Mab1536

Protein samples were extracted with non-reducing Laemmli buffer without bromophenol blue and quantified by the BCA assay. Extracts were then loaded onto 10% standard polyacrylamide gel electrophoresis after adding 5% 2-mercaptoethanol, and transferred to nitrocellulose membranes or PVDF membranes for BN-PAGE. The following antibodies were used: monoclonal anti-HIF-1α antibody (#MAB1536; R&D Systems), monoclonal anti-NDUFS4 antibody (ab87399; Abcam), monoclonal anti-NDUFS2 antibody (ab110249; Abcam), anti-NDUFB6 antibody (16037-1-ap, Proteintech), anti-ubiquinol-cytochrome c reductase core protein I antibody (ab110252; Abcam), anti-PINK1 (sc-33796, Santa Cruz Biotechnology) and monoclonal anti-α-tubulin antibody (T6199, Sigma). Antibody binding was detected by chemiluminescence with species-specific secondary antibodies labelled with horseradish peroxidase (HRP), and visualized on a digital luminescent image analyzer (Fujifilm LAS-4000).

Immunofluorescence was performed on MiaPaCa-2 3D spheroids to measure the expression of hypoxia-inducing factor 1α (HIF1α). To generate spheroids, 250 nL of Matrigel-cell mixture (at a concentration of 1 × 10⁷ cells/mL) was spotted on a glass bottom confocal dish using the ASFA^TM spotter. The spots were gelled by incubating the dish upside down in a humidity chamber for 15 min at 37 °C. Fresh DMEM was added to the confocal dish to cover the spots and the spheroids were cultured for 5 days, and then the spheroids were washed with DPBS and fixed with 4% (w/v) paraformaldehyde and 0.25% glutaraldehyde (w/v) in DPBS for 20 min. The spheroids were permeabilized with 0.25% (v/v) Triton X-100 in DPBS for 30 min and quenched with 2 mg/mL sodium borohydride in DPBS. The spheroids were then blocked with 5% (w/v) bovine serum albumin and 1% (v/v) goat serum in DPBS overnight at 4 °C and stained with mouse 10 µg/mL anti-HIF1α antibody (R&D systems MAB1536) in 1% (w/v) BSA in DPBS overnight at 4 °C. An Alexa Fluor 488 goat anti-mouse (Invitrogen A28175) antibody was used as a secondary antibody with staining done in 1% BSA in DPBS overnight at 4 °C at 1:500 dilution. The cells were then stained with 5 µg/mL Hoechst 33342 for 10 min at room temperature and imaged using a Leica TCS SP8 STED Confocal Microscope. Z-stacks of the spheroids were constructed using Imaris Viewer.

Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 (1:100, MAB2018, R&D Systems, USA), OCT-4 (1:100, MAB1759, R&D Systems, USA), Nanog (1:100, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:100, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:150, MBS462020, MyBiosource, USA), CD15 (1:100, MAB2155, R&D Systems, USA), NESTIN (1:100, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:100, ab130244, Abcam, USA), VEGF (1:100, MAB293, R&D Systems, USA) and HIF1α (1:100, MAB1536, R&D Systems, USA). Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Germany).

Cells cultured in hypoxia for 0, 12, 24, 48 and 72 h were collected, subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with antibodies against SOX-2 (1:1000, MAB2018, R&D Systems, USA), OCT-4 (1:1000, MAB1759, R&D Systems, USA), Nanog (1:1000, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:1000, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:1000, MBS462020, MyBiosource, USA), CD15 (1:1000, MAB2155, R&D Systems, USA), NESTIN (1:1000, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:1000, ab130244, Abcam, USA), VEGF (1:1000, MAB293, R&D Systems, USA) and HIF1α (1:1000, MAB1536, R&D Systems, USA). Enhanced chemiluminescence was conducted for visualization.