Lentivirus

In order to overexpress HSPB7, the lentivirus expressing HSPB7 and the scramble negative control (NC) were purchased from Cyagen Company (Guangzhou, China). For HSPB7 knockdown, recombinant lentiviruses targeting HSPB7 (shHSPB7-1 and shHSPB7-2) and the non-targeting negative control (shNC) were purchased from GenePharma Co. (Shanghai, China). The shRNA sequences were as follows: shNC, TTCTCCGAACGTGTCACGT; shHSPB7-1, ACAGAACCUCUUCCACCUUTT; and shHSPB7-2, GAACACCUUCGCUCACAAGTT. For virus transfection, cells were exposed to the lentiviral supernatant with the addition of polybrene (5 μg/mL, Sigma-Aldrich) for 24 h. After 72 h, antibiotic selection was conducted by adding puromycin (5 μg/mL, Sigma-Aldrich) to transfected cells.

L1210 murine leukemia cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin. Cells were maintained in 5% CO₂ to 95% air. To culture bone marrow-derived macrophages (BMDMs), mononuclear cells were isolated from tibias and femurs of C57BL/6 mice. Cells were cultured at a density of 2 × 10⁶/mL in DMEM containing 10% FBS and 25 ng/mL murine macrophage-colony stimulating factor (M-CSF) (PeproTech, Rocky Hill, NJ, USA) for 7 days. In some experiments, IFNγ (20 ng/mL, PeproTech), LPS (50 ng/mL, Sigma, St. Louis, MO, USA), or IL4 (20 ng/mL, PeproTech) was added and cultured for 24 h before further analyses. Macrophages treated with PBS, LPS + IFNγ, or IL4 were named as M^PBS, M^LPS, or M^IL4, according to Epelmann et al. (5 (link)). Cells were transfected siRNA with Lipofectamine LTX (Invitrogen) according to the recommended protocol. Three pairs of siRNA for each target were designed and their knockdown efficiency was determined by qRT-PCR and Western blotting. The siRNA with highest efficiency was chosen for further experiments. Lentivirus was packaged by Cyagen Biosciences with commercial service (Guangzhou, China).

To trace the cell fate of Prx1⁺ MSCs and Prx1⁻ MSCs, they were labelled with GFP using lentivirus (Cyagen Biosciences) and transplanted into the mice (n = 3 per group) with femoral bone defect. At 2 weeks after surgery, femurs were harvested, and fixed samples were decalcified, dehydrated and embedded in Tissue‐Tek^® OCT Compound (SAKURA). The 10 μm thickness of sagittal sections was cut with a freezing microtome (Thermo Scientific). The sections were blocked in 5% BSA for 40 minutes at room temperature and incubated with the primary antibodies anti‐DMP1 (1:400; Abcam) and anti‐GFP (1:400; Abcam) at 4°C overnight. After washing, the sections were then incubated with the respective secondary antibodies (1:500; Abcam) for 1 hour at room temperature and sealed with DAPI. The images were captured with a fluorescence microscope (Zeiss). For DMP1 quantification, Image J (1.52 version) was used to calculate the area percentage of DMP1 in the bone defect healing area.