All animals were housed in an air-conditioned room (22–25°C; 12-h light/dark cycle; 50% humidity) with free access to food pellets and tap water. Ocn-Cre transgenic mice used in BM niches (18) and Sirt1 mice (19) have been described (Fig. S1). Ocn-Cre, floxed Sirt1 and wild-type C57BL/6 mice were purchased from Jackson Laboratory and employed for experiments. The Ocn-Cre mice are widely used to target osteoblasts in the BM niche (18). To investigate the role for HSC maturation of Sirt1 in the BM niche, such as in osteoblast, Sirt1^{floxed/floxed} mice were crossed with Ocn-Cre. A total of 70 mice (female; weight, 15–18 g; age, 8–10 weeks) were used for experiments.

Transplantations of BM cells were performed as described previously (20). Briefly, mice were euthanized then tibias and femurs were recovered. Scalpel was used to cut the ends of bone off and then a syringe with 27G needle (filled with PBS supplemented with 2% FBS and 1% P/S) was used to flush into a 50 ml tube covered with 40 µM nylon filter. Recovered BM cells were lysed in RBC lysis buffer (Lonza Group, Ltd.) for 5–10 min on ice. Samples were then washed with 10 ml PBS supplemented with 2% FBS and 1% P/S and centrifuged 5 min at 1,500 rpm. Supernatant was aspirated and the cell pellets were collected for the experiments.

Control and Sirt1^Δ/Δ mice received lethal doses of irradiation (9.5 Gy, Gammacell 3000) prior to the transplantation of 200 µl 1×10⁶ BM cells by retro-orbital injection on the same day as previously described (21). For serial 5-FU treatments, 5-FU (150 mg/kg) was injected intraperitoneally every seven days until 100% animal mortality was achieved (22). For the duration of the present study, the following criteria were applied to define a mouse as moribund, which would require immediate euthanasia (23,24): i) Ruffled and/or matted fur; ii) weight loss of >15%; iii) hypothermia (detected by touching); iv) hunched posture; v) unable to eat or drink freely; vi) barrel rolling. No animals satisfied these criteria in the present study.

Reverse transcription and quantitative PCR were performed as previously described (14,25). Briefly, total RNAs were extracted from BM cells using QIAGEN RNeasy-Plus Mini-columns according to manufacturer's protocol (Qiagen, Inc.). cDNA synthesis were performed following the protocols of the manufacturer of the kit (Bio-Rad Laboratories, Inc.). qPCR was performed with SYBR Green Mix (Roche Diagnostics) and LightCycler 96 instrument (Roche Diagnostics) and data were normalized to the housekeeping gene GAPDH. The mRNA levels were measured using the 2^−ΔΔCq method of quantification (26). RT-qPCR was performed using the following primers: Sirt1 forward, 5′-CTGAAAGTGAGACCAGTAGCA-3′ and reverse, 5′-GATGAGGCAAAGGTTCCCTA-3′ and GAPDH forward, 5′-GCACAGTCAAGGCCGAGAAT-3′ and reverse, 5′-GCCTTCTCCATGGTGGTGAA-3′.

Flow cytometry was performed as described previously (20). Briefly, BM cells were collected from femurs and tibias of the mice by flushing using fluorescence-activated cell sorting buffer, which consisted of phosphate buffered saline containing 2% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) and 0.1% sodium azide. Peripheral blood cells were collected from the tail vein. Flow cytometry was performed using antibodies listed in Table S1. Data acquisition and analysis were performed with Cell Quest v.3.3 or Diva software v.6.1.3 (BD Biosciences) and with FlowJo software v.10.3 (FlowJo LLC) respectively.

The statistical significance of differences in the means between two populations were assessed using two-tailed unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference for pairwise comparisons (between indicated genotypes in the same population). The Kaplan-Meier log-rank test was used to analyze survival data.

SIRT1 is a critical regulator of homeostatic adult HSCs in mice (13,14). Based on these observations, it is tempting to speculate that SIRT1 may also serve a role for HSC maturation in the BM niche. To investigate the role for HSC maturation of SIRT1 in the BM niche, Sirt1^{floxed/floxed} mice were crossed with Ocn-Cre transgenic mice, resulting in Sirt1^Δ/Δ mice. A previous study showed that the loss of Sirt1 in the hematopoietic system resulted in anemia and specified lineage decision (myeloid versus lymphoid lineage specification) (14). However, Sirt1 deletion in the BM niche demonstrated no differences in the numbers of white blood cells (WBCs), lymphocytes (LY), neutrophils (NEU), monocytes (MO), hemoglobin (HGB), and platelets (PLT) (Fig. 1A). It was also found that Sirt1^Δ/Δ mice did not result in significant differences compared with wild-type mice in their ability to produce mature myeloid cells (Mac1⁺ cells), T cells (CD3⁺ cells) and B cells (B220⁺ cells) in the peripheral blood (PB) and bone marrow (BM) (Fig. 2 and Fig. S2). The expression of Sirt1 was decreased in the CD31⁻CD45⁻Ter119⁻ BM niche cells of Sirt1^Δ/Δ mice compared with Sirt1^+/+ mice, which confirmed Sirt1 knockdown (Fig. 1B).

No differences in the numbers of HSCs (Lin⁻Sca1⁺cKit⁺CD150⁺CD48⁻, SLAM cells), Lin⁻Sca1⁺cKit⁺ (LSK) progenitor cells, common myeloid progenitor (CMP) cells (Lin⁻Sca1⁻cKit⁺CD34⁺CD16/32⁻), granulocyte-macrophage progenitor (GMP) cells (Lin⁻Sca1⁻cKit⁺CD34⁺CD16/32⁺), megakaryocyte-erythroid progenitor (MEP) cells (Lin⁻Sca1⁻cKit⁺CD34⁻CD16/32⁻) and common lymphoid progenitor (CMP) cells (Lin⁻Sca1^lowcKit^lowCD127⁺) were observed in the Sirt1^Δ/Δ mice compared with control mice (Fig. 3 and Fig. S3).

To assess the role of HSC maturation following Sirt1 deletion in the BM niche under stress condition, wild-type BM cells were transplanted into Sirt1^Δ/Δ mice, which had been lethally irradiated to remove endogenous blood cells (Fig. 4 and Fig. S4). Compared with control mice, Sirt1^Δ/Δ mice exhibited no detectable effects on peripheral blood (PB) donor chimerism (Fig. 4A), bone marrow (BM) donor chimerism and the numbers of mature hematopoietic cell types up to 16 weeks post-transplantation (Fig. 4B). In addition, Sirt1^Δ/Δ mice demonstrated no differences in the numbers of WBCs, Lys, NEUs, MOs and the levels of hemoglobin (HGB) four weeks post-transplantation compared with control mice (Fig. S5).

Next the Sirt1^Δ/Δ mice were repeatedly treated with 5-FU weekly to assess their sensitivity to stress under non-transplantation stress. Exposure of Sirt1^Δ/Δ mice to serial proliferative stress induced by 5-FU (22) exerted in no significant differences in animal mortality compared with control mice, according to the Kaplan-Meier curve (Fig. 5).