Figure 2. ARRDC4 as a metabolic stress inducer.

A. Adenoviral overexpression of Arrdc4 increased lactate dehydrogenase (LDH) release into culture media compared to empty vector (EV) control in mouse neonatal cardiomyocytes (t-test). B-F. After adenoviral induction of ARRDC4 in HT1080 cells, cellular damage was detected by trypan blue staining (B), activation of caspase-3/7 (C), FITC-Annexin V-positive apoptosis (D), nuclear green DCS1-positive necrosis (E) and cellular ATP levels (F) (A and C-F, t-test; B, Mann-Whitney U test). G and H. The amount of glutathione was estimated in live cells. NRF2 activation was quantified as a cellular response to oxidative stress. HT1080 cells were transfected with a plasmid containing a reporter gene in which luciferase was fused with the NRF2 ubiquitination domain; thus, oxidative stressor would stabilize the luciferase-fusion protein and increase luciferase activities. H₂O₂ (500 μM for 2 hr) as a positive control. P=N.S. (t-test). I. The induction of major endoplasmic reticulum (ER) stress markers was quantified by quantitative PCR in Arrdc4-overexpressing mouse neonatal cardiomyocytes and HT1080 cells. Tunicamycin (1 μg/ml, 2 hr) was added to aggravate ER stress (St-t, t-test; MW-U, Mann-Whitney U test). J. XBP1s activation was quantified by luciferase assay in HT 1080 cells (t-test). K and L. Protein expression of ATF4 or CHOP was analyzed by Western blot analysis and quantified by densitometry (St-t, t-test; MW-U, Mann-Whitney U test). M and N. N-linked protein glycosylation was detected by fluorescently-labeled concanavalin A. The addition of N-acetylglucosamine (GlcNAc, 10 mM, 8 hr) or uridine diphosphate-GlcNAc (UDP-GlcNAc, 1 mM, 8 hr) partially restored ARRDC4-mediated induction of CHOP (one-way ANOVA post-hoc Bonferroni test) in Arrdc4-overexpressing mouse neonatal cardiomyocytes.