1000, Invitrogen #A11034) was incubated for 10 min at RT after removing primary antibody and washing three times. Finally, fluorescence intensity was assessed by flow cytometry. To avoid unspecific interactions, control cells were only incubated with the secondary labeled-antibody and compared fluorescence intensities between cells stained with both primary and secondary antibodies. Data obtained was analyzed using FlowJo software.

To verify the expression of HIF-1α and VEGF proteins, the total protein in the tissue samples was homogenized and lysed with ice-cold RIPA buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% NP40, protease, and phosphatase inhibitor). Protein extracts were separated using 10% polyacrylamide gel and were electro-transferred to nitrocellulose membranes. After blocking with 5% milk in TBST buffer (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% Tween-20), the membranes were probed with primary antibodies: HIF-1α (NB100-449, Novus Biologicals, Littleton, CO, USA); VEGF (ab46154, Abcam, CA, MA, USA); and actin (A2066, Sigma-Aldrich, St. Louis, MO, USA), followed by incubation with HRP-conjugated secondary antibody (T6778, Sigma-Aldrich, St. Louis, MO, USA). The target bands were visualized using enhanced chemiluminescence reagent (Amersham Biosciences), and the band images were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).