Lc3a b 2

An RNeasy Plus kit (Qiagen, Germantown, MD, USA), a high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA), and a Power SYBR Green PCR master mix kit (Applied Biosystems) were employed with listed PCR primers (Table 1). For detecting GFP-labeled 4T1.2 cells in the right femur, total DNA was isolated with QIAamp DNA mini kit (Qiagen) for qPCR.
For Western blotting, cells were lysed by a radio-immunoprecipitation assay buffer (Santa Cruz). Proteins were fractionated by 10-15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Antibodies against ATF4, caspase 3, cathepsin K, eIF2α, p-eIF2α (Ser51), LC3A/B II, NFATc1, Chk1, p-Chk1 (Ser296) (Cell Signaling, Danvers, MA, USA), TRAP (Abcam, Cambridge, MA, USA), and β-actin (Sigma) were utilized.

Cells were lysed in a radio-immunoprecipitation assay (RIPA) buffer with protease inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phosphatase inhibitors (Calbiochem, Billerica, MA, USA). Isolated proteins were fractionated using 10–15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membrane was incubated for 1 h with primary antibodies followed by 45 min incubation with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling, Danvers, MA, USA). We used antibodies against ATF4, caspase 3, cathepsin K, DRD1, eIF2α, p-eIF2α, lamin B, LC3A/B II, NFATc1 (Cell Signaling), and β-actin (Sigma). Protein levels were assayed using a SuperSignal west femto maximum sensitivity substrate (Thermo Scientific, Waltham, MA, USA), and signal intensities were quantified with a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan).