Reagents
Anti-mouse CD117 (c-kit)-APC (clone 2B8), anti-mouse Ly-6 A/EA (Sca-1)-PE/Cy7 (clone D7), biotin-conjugated anti-mouse CD4 (clone GK1.5), anti-mouse CD8 (clone 53–6.7), anti-mouse CD45R/B220 (cloneRA3-6B2), anti-mouse Ly6G/Gr-1 (clone RB6-8C5), anti-mouse CD11b (clone M1/70), anti-mouse Ter-119 (clone Ter-119), and APC-Cy7-conjugated streptavidin were obtained from eBioscience (San Diego, CA, USA). Anti-mouse CD45.1-FITC (clone A20, Ly5.1), anti-mouse CD45.2-PE (clone104, Ly5.2), anti-mouse Ly6G/Gr-1-PE/Cy7 (cloneRB6-8C5), anti-mouse CD45R/B220-PerCP (cloneRA3-6B2), anti-mouse CD11b-PE/Cy7 (cloneM1/70), anti-mouse CD3-APC (clone145-2C11), and streptavidin-PerCP (405213) were obtained from Biolegend (San Diego, CA, USA). Anti-mouse γH2AX and anti-mouse cleaved CASPASE 3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-mouse HO-1, anti-mouse NQO1, anti-mouse cytochrome C, anti-mouse β-ACTIN, anti-mouse NRF2, anti-mouse BAX, anti-mouse BAK, and anti-mouse BCL-XL antibodies were obtained from Abcam (Cambridge, UK). FITC-conjugated goat anti-rabbit/mouse antibodies were obtained from ZSGB-BIO (Beijing, China). Cytofix/Cytoperm buffer and Perm/Wash buffer were obtained from BD Pharmingen (San Diego, CA, USA). ATX was obtained from Aladdin Co. (Shanghai, China).
Mice
Male C57BL/6 (CD45.2) mice were purchased from the Beijing HFK Bioscience Co. Ltd. (Beijing, China). Male C57BL/6 (CD45.1) mice were purchased from the Institute of Hematology and Blood Disease Hospital (PUMC, Tianjin, China). Nrf2
−/− mice were generously provided by Dr. Thomas W. Kensler of the University of Pittsburgh, USA. All mice were approximately 6–8 weeks old (20–22 g) and housed under specific pathogen-free conditions at the Experimental Animal Centre of the Institute of Radiation Medicine of PUMC. All animal studies were approved by the Animal Care and Ethics Committee of the Institute of Radiation Medicine of PUMC (SYXK-2014-0002).
TBI and ATX administration
Two batches of CD45.2 mice were respectively divided into (1) five groups: control group, TBI group, TBI + 25 mg/kg ATX group, TBI + 50 mg/kg ATX group, and TBI + 100 mg/kg ATX group; and (2) four groups: control group, ATX group, TBI group, TBI + ATX group (the ATX concentration was 50 mg/kg/day). Nrf2
−/− mice were divided into three groups: control, TBI alone group, and TBI + ATX group. Each group had five mice. All mice that received ATX were administrated the compound by gavage 3 days before irradiation and 7 days after irradiation. Mice in all the TBI groups received 4 Gy γ-ray at a dose rate of 0.99 Gy/min. Control mice were sham-irradiated.
Weight and organ index
The mean body weights of the groups were plotted to determine the weight gain or loss in the control and test groups. Then the mice were sacrificed and the organs were excised and weighed. Organ weights were recorded, and indices (in g/g) were calculated by the ratio of the wet weights of the individual organs to the whole body weights.
Peripheral blood cell and bone marrow cell counts
Blood was obtained from the mice via the orbital sinus and was collected in micropipettes coated with the ethylenediaminetetraacetic acid (EDTA.K3). The cell counts included white blood cells (WBCs), percentage of lymphocytes (LY%), and percentage of neutrophil granulocytes (NE%). Bone marrow cells were flushed from both the tibias and femurs with sterile phosphate-buffered saline (PBS), and the cell numbers were counted using a Celltac E hemocytometer (Nihon Kohden, Japan).
Flow cytometry analysis
For B cell, T cell, and myeloid cell analysis in peripheral blood, 50 μl peripheral blood was first incubated with B220, CD3, CD11b, and Gr1 at room temperature, and then the red blood cells were removed with BD FACS™ Lysing Solution. For HPC and HSC analysis, bone marrow cells were isolated as described above. They were then filtered and counted prior to staining with antibodies. Bone marrow cells (5 × 106) were incubated with biotin-labeled antibodies specific for murine Ter119, B220, Gr1, CD11b, CD4, and CD8, and were then stained with streptavidin, c-kit, and sca1. For HO-1, NQO1, γH2AX, and cytosol cytochrome C (cyt C) analysis, bone marrow cells (5 × 106) were stained with c-kit antibody, fixed, and permeabilized using BD Cytofix/Cytoperm buffer according to the manufacturer’s protocol, and finally stained with respective antibodies and FITC-conjugated secondary antibodies. Data acquisition was performed using a BD Accuri C6 and analyzed using BD Accuri C6 software.
Competitive repopulation assay
Bone marrow cells (1 × 106) from C57BL/6 (CD45.2) mice after the various treatments and 1 × 106 bone marrow cells from C57BL/6 (CD45.1/45.2) mice were mixed and transplanted into lethally irradiated C57BL/6 mice (CD45.1). The percentage of donor-derived (CD45.2) cells in the recipients’ peripheral blood was examined 2 months after transplantation.
Colony of granulocyte macrophage cells (CFU-GM) assay
Bone marrow cells (1 × 104) from the control groups and 1 × 105 bone marrow cells from the TBI groups were cultured in M3534 methylcellulose medium (Stem Cell Technologies) for 5 days. The colonies of CFU-GM with more than 30 cells were counted according to the instructions. The results are expressed as the numbers of CFU-GM per 105 bone marrow cells.
Analysis of intracellular ROS levels
Bone marrow cells (5 × 106) from wild mice or Nrf2
−/− mice were first stained with c-kit antibody, and were then incubated with 2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA; Beyotime Biotechnology, Nanjing, China; 10μM), MitoSOX (Life Technologies, Grand Island, NY, USA; 10μM); and dihydroethidium (DHE; Beyotime Biotechnology; 5μM) for 20 min, 30 min, and 10 min, respectively, at 37 °C in a water bath according to the manufacturer’s protocol. The levels of intracellular ROS in c-kit-positive cells were analyzed by measuring the mean fluorescence intensity (MFI) of DCFDA, DHE, and oxidized MitoSOX using a flow cytometer.
Analysis of SOD2, CAT, and GPX1 activity
To isolate c-kit-positive cells, bone marrow cells were stained with CD117 microbeads (Miltenyi Biotec, Teterow, Germany) for 20 min at 2–8 °C, and the c-kit-positive cells were collected according to the manufacturer’s protocol. SOD2, CAT, and GPX1 enzymatic activities in c-kit-positive cells were analyzed using SOD, CAT, and GPX1 assay kits (Beyotime Institute of Biotechnology, Jiangsu, China), respectively, following the manufacturer’s instructions.
Immunofluorescence staining of NRF2
c-kit-positive cells were sorted as described above, and fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized in 0.2% Triton-X-100/PBS and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and then incubated with anti-NRF2 antibody (1:300; overnight at 4 °C). Cells were washed three times with PBS for 5 min, and incubated with FITC-conjugated goat anti-rabbit IgG (1:300) antibody in the dark for 1 h at room temperature. Finally, a fluorescent sealing liquid solution (ZSGB-BIO, Beijing, China) containing 4′,6-diamidino-2-phenylindole (DAPI) was added to stain the nuclei. Fluorescence images were visualized with a fluorescence microscope.
Apoptosis assay
Bone marrow cells (5 × 106) were first incubated with a c-kit antibody, and cell apoptosis was then evaluated using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) followed by flow cytometry analysis according to the manufacturer’s protocol. Finally, samples were evaluated by BD Accuri C6 and analyzed using BD Accuri C6 software.
Analysis of cytochrome C release from mitochondria in c-kit-positive cells
c-kit-positive cells were sorted as described above. The cytochrome C release from mitochondria in the c-kit-positive cells was determined by analysis of the MFI by flow cytometry according to the method described previously [20].
Western blotting analysis
c-kit-positive cells were collected as described above and then lysed using RIPA reagent (Boster Biological, Wuhan, China) supplemented with a protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and phenylmethylsulfonyl fluoride (PMSF; Sigma). Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Sigma), and detected using specific antibodies as follows: β-ACTIN antibody (1:1000), NRF2 antibody (1:1000), BAX antibody (1:1000), BAK antibody (1:1000), BCL-XL antibody (1:1000), and cleaved CASPASE 3 antibody (1:1000). Blots were developed using Molecular Imager Chemi Doc™ XRS+ with Image Lab™ Software (Bio-Rad, Richmond, CA, USA).
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 5 software with a t test and Welch’s correction. Differences were considered significant at P < 0.05.