U87MG revealed an intermediate resistance (42.51 2.742 mM). MF, DCA and DON in GBM27 and DON in all other cell lines. MF and DCA shifted the metabolism of treated cells towards glycolysis or oxidation, respectively. DON consistently decreased total ATP production. Our study highlights the need for a better characterization of GBM from a metabolic perspective. Metabolic therapy should focus on both glycolytic and oxidative subpopulations of GSCs. or 30 mM; G8769, Sigma-Aldrich, St. Louis, MI, USA), BSA-FV (0,01% and were used as housekeeping genes. For relativization and Galangin comparison with a non-tumoral control, we compared our samples with a pool of retrotranscribed RNA from brain tissue obtained from epileptic patients, provided courtesy of Hospital Universitario y Politcnico La Fe (Valencia). Table Ets2 1 Forward (FW) and reverse (RV) primers for real-time quantitative reverse transcription PCR (RT-qRT-PCR). FWTTCTACAATGAGCTGCGTGTGRVGGGGTGTTGAAGGTCTCAAAFWTCCTCCACCTTTGACGCTGRVACCACCCTGTTGCTGTAGCCFWGCCCGCTTTGTGTGACTAAARVCAGGGGTAAATAACGGCACAFWGCACTAAAGGCCACTGGACRVCCAAGAGGCCACCACTACTGFWCTGACCGCTAGTAGGGAGGTRVAACATCCCAGAACCCTGCTGFWATCCTCCTGCCTGAGTCTCTRVCAAATGCCAAGGACTGCTGTFWTGCCTACGACATGGCTAAGCTCRVGACGTAGACCATGTGAATCGGCFWTGGAAGGAGTGGGTACTGATGCRVGGATTGCTCCAATCATCGGCTTCFWAACTCGGGATGTTGGGGATTRVAGAGAAAAGCCCTTCCTACTGAFWGTCCAGGGCTTGTTCTATTCARVATGCTGCACTTAGAGACCCTFWTGGAACATTGTTACAGCAGGCRVAGCTCTTCTCCCGTGTCTTC Open in a separate window 2.5. Antibodies All primary and secondary antibodies were purchased from commercial sources, listed as follows: AMPK Antibody (2532, Cell Signaling, Danvers, MA, USA), phospho-AMPK (Thr172) (2535, Cell Signaling), Anti-Pyruvate Dehydrogenase E1-alpha Galangin subunit antibody (ab110334, Abcam, Cambridge, UK), Anti-PDHA1 (phospho S293) antibody (ab177461, Abcam), -Actin (A5441, Sigma-Aldrich), -Tubulin (sc-8035, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies for horseradish peroxidase (HRP) detection were anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology) and anti-mouse IgG (PI-2000, Vector Laboratories, Burlingame, CA, USA). 2.6. Protein Isolation/Quantification and Western Blotting Centrifuged and pelleted U87MG and GSCs were resuspended in 100 L of radioimmunoprecipitation buffer [RIPA; 100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM EDTA and 0.2% SDS, with phosphatase and a protease inhibitor cocktail and stored at ?80 C for a minimum of 24 h. Samples were then centrifuged at 13,200 RPM for 20 min at 4 C; protein-containing supernatant was conserved. Total protein concentration was determined using Bio-Rad Protein Assay according to the manufacturer instructions; Galangin after corresponding incubation, absorbance was read at 595 nm. In phosphorylation experiments, treatments dissolved at 1:1 concentration in serum-free medium were added 3C4 days after seeding GSCs, and 24 h in the case of U87MG. U87MG cells were washed twice with PBS and serum-deprived for 1 h prior to sample collection. Protein was subsequently recovered at the indicated timepoints (30 min, 60 min, 2 h, 6 h). Western blotting experiments were performed adapting the protocol from Mahmood et al. [49]. Briefly, protein extracts were separated by 8%C12% SDS-PAGE and transferred to nitrocellulose Galangin membranes. After blocking for 1 h with 5% Bovine Serum Albumin (BSA) in Tween-Tris Buffered Saline 1 [T-TBS; 10 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.1% Tween-20], membranes were incubated with the corresponding primary antibody O/N at 4 C. After washing three times for 10 min with T-TBS, membranes were incubated with HRP-linked secondary antibody for 1 h at room temperature (RT). Detection was performed using ECL reagents (GE Healthcare) according to the manufacturers guidelines and revealed in a BioRad ChemiDoc chemiluminescence system. The same membranes were then incubated with a housekeeping primary antibody O/N at 4 C, washed the next day and incubated with an HRP-linked secondary antibody for 1 h RT before ECL detection. 2.7. Seahorse XFp Protocol for Real-Time Metabolic Evaluation of U87MG Adherent Cells and GSCs Neurospheres Experiments were performed in an XFp 8-well microplate using the Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA). Briefly, GSCs were seeded at a density of 10,000 Galangin cells/well and allowed to.
