Journal: aging and disease

Article Title: DNA Demethylation of Promoter Region Facilitates Atoh-1-Induced Interleukin-19 Expression Activation in Bone Marrow Monocytes of Old Mice

doi: 10.14336/ad.2024.0108

Figure Lengend Snippet: Figure 1. IL-19 expression up-regulation in BMMs from old mice. (A) IL-19 mRNA expression in BMMs from young and old mice determined by RT-qPCR. (B) IL-19 secretion levels in the supernatant of BMMs from young and old mice determined by ELISA. (A, B, n=6 per group; one technical replicate of six biological replicates for each group). β-actin was used as internal control for RT-qPCR. Data are representative of three independent experiments. Data were shown as the means ± s.d. P-values were determined by unpaired two-tailed Student’s t-test (A, B). **: p<0.01, ***: p<0.001, Old vs. Young. Statistical analysis All data are expressed as mean ± s.d. All data were tested for normality using the Shapiro-Wilk test. For data that conformed to normal distribution, data between two groups were compared using unpaired Student's t-test, and data from three and more groups were analyzed by one- way analysis of variance (ANOVA) for multiple comparisons. ANOVA was analyzed using the Turkish test for statistically significant differences. For data that did not conform to normal distribution, we used nonparametric tests and Mann-Whitney U-test for data between two groups. Normality tests were performed

Article Snippet: IL-19 levels in the peripheral blood serum, bone marrow plasma or the supernatants of in vitro BMMs culture of mice were measured with ELISA kits (NBP3-06800, Novus Biologicals) according to the manufacturer’s instructions, and the steps were as follows.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test, MANN-WHITNEY

Journal: aging and disease

Article Title: DNA Demethylation of Promoter Region Facilitates Atoh-1-Induced Interleukin-19 Expression Activation in Bone Marrow Monocytes of Old Mice

doi: 10.14336/ad.2024.0108

Figure Lengend Snippet: Figure 2. Alleviation of trabecular bone loss of old mice in response to in vivo deletion of IL-19 from BMMs. (A) IL-19 secretion levels in the supernatant of BMMs from young and old mice with and without in vivo IL-19 deletion from BMMs determined by ELISA. (B) IL-19 levels in the bone marrow of young and old mice with and without in vivo IL-19 deletion from BMMs determined by ELISA. (C) IL-19 levels in the peripheral blood serum of young and old mice with and without in vivo IL-19 deletion from BMMs determined by ELISA. (D) Representative Micro-CT 3D reconstruction images of trabecular bone in the distal femur from young and old mice with and without in vivo IL-19 deletion from BMMs. (E) Quantification of trabecular bone volume fraction (BV/TV), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp) of trabecular bone in the distal femur from young and old mice with and without in vivo IL-19 deletion from BMMs determined by Micro-CT. (F) Representative Micro-CT 3D reconstruction images of trabecular bone in lumbar #1 (L1) vertebrae from young and old mice with and without in vivo IL-19 deletion from BMMs. (G) Quantification of BV/TV, Tb.N, and Tb.Sp of trabecular bone in L1 vertebrae from young and old mice with and without in vivo IL-19 deletion from BMMs determined by Micro-C. (H) Representative Trap staining and quantification of osteoclast number (Oc.N) of in the distal femur from young and old mice with and without in vivo IL-19 deletion from BMMs. (A-H, n=6 per group; one technical replicate of six biological replicates for each group). The arrows point at the positive staining. Data are representative of three independent experiments. Data were shown as the means ± s.d. P-values were determined by unpaired two-tailed Student’s t- test (B, E, G, H), and unpaired two-tailed Mann–Whitney U-test (A, C). *: p<0.05, **: p<0.01, ***: p<0.001, ns: no significance, IL-19LysM-Cre vs. IL-19fl/fl.

Article Snippet: IL-19 levels in the peripheral blood serum, bone marrow plasma or the supernatants of in vitro BMMs culture of mice were measured with ELISA kits (NBP3-06800, Novus Biologicals) according to the manufacturer’s instructions, and the steps were as follows.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Micro-CT, Staining, Two Tailed Test, MANN-WHITNEY

Journal: aging and disease

Article Title: DNA Demethylation of Promoter Region Facilitates Atoh-1-Induced Interleukin-19 Expression Activation in Bone Marrow Monocytes of Old Mice

doi: 10.14336/ad.2024.0108

Figure Lengend Snippet: Figure 5. Atoh-1 binding on IL-19 promoter to activate IL-19 expression due to DNA demethylation of IL-19 promoter. (A) Atoh-1 mRNA expression in BMMs from young and old mice determined by RT-qPCR; n=6 per group. (B) Atoh-1 protein expression in BMMs from young and old mice determined by western blot. (C) Exogenous HA-tagged Atoh-1 protein expression in mouse BMMs in response to Atoh-1 over-expression determined by western blot. (D) IL-19 mRNA expression in mouse BMMs in response to exogenous Atoh-1 over-expression determined by RT-qPCR. (E) IL-19 secretion levels in the supernatant of BMMs in response to exogenous Atoh-1 over-expression determined by ELISA. (F) Atoh-1 protein expression in mouse BMMs in response to siRNA- mediated Atoh-1 knockdown determined by western blot. (G) IL-19 mRNA expression in mouse BMMs in response to siRNA-mediated Atoh-1 knockdown determined by RT-qPCR. (H) IL-19 secretion levels in the supernatant of BMMs in response to siRNA-mediated Atoh-1 knockdown determined by ELISA. (I) Atoh-1 binding on mouse IL-19 promoter in BMMs from young and old mice determined by ChIP. (J) Atoh-1 binding on mouse IL-19 promoter in BMMs with or without 5’-aza (10 μM) determined by ChIP. (K) Atoh-1 binding on mouse IL-19 promoter in BMMs in response to exogenous Dnmt1 over-expression determined by ChIP. (A, D, E, G, H, n=6 per group; one technical replicate of six biological replicates for each group; B, C, F, I-K, representative results from two independent biological experiments). β-actin was used as internal control for RT-qPCR and western blot. Data are representative of three independent experiments. Data were shown as the means ± s.d. P-values were determined by unpaired two-tailed Student’s t-test (A, D, E, G, H). **: p<0.01, ***: p<0.001, ns: no significance, Old vs. Young, HA-Atoh-1 vs. Empty vector, Atoh-1 siRNA vs. Ctrl.

Article Snippet: IL-19 levels in the peripheral blood serum, bone marrow plasma or the supernatants of in vitro BMMs culture of mice were measured with ELISA kits (NBP3-06800, Novus Biologicals) according to the manufacturer’s instructions, and the steps were as follows.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay, Knockdown, Control, Two Tailed Test, Plasmid Preparation