Fig. 6

NF-YAx promotes KIF1Bβ-dependent necroptosis. a Evidence of vacuolation, swelling and necrotic-lysis in HEK-293, SH-SY5Y and ST14A cells follwoing 48 h transfection with NF-YAx expression vector (bar = 10 μm). b Western blots demonstrating the presence of both NF-YAl and NF-YAx in adherent (AD) and NF-YAx in dead/dying suspension (SUS) HEK-293 (upper panel) and SH-SY5Y (lower panel) extracts (20 μg), following 48 h transfection. c RT-PCRs showing reduced Bmi-1 expression, induction of KIF1Bβ expression but no change in EglN3 expression in HEK-293 and SH-SY5Y cells, following 24 h transfection with NF-YAx but not NF-YAl expression vector, together with NF-YA isoform and 18S rRNA levels. d Western blots showing stimulation of KIF1Bβ expression but no changes in ALIX, BclxL, Mcl1, Bcl2, EglN3, JNK, phosphorylated JNK levels and no cleavage of caspase 3 (Casp 3), caspase 9 (Casp 9) or PARP in HEK-293 and SH-SY5Y cells following 48 h transfection with NF-YAx but not NF-YAl expression vector (30 μg). e RT-PCR demonstrating KIF1B-specific (KIF1B) but not scrambled siRNA (Con) knockdown of NF-YAx-induced KIF1Bβ expression in HEK-293 cells compared to 18S RNA levels plus micrographs and a histogram showing prevention of NF-YAx-induced HEK-293 death by KIF1B-specific siRNAs (bottom 2nd panel) but not scrambled siRNAs (Con siRNA, bottom 3rd panel), compared to sham-transfected (bottom 1st panel) and siRNA-transfected controls (top 1st, 2nd and 3rd panels) (bar = 100 μm). Data are displayed as mean (± SD) percent adherent vs suspension cells in two independent experiments performed in duplicate (* = significant). f Western blots and histograms demonstrating significant increases in the ratio of ubiquitinated protein to β-actin in HEK-293 adherent plus suspension cell extracts (left panels and histogram) and ratio of p62 to β-actin in HEK-293 adherent plus suspension cell extracts (right panels and histogram), following 48 h transfection with NF-YAx compared to pcDNA, NF-YAl and NF-YAs expression vectors. Data are displayed as the mean (± SD) densitometric ratio in three independent experiments (* = significant)