Article Figures & Data
Figures
- Fig. 1.
Conditional knockdown of Nox4 expression in long bones. (A) Model for PrxCre-mediated excision of exons 1 and 2 of the Nox4 gene. Indicated are genotyping amplicons for distinguishing the various Nox4 alleles. (B–D) The concentrations of Nox4 mRNA from the knockout (K/O) region, total Nox4 mRNA, and Nox2 relative to β-actin mRNA were determined in the femoral shaft and femoral bone marrow from 13-week-old mice by qRT-PCR. There were 5–11 mice per group. (E and F) The concentration of Nox4 mRNA was determined in liver and kidney aliquots from 32-week-old female mice. There were four mice per group. *P < 0.05; ***P < 0.001 vs. the Nox4 fl/fl genotype. PCR, polymerase chain reaction.
- Fig. 2.
Sex and age affect trabecular bone of mice maintained on standard chow. Males and females of genotypes Nox4 fl/fl, PrxCre Nox4 fl/fl, and Nox4 0/0 at 13 and 32 weeks of age were analyzed. There were 5–12 mice per group. Three-way ANOVAs were conducted with sex, age, and genotype as the three factors. The panels indicate significant main effects and important interaction effects. (A–H) BMD, bone mineral density; Trab., trabecular; Vol., volume.
- Fig. 3.
Age, sex, and the Nox4 genotype affect cortical bone of mice maintained on standard chow. Males and females of genotypes Nox4 fl/fl, PrxCre Nox4 fl/fl, and Nox4 0/0 at 13 and 32 weeks of age were analyzed. There were 5–12 mice per group. Three-way ANOVAs were conducted with sex, age, and genotype as the three factors. The panels indicate significant main effects and important interaction effects. (A–E) BMD, bone mineral density;
- Fig. 4.
Chronic ethanol feeding for 3 months affects body composition and bone turnover. (A) The diagram illustrates the time course of the body weights for males and females fed either the ethanol diet or control diet for 3 months. (B–D) Body weight, relative liver weight, and the concentration of liver triglycerides were determined at the end of the chronic ethanol feeding experiment. (E–G) Serum concentrations of ALT, Osteocalcin and CTX were determined. Three-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. For ALT and CTX, a separate two-way ANOVA for just the data for females was calculated. EtOH, ethanol.
- Fig. 5.
Trabecular data for mice from the chronic ethanol feeding experiment. (A–H) Trabecular bone parameters for a region of the tibiae 0.4–0.7 mm distal to the proximal growth plate. Three-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. BMD, bone mineral density; EtOH, ethanol;
- Fig. 6.
Cortical data for mice from the chronic ethanol feeding experiment. (A–E) Cortical bone parameters for the tibiae. Three-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. BMD, bone mineral density; EtOH, ethanol.
- Fig. 7.
RNA-Seq analysis of femoral shafts of mice from the chronic ethanol feeding experiment. RNA-Seq was conducted on femoral shaft RNA isolated from male mice. A two-way ANOVA was conducted for each gene with diet and genotype as the two factors. The test probabilities were not adjusted for the multiplicity of genes. Volcano plots depicting the test probability for each gene against the fold change in gene expression are shown for the main effect of diet (A), the main effect of genotype (B), and the interaction effect between diet and genotype (C). Points above the red horizontal dashed line represent genes with test probabilities P < 0.05. Red points indicate genes with at least a 2-fold change in gene expression and a test probability P < 0.05. (D) Among the 100 genes with the highest expression levels in Nox4 fl/fl mice on the control diet, the change in gene expression caused by ethanol is depicted for the 16 genes that show a significant (P < 0.05) main diet effect. EtOH, ethanol.
- Fig. 8.
qRT-PCR gene expression analysis replicates findings from RNA-Seq. Gene expression was determined for RNA isolated from the femur shafts of mice from the chronic ethanol feeding experiments by qRT-PCR. The expression levels were normalized to the concentration of total RNA. The expression levels were determined for total Nox4 mRNA (A), Frzb (B), Myh3 (C), Col2a1 (D), osteocalcin (E), RANKL (F), Nox2 (G), GAPDH (H), and Cyp2s1 (I). 3-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. EtOH, ethanol.
Tables
Gene Gene Symbol Forward Primer Reverse Primer Nox4 knockout region Nox4 acc tct gcc tgc tca ttt gg cct agg ccc aac att tgg tga Total Nox4 Nox4 gga tca cag aag gtc cct agc gtt gag ggc att cac caa gtg Nox2 Cybb ggg atg aat ctc agg cca at gcc gtc cat aca gag tct tc β-Actin Actb aga tga ccc aga tca tgt ttg aga cca gag gca tac agg gac agc GAPDH Gapdh cat ctt cca gga gcg aga cc cct tca agt ggg ccc cg RANKL Tnfsf11 cag cat cgc tct gtt cct gta ctg cgt ttt cat gga gtc tca Cyp2S1 Cyp2s1 ttg gca tcc gtt tgc cct at gag aac atc tcg tag gcc tgg Osteocalcin Bglap agc ctt cat gtc caa gca gga g gac tga ggc tcc aag gta gcg Col2a1 Col2a1 ggc cag gat gcc cga aaa tta cgc acc ctt ttc tcc ctt gt Frzb Frzb aca act atg tca tcc ggg ct tga cgg tgt ccc ttg gaa tg
Data Supplement
- Supplemental Figures -
1. (A)Nox4 and Cyp2s1 mRNA expression were compared in two Nox4 knockout models.
2. (A)Western blot for detection of nitrotyrosinewith 0 –50 ng of nitrotyrosinebovine serum albumin ...
- Supplemental Table -
mRNAseq data set.
- Supplemental Figures -
In this issue
Independent Effects of Nox4 and Ethanol on Bone Biology
