Jetpei macrophage

The following media were used: Minimal Essential Medium Eagle (αMEM, Sigma), Dulbecco's modified Eagle medium (DMEM, Sigma), foetal calf serum (HyClone Laboratories) and L-Glutamine (Invitrogen). Complete media were supplemented with 10% foetal calf serum (FCS), 2 mM L-Glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.
The following antibodies were used: rabbit anti-PML (sc-5621; 1:500; SantaCruz Biotechnology), rabbit anti-actin IgG (AA20-33; 1:1000; Sigma). The cytokines murine M-CSF (Prospec Bio), human recombinant RANK-L (R&D Systems) and IFN-γ (Thermo Fisher Scientific) were used.
Other reagents, kits and cell lines used were: penicillin (Invitrogen), streptomycin (Invitrogen), geneticin (G418; Thermo Fisher Scientific), cell dissociation buffer (Thermo Fisher Scientific), jetPEI-Macrophage (Polyplus transfection), Alamar Blue reagent (Invitrogen), GenElute Mammalian Total RNA kit (Sigma), RNeasy kit and DNeasy blood and tissue kit (Qiagen), qScript cDNA SuperMix kit (QuantaBioscience), SensiFAST Probe No-ROX kit (Bioline), Ambion Ribopure Blood RNA isolation kit (Thermo Fisher Scientific), Osteo Assay plates (Corning) and RAW 264.7 (ATCC). All cultures were performed in standard conditions of 5% CO₂ and 37°C in a humidified atmosphere.

For transient transfection, cells were transfected with ASO or siRNA by means of Lipofectamine-RNAi MAX (Invitrogen, catalog 13778150) or transfected with plasmid by means of Lipofectamine 2000 (Invitrogen, catalog 11668019) according to the manufacturers’ instruction. The sequences of ASO and siRNA are described in Supplemental Table 1. For stable transfection, RAW264.7 cells were transfected with pGV-mascRNA or empty vector (pGV-NC) by means of jetPEI-Macrophage (Polyplus, catalog 103-05N) according to the manufacturers’ instruction. Twenty-four hours after transfection, cells were screened by G-418 (400 ng/mL; Beyotime Biotechnology; catalog ST081) for 10 days. The stably transfected cell pools were then split and plated for subsequent experiments.

The Dual-Luciferase^® Reporter Assay System (Promega, Madison, United States) was used to measure reporter luciferase activities using a GloMax^® 20/20 tube luminometer (Promega, United States). For NF-κB/AP-1 activity detection, RAW264.7 cells were co-transfected with pGL3-NF-κB-luc/pGL3-AP-1-luc, a Renilla luciferase reporter plasmid (pRL-TK, Promega) and pcDNA3.1-Myc-PPE68 plasmid using jetPEI-Macrophage (Polyplus). Then, 24 h post-transfection, cells were treated with LPS (1000 ng/mL, Sigma-Aldrich) to stimulate NF-κB or AP-1 activation.
HEK293T cells were co-transfected with pGL3-NF-κB-luc/pGL3-AP-1-luc, a Renilla luciferase reporter plasmid, pcDNA3.1-Myc-PPE68/pcDNA3.1, and pcDNA3.1-Flag-MyD88/TRAF6/TAK1/TAB1-3, respectively, for 24 h.
Luciferase activities were measured using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega). All data were normalized for transfection efficiency based on Renilla luciferase activity.

RAW264.7 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS. After reaching 70%-90% confluence, transient transfection was performed with jetPEI-Macrophage (Polyplus) following the manufacturer’s instructions. For siRNA transfection, the silencing RNA (siRNA) of E3 ubiquitin ligase FBW7 and control siRNA were synthesized from TSINGKE company (Wuhan, China). RAW264.7 cells were transfected with 100 nM siRNA. Transfection efficiency was measured by Western blot after 48 h post transfection. The sequences of siRNAs were listed in Supplementary Table 2.