Journal: The Journal of Experimental Medicine

Article Title: Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

doi: 10.1084/jem.20151100

Figure Lengend Snippet: IGF1 injection reverts the increases in HSC quiescence but not the suppression of lymphopoiesis in mice exposed to DR. (A) ELISA-determined serum level of IGF1 in mice treated with DR or AL for 4 d or refed with an AL diet for 3 d after 4 d of DR ( n = 3–4 mice per group; n = 2 independent experiments). (B) ELISA-determined serum level of IGF1 in 2-mo-old p53 −/− mice and p53 +/+ mice (littermates) that were treated with DR or AL for 2 wk ( n = 3–4 mice per group; n = 2 independent experiments). (C–G) Mice were treated with a DR or AL diet for 8 d. In parallel, recombinant human IGF1 protein or vehicle control was subcutaneously injected twice per day at a dose of 500 µg/kg ( n = 4 mice per group; n = 2 independent experiments). FACS analysis was performed 24 h after the last injection on freshly isolated BM cells: percentage of HSCs in the indicated cell cycle stages (C), total numbers of CLPs (D), pro–B cells (E), MEPs (F), and proEry’s (G) per million total BM cells are show. AL con, control-injected AL mice; AL IGF1, IGF1-injected AL mice; DR con, control-injected DR mice; DR IGF1, IGF1-injected DR mice. Data are displayed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA. ns, not significant.

Article Snippet: ELISA for detecting IGF1, IL-6, IL-7, and total corticosterone was performed using the IGF-I mouse/rat ELISA kit (Mediagnost), the Mouse IL-6 Quantikine ELISA kit (R&D Systems), the Mouse IL-7 Quantikine ELISA kit (R&D Systems), and Corticosterone EIA kit (ARBOR ASSAYS), respectively, according to the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

doi: 10.1084/jem.20151100

Figure Lengend Snippet: IL-7 or IL-6 injection reverts suppression of lymphopoiesis in mice exposed to DR. (A) ELISA-determined serum level of IL-7 in mice treated with DR or AL for 7 d or refed with AL diet for 3 d after 7 d of DR ( n = 3–4 mice per group; n = 2 independent experiments). One-way ANOVA. (B–G) 2-mo-old mice were treated with DR or AL diet for 9 d. In parallel, mouse IL-7 protein or vehicle control was subcutaneously injected for 9 d at a dose of 50 µg/kg once per day ( n = 5 mice per group; n = 2 independent experiments). Mice were sacrificed for analysis 24 h after the last injection. (B–D) FACS analysis was performed on freshly isolated BM cells to determine the total number of CLPs (B) and MEPs (C), and the percentage of CLPs (D) of DR mice in the indicated phases of the cell cycle (FACS analysis by Ki67 and DAPI staining). (E and F) the ratio of CLPs versus lymphoid-biased HSCs (E) and of pro–B cells versus CLPs (F). (G) The mRNA expression levels of genes that are associated with lymphoid cell differentiation and erythroid cell differentiation were determined by qRT-PCR in freshly isolated lymphoid-biased HSCs under the indicated treatment conditions. One-way ANOVA (B, C, and E–G) or unpaired two-tailed Student’s t test (D) was used. (H) ELISA-determined serum level of IL-6 in mice treated with DR or AL for 7 d or refed with AL diet for 3 d after 7 d of DR ( n = 3–4 mice per group; n = 2 independent experiments). One-way ANOVA was used. (I) 2-mo-old mice were treated with a DR or AL diet for 5 d. In parallel, mouse IL-6 protein or vehicle control was subcutaneously injected into mice daily at a dose of 50 µg/kg ( n = 4 mice per group; n = 2 independent experiments). The total number of CLPs was determined by FACS analysis, 24 h after the last injection. One-way ANOVA analysis. (J–P) ADX or sham surgery was performed on 2-mo-old mice. 10 d after operation, mice were treated with DR or AL for 9 d ( n = 4–5 mice per group; n = 2 independent experiments). Mice of the indicated cohorts were analyzed as follows: (J, O, and P) ELISA-determined serum levels of total corticosterone (J), IL-7 (O), and IL-6 (P); (K–M) FACS analysis of the total number of CLPs (K) and MEPs (M) per million BM cells and of the percentage of CLPs (L) of DR mice in different stages of the cell cycle (by Ki67 and DAPI staining); (N) expression of lymphoid- and erythroid-specific genes in lymphoid-biased HSCs of mice of the indicated cohorts. One-way ANOVA (J, K, and M–P) or unpaired two-tailed Student’s t test (L) was used. AL con, control-injected AL mice or sham-operated AL mice; AL IL-7, IL-7–injected AL mice; DR con, control-injected DR mice or sham-operated DR mice; DR IL-7, IL-7–injected DR mice; AL IL-6, IL-6–injected AL mice; DR IL-6, IL-6–injected DR mice; AL ADX, AL mice with ADX; DR ADX, DR mice with ADX. Data are displayed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant.

Article Snippet: ELISA for detecting IGF1, IL-6, IL-7, and total corticosterone was performed using the IGF-I mouse/rat ELISA kit (Mediagnost), the Mouse IL-6 Quantikine ELISA kit (R&D Systems), the Mouse IL-7 Quantikine ELISA kit (R&D Systems), and Corticosterone EIA kit (ARBOR ASSAYS), respectively, according to the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control, Isolation, Staining, Expressing, Cell Differentiation, Quantitative RT-PCR, Two Tailed Test