T5076

U87NS at day 14 were washed, centrifuged at 200× g, resuspended in DMEM supplemented with 10% FBS in presence or absence of 100 µM F-NANA and allowed to differentiate for 5 days in 60 mm diameter tissue culture petri dishes. Total RNA was extracted, and RT-PCR was performed to evaluate mRNA expression of the stemness markers oct-4, sox-2, and nanog. For immunofluorescence analysis, the assay was performed in 8-well chamber slides (Nunc Lab-Tek) and at the end of the experiment cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% PBS-triton X-100, blocked for 1 h with 0.2% PBS-gelatin and incubated overnight at 4 °C with the following primary antibodies: rabbit monoclonal anti-PSA (MBS488177, MyBioSource—dilution 1:200), mouse monoclonal anti-GFAP (sc-16648, Santa Cruz Biotechnology—dilution 1:500), mouse monoclonal anti-Nestin (sc-23927, Santa Cruz Biotechnology—dilution 1:200), mouse monoclonal anti-βIII-tubulin (T5076, Sigma-Aldrich—dilution 1:200). The secondary antibodies used were goat anti-mouse Alexa-fluor 594 conjugate and goat anti-rabbit Alexa-fluor 488 conjugate (dilution 1:1000, Life Technologies). Nuclei were counterstained by DAPI incorporation for 3 min in the dark and Mowiol was used to mount coverslips on chamber slides. Images were acquired with a Nikon Eclipse Ni motorized microscope system at 20× magnification.

U87-MG cells (5 × 10³) were plated on 8-well chamber slides (Nunc Lab-Tek, Waltham, MA, USA) and maintained overnight in DMEM supplemented with 10% FBS either under hypoxia or normoxia, in presence or not of 100 μM F-NANA. For immunofluorescence staining we followed the protocol previously described by our group [60 (link)]. Briefly, cells were fixed by 4% paraformaldehyde, permeabilized with 0.3% PBS-triton X-100, blocked for 1 h with 0.2% PBS-gelatin and incubated overnight at 4 °C with the following primary antibodies: rabbit monoclonal anti-PSA (MBS488177, MyBioSource—dilution 1:200) and mouse monoclonal anti-βIII-tubulin (T5076, Sigma-Aldrich—dilution 1:300). Goat anti-rabbit Alexa-fluor 488 and goat anti-mouse Alexa-fluor 594 secondary antibodies (dilution 1:1000, Life Technologies) were used and nuclei were counterstained by incubating cells with DAPI for 3 min in the dark. A Nikon Eclipse Ni motorized microscope was used to acquire the images at 20× magnification.