Heat inactivated fbs

Osteoblasts (MG-63 cell line; American Type Culture Collection, Rockville, MA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS (Equitech-Bio Inc., TX, USA), 2 mM glutamine and 100 units/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO₂ in air.

Murine macrophage RAW264.7 cells were obtained from RIKEN. RAW264.7 cells were cultured in DMEM (Sigma) supplemented with 10% heat-inactivated FBS (Equitech-Bio Inc., Kerrville, TX, USA). The translation of GADD34 mRNA in RAW264.7 cells was knocked down (shGADD34) as previously described.^{22 (link)} Non-target control shRNA (Sigma) was used as a control (shControl). Human monocytic THP-1 cells were obtained from RIKEN. THP-1 cells were cultured in RPMI-1640 (Sigma) supplemented with 10% heat-inactivated FBS (Hyclone, Logan, UT, USA). THP-1 was transfected with 10 nM siRNA using Lipofectamine RNAiMax (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. siRNAs were obtained from Ambion (Waltham, MA, USA). The sequence of siRNA used to knockdown GADD34 is 5′-GGAUCAGCCCGAGGAUGAAA-3′. Non-targeted control siRNA was used as a control. Recombinant experiments were approved by the Committee of Nagoya University Graduate School of Medicine.