Sca 1

Cell surface markers analysis was performed using phycoerythrin-conjugated antibodies against Sca-1 (eBioscience 11:5781-81), CD29 (eBioscience 12:0291-81), CD105 (eBioscience 12:1051-82), CD140, (eBioscience 14:1401-82) CD11b (eBioscience 12:0112-81), CD34 (eBioscience 14:0341-81) and CD45 (eBioscience 14:0451-81), followed by flow cytometry using a FACSAria II system (BD Biosciences, San Jose, CA, USA) in the CGMH Core facility. Voltages and gates were set based on unstained periosteum samples from Cre-negative animals.

Reagents for inducing MSC differentiation, including 3-isobutyl-1-methylxanthine, l-ascorbic acid, indomethacin, dexamethasone, insulin, β-glycerophosphate, Alizarin Red S, and Oil Red O were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Antibodies used in Western blotting analysis were: AKT, pAKT, pP38, pP65, pJNK, pERK and GAPDH (Cell Signaling Technology, Danvers, MA, U.S.A.). Fluorescence antibodies used in flow cytometry were: CD45, CD31, F4/80, MHCII I-A, MHCI KbDb, Sca1 and CD44 (eBioscience, Thermo Fisher, U.S.A.).

To construct the LEMP-HA, Flag-LEMP plasmids, the corresponding sequence was HA or Flag tagged and inserted into the pHAGE-fEF1a-IRES-ZsGreen vector. LEMPΔATG-HA plasmid was obtained using mutagenesis. To construct LEMP-Flag-px330, a single-guide RNA (sgRNA) specific to the C-terminal coding sequence of mLEMP locus was cloned into the px330. The donor plasmid was made with a Flag tag in-frame with the LEMP coding sequence and flanked by ~500 base pair homology arms specific to the LEMP locus. The zebrafish cDNA of LEMP gene was amplified and cloned into pCS2+ vector. To construct the plasmid used for synthesis of antisense RNA probe, the relevant sequence of LEMP was cloned into pCS2+ vector. To make the construct of LAT-EGFP, the zLEMP-AMO targeting sequence was inserted into the corresponding start codon region of EGFP in pCS2+ vector.
The antibodies against Flag (Sigma, F3165), HA (Sigma, H6908), GAPDH (Abcam, ab8245), MHC (Upstate, 05-715), Mitotracker (Invitrogen, M7510), Gm130 (BD, 610823), Calnexin (Santa Cruz, SC-6465), LEMP (ABclonal, A18310), CD31 (BD, 562861), CD45 (eBioScience, 45-0451-82), CD11B (eBioScience, 45-0112-82), Sca1 (eBioScience, 56-5981-82), CD34 (BD, 553733), and integrin-α-APC (R&D, FAB3518A) were purchased.

BM cell suspensions were prepared from the femur whose cavity was washed and then crushed for cell collection. The LSK (Lin⁻Sca-1⁺c-Kit⁺) cells were prepared by labeling BM lineage⁻ cells suspensions obtained by magnetic bead (STEMCELL) with a mixture of monoclonal antibodies against Sca-1 (1:200, eBioscience), c-Kit (1:200, eBioscience), CD11b (1:400, eBioscience), Gr-1 (1:400, eBioscience), B220 (1:200, eBioscience), CD3 (1:200, eBioscience), TER-119 (1:200, eBioscience), Annexin V (1:50, BD), Propidium Iodide (PI, 1:200, BD). Fluorescence-activated cell analysis was performed on an LSRFortessa cell analyzer (Becton Dickinson). Fluorescence-activated cell sorting was performed on a FACSAriaII Flow Cytometer (Becton Dickinson). Data were analyzed by using FlowJo™ (Tree Star).
CFC was conducted as previously described [31 ]. It was performed in MethoCult GF M3434 (STEMCELL) containing with 600∼700 LSK cells. After incubating the cells for 14 days, the scored for colony formation was conducted.

BM samples were collected from the femurs and tibias of mice and then lysed with erythrocyte lysing buffer. The remaining cells were first stained with Fixable Viability Stain 510 (FVS510; BD Bioscience). For blood cell lineage detection, the cells were stained with lineage antibodies against CD3e, CD11b, Ter-119, B220, Gr-1 and matched isotype controls (eBioscience). To examine the percentages of LSK (Lin^–Sca-1⁺c-Kit⁺) cells, BM cells were stained with biotin-labelled lineage antibodies and then incubated with streptavidin APC-eFluor® 780-labelled secondary antibody (eBioscience), along with BV605-labelled anti-mouse Sca-1 (BD Bioscience) and APC-labelled c-Kit (eBioscience). For the detection of engraftment and chimerism, the BM cells were stained with PE or FITC-labelled antibodies against CD45.1 or CD45.2 (eBioscience). All samples were detected by BD FACS Aria (BD Bioscience).