Fig. 3
Loss of Regnase-1 expression results in accelerated CD34− HSC cell cycle progression. a Cell-cycle analysis of control (fl/fl) or Regnase-1-KO (Δ/Δ) BM CD34− HSC and CD34+ HSC populations using EdU/7AAD staining. Dot plots indicating the frequency of HSPCs in G0/G1 (EdU− 7AAD−), G2/M (EdU+), and S (EdU+ 7AAD+) phase of cell cycle (n = 3 per group; 3 independent experiments). b Quantification of HSPCs as shown in a. c Detection of proliferative cells in BM section of control (fl/fl) or Regnase-1-KO (Δ/Δ) mice by EdU labeling. Representative image showed EdU staining together with c-Kit and Nuclei (TOPRO3) counter-staining. Scale bars represent 50 μm. d Quantification of the ratio of EdU-positive proliferating cells in c-Kit-positive cells. e Representative cell cycle plots of CD34− HSC populations in BM from control (fl/fl) or Regnase-1-KO (Δ/Δ) mice using Hoechst 33342/pyronin Y staining (n = 3 per group; 3 independent experiments). f Representative data showing number and ratio of cells in the G0, G1, and S/G2/M phases as shown in e. g qRT-PCR analysis of cell cycle regulatory genes in CD34− HSCs from control (fl/fl) and Regnase-1-KO (Δ/Δ) mice (n = 3 per group). h Flow cytometric analysis of apoptotic populations in CD34− HSCs and CD34+ HSCs of control (fl/fl) or Regnase-1-KO (Δ/Δ) mice BM with a combination of annexin V and SYTO Green staining (n = 3 per group; 3 independent experiments). i Ratio of apoptotic cells (annexin V+ SYTO Green+). Data are the means ± SD. j Kaplan-Meier survival curve of control (fl/fl) and Regnase-1-KO (Δ/Δ) mice after weekly administration of 5-FU (150 mg/kg) (n = 7). P values were calculated by the logrank test. k Representative dot plot depicting frequency of phenotypically defined HSPCs at the indicated days following a single injection of 5-FU. l Absolute LSK cell numbers from control (fl/fl) and Regnase-1-KO (Δ/Δ) mice at the indicated days after 5-FU treatment (n = 3). m Expression Regnase-1 mRNA in LSK cells of control (fl/fl) mice at the indicated days after 5-FU treatment (n = 3). Error bars indicate mean ± SD. *p < 0.05; **p < 0.01, ***p < 0.005, two-sided t-test in b, d, f, g, i, m, l
