Fig. 2

NF-YAx is development-regulated and induced by doxorubicin in neuroblastoma cells. a Mouse embryo upper (U), mid (M) and lower (L) regions used for RT PCR plus representative agarose gels demonstrating NF-YA isoform (upper panels) and GAP (lower panels) RT-PCR levels in stage E8.5 - E18.5 mouse embryo U, M and L regions (* indicates NF-YAx). b Representative micrographs (bar = 50 μm) of SH-SY5Y death induced by doxorubicin (10 μM) at 6, 24 and 48 h, a representative AO/EtBr assay demonstrating dead (red) and living (green) SH-SY5Y cells following incubation without (Con 48 h) or with 10 μM doxorubicin (Dox 48 h) (bar = 100 μm) plus a histogram demonstrating time-dependent SH-SY5Y cell death induced by 10 μM doxorubicin at 6, 24 and 48 h, displayed as mean (± SD) percent death in three independent assays, performed in duplicate, and RT-PCR demonstrating the induction of NF-YAx expression in SH-SY5Y cells, following 48 treatment with 10 μM doxorubicin (Dox) compared to untreated controls (Con). c RT-PCR Southern blots demonstrating time and concentration-dependent doxorubicin induction of NF-YAx expression in SH-SY5Y cells together with NF-YA isoform cDNA standards (NF-YA cDNA), in blots hybridized with NF-YA exon C probe (Exon C, upper panel) but not with NF-YA exon D probe (Exon D, lower panel). d Western blot demonstrating doxorubicin (10 μM for 24 and 48 h) induction of endogenous NF-YAx protein expression in SH-SY5Y nuclear extracts (50 μg). e) Representative micrographs of SH-SY5Y cells untreated (Con) or treated for  48-h with 100 μM necrostatin- 1 (Nec), 10 μM doxorubicin (Dox) or with 100 mM necrostatin-1 plus 10 mM doxorubicin (Nec/Dox) and micrograph demonstrating cellular swelling, vacuolization and lysis of doxorubicin-treated (10 μM for 48 h) SH-SY5Y cells (bar = 50 μm) plus a histogram demonstrating significant inhibition (*) of SH-SY5Y cell-death induced by 10 μM doxorubicin (Dox) in the presence of 100 μM necrostatin-1 (Nec/Dox) at 48 h, compared to untreated controls and in the presence of 100 μM necrostatin-1 (Nec) alone, displayed as mean (± SD) percent death in three independent assays, performed in duplicate. f RT-PCRs of NF-YA isoform (upper panels) and GAP (lower panels) levels in SH-SY5Y cells treated for 48 h without (CON) or with TPA (10 ng/ml), retinoic acid (RA,10 μM), dibutyryl cAMP (cAMP, 0.5 mM), CoCl₂ (150 μM), DTT (5 mM), thapsigargin (THA, 10 ng/ml) and tunicamycin (TUN, 1 μM) plus NF-YAx cDNA standard (NF-YAx) (upper left panel), and NF-YA isoform and GAP levels in stable TrkT3 and TrkAIII SH-SY5Y-transfectants (bottom 2 panels).