Figure 3

Differentiation agent PMA increases mitochondrial and nuclear GLS2 protein levels in SH-SY5Y and HepG2 cancer cells. (A) Representative Western blot of nuclear fractions isolated from SH-SY5Y cells after incubation with 1 µM PMA for 12 h and 24 h or dimethyl sulfoxide (DMSO). Blots were probed with antibodies against GLS2, p21, p53 and TATA-binding protein (TBP) used as a loading control. Antibodies against COX IV were used to assess cytoplasmic (mitochondrial) contamination. (B) Representative Western blot of nuclear fractions isolated from human GBM T98G cells: wild-type (WT), stably-transfected with vector alone (pcDNA3) and stably-transfected with GLS2 (GAB). Blots were probed with antibodies against GLS2, p53, c-Myc and the loading control TBP. Antibodies against COX IV were used to assess cytoplasmic (mitochondrial) contamination. (C) Representative Western blot of whole cellular extracts isolated from human GBM T98G cells: wild-type (WT) and stably-transfected with GLS2 (GAB). Blots were probed with antibodies against p53 and c-Myc using β-actin as loading control. (D) Confocal microscopy images of GLS2 immunofluorescence in HepG2 cells treated with PMA. Cells grown on cover slips were treated with vehicle DMSO (I–III) or 1 µM PMA in DMSO (II–IV) for 6 h. After incubation, cells were stained with rabbit antibody to GLS2 and Alexa 488 conjugated secondary antibody. Induction of differentiation (II–IV) clearly increased the nuclear (asterisk) and cytoplasmic accumulation of GLS2 protein (arrows) versus untreated cells (I-III). GLS2 also showed some minor nuclear localization without treatment (asterisk in III). Open arrows in IV indicate an intense perinuclear immunostaining of GLS2. Scale bars: 100 μm (I,II) and 10 μm (III, IV). Original Western blots of Fig. 3-A, B and C are shown in Supplemental Fig. S1. Densitometric analysis of the protein bands, normalized to the loading control, are shown on the right of panels A, B and C. For all panels, the values represent the mean (n ≥ 2) and the error bars represent ± SD, except for p21 in A and c-Myc in B (n = 1). Paired Student’s t test was done between PMA treated and untreated cells, and T98G-GAB vs T98G-WT or T98G-pcDNA3.*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.