Fig 1: SH-SY5Y cells differentiate into a more glutamatergic-like phenotype following treatment with B-27. (A) SH-SY5Y cells were cultured in DMEM/F-12 supplemented with either 10% FBS, 2% B-27 or 5% B-27 for 96 h. qRT-PCR was performed to measure mRNA expression of cholinergic marker SLC18A1 (VMAT1), dopaminergic marker TH and glutamatergic markers GLUL, SLC17A7 (VGLUT1) and GLS. Relative expression of SLC18A1, TH, GLUL, SLC17A7 and GLS (standardized to GAPDH and POLR2A) normalized to the DMEM/F-12 + 10% FBS culture conditions. Data presented as mean ± SEM; N = 3 independent biological samples with two to three technical replicates each. Statistical significance was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com. ns = not significant; *p < 0.05; **p < 0.01; ***p < 1 × 10–3; ****p < 1 × 10–4. (B) Western blot of lysate from SH-SY5Y cells cultured in DMEM/F-12 supplemented with either 10% FBS or 5% B-27 for 96 h. Membranes were probed with rabbit anti-GLS (Proteintech #29519-1-AP), mouse anti-TH (Proteintech, #66334-1-Ig), mouse anti-GLUL (Proteintech, #66323-1-Ig) and mouse anti-GAPDH (Santa Cruz Biotechnology, #sc-47724). GAPDH was used as a loading control. Molecular weights are as follows GLS = 58 kDa and 65 kDa; TH = 55 kDa; GLUL = 42 kDa; GAPDH = 36 kDa. (C) Levels of protein expression relative to GAPDH were quantify through densitometric analysis using the FIJI software (Schindelin et al., 2012). Three independent experiments were performed and densitometric analysis performed using seven independent blots. Data presented as mean ± SEM. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using Unpaired t tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com. ****p < 1 × 10–4. DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GLS, Glutaminase; GLUL, Glutamate-ammonia ligase; mRNA: messenger RNA; POLR2A, RNA polymerase II subunit A; SEM, standard error of the mean; SLC17A7, Solute carrier family 17 member 7; SLC18A1, Solute carrier family 18 member A1; TH, tyrosine hydroxylase; VGLUT1, vesicular glutamate transporter 1; VMAT1, Vesicular monoamine transporter 1.