Fig 1: Amount of protein superoxide dismutase 1 (SOD1) (A, catalase (CAT) (B), thyredoxin (TRX) (C), forkhead box O3 (D), myoblast determination protein 1 (MYOD) (E), heat shock protein 70 (HSP70) (F), and housekeeping proteins: hypoxanthine-guanine phosphoribosyltransferase (HPRT) and actinin (ACT) in non-treated (WT) and PBN-conditioned (PBN) SkMDS/PCs populations cultured under standard (N) and hypoxic (H) in vitro conditions. Abbreviations: SkMDS/PCs = skeletal muscle derived stem/progenitor cells; WT = wild-type non-treated SkMDS/PCs; PBN = SkMDS/PCs cultured in medium conditioned with tert-butyl-alpha-phenyl nitrone. * significant at p value <0.05, ** significant at p value <0.01, *** significant at p value <0.001.
Fig 2: Castration induces messenger RNA (mRNA) expression of fetal genes in the heart. Male mice were sham-operated (Sham; n = 18-20) or castrated (ORX; n = 18-20) at age 11 to 13 weeks and the left ventricle was collected 3 weeks later. A, mRNA levels of genes involved in lipid (Cd36) and glucose transport (Glut1 and Glut4) and B, expressed in the fetal gene program (b-Mhc, Anp, and Bnp) as well as a-Mhc (that replaces b-Mhc during postnatal development) were analyzed by quantitative polymerase chain reaction. Expression levels were normalized against the reference gene Hprt1. Bars indicate means, error bars indicate SEM. LV, left ventricle. Data were analyzed by t test or Mann-Whitney U test. *P < .05, **P < .01, ***P < .001 vs Sham.
Fig 3: Effects of DOX on cardiomyocyte apoptosis, LV superoxide production and Nox2 mRNA and protein expression in WT and Nox2-/- (KO) mice. (A) TUNEL-positive cardiomyocyte nuclei in LV sections quantified by digital image analysis (n = 6, 80 cells in each). (B) Caspase 3/7 activity in LV tissue, expressed as relative light units (RLU; n = 5). (C) NADPH-dependent superoxide production analysed in LV membrane fractions by lucigenin-enhanced chemiluminescence (n = 6). (D) Effects of selective ROS inhibitors on superoxide production in WT samples (n = 6). (E) Nox2 mRNA expression assessed in LV tissue by real-time TaqMan RT-PCR (n = 15-WT, n = 9-KO). (F) Representative western blot of Nox2 protein expression in LV homogenate (M, MCF-7 cell lysate positive control; WTC, WT control; WTD, WT DOX-treated; KOC, KO control; KOD, KO DOX-treated; HPRT, hypoxanthine-guanine phosphoribosyltransferase endogenous control) and its quantification (n = 6). Data are shown as mean ± SEM and analyses performed using a two-factor ANOVA followed by Student's unpaired t-test (A–C) or Kruskal–Wallis test with Dunn's post hoc test for Control or DOX (D) or Student's unpaired t-test with Walsh's correction to account for unequal variances (E, F). *P < 0.05 versus WT Control; # P < 0.05 versus WT DOX.
Fig 4: Abnormal Paneth cells show ER stress.(A, B) Representative transmission electron microscopy images of Paneth cells at the base of ileal crypts in (A) ICR and (B) SAMP1/YitFc mice. Scale bars indicate 2 µm. (C, D) Quantitative analysis of (C) granule number and (D) ER lumen diameter in Paneth cells (n = 3/each week for SAMP1/YitFc mice). For the measurements, three Paneth cells were randomly selected from each mouse. (E) SDS–PAGE Western blot analysis of ER stress markers, pIRE1a, ATF4, cleaved-ATF6, and GRP78 in ileal crypts (n = 4/each group). Total-IRE1a and HPRT1 was used as loading control. (F) Relative expression level of ER stress markers calculated from the band intensity. Error bars represent mean ± SEM. (C, D, F) Statistical significance was evaluated by t test in (C, D), and one-way ANOVA followed by Tukey’s post hoc test in (F). P < 0.05 was considered statistically significant. *P < 0.05, †P < 0.01, §P < 0.001. E, ER; G, granules; N, nucleus; n.s., not significant.
Fig 5: Effect of DOX concentration and time of incubation on superoxide production, gene and protein expression in HL-1 cardiomyocytes. (A) Representative western blot showing effects of DOX concentration on Nox2 protein expression at 24 h and its quantification (n = 5); (B) NADPH oxidase activity in the presence of (i) 0.5 µM and (ii) 5 µM DOX assessed by lucigenin-enhanced chemiluminescence over 24 h (both n = 5); (C) Representative western blot showing effects of DOX concentration on Mfn2 protein expression at 24 h and its quantification (n = 5). HPRT, hypoxanthine-guanine phosphoribosyltransferase endogenous control; H, HeLa cell lysate positive control; M, MCF-7 cell lysate positive control; C, Control (normal medium). Data are shown as mean ± SEM and analyses performed using one-factor ANOVA (Kruskal–Wallis test) and Dunn's post hoc test (A, C) or two-factor ANOVA with Student's paired t-test, as indicated (B). *P < 0.05 versus Control.
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