Hochst33342

Cy3-labeled FISH probes for 887L, β-ACTIN (ACTB), and Cy5 labeled FISH probes for 887S, β-ACTIN (ACTB) RNAs were designed and synthesized by GenePharma (Shanghai, China). The FISH assay was conducted according to the instruction of Fluorescent In Situ Hybridization Kit (GenePharma, Shanghai, China). Briefly, cells growing on glass coverslips were fixed with ice-cold 4% paraformaldehyde for 15 min and blocked with pre-hybridization buffer for 30min. The desired RNA probes (4μM) were incubated with hybridization solution for 5min at 73°C before hybridization. The cells were then incubated with the RNA probes (4μM) in the dark at 37°C for 12~16h, followed by three times of wash with 4×SSC containing 0.1% Tween-20. Finally, the cell nuclei were stained with Hochst33342 (Thermo, America). Images were taken by confocal fluorescence microscope (FV1200MPE-share, Olympus, Japan).

Doxorubicin was purchased from Merck (Doxorubicin, Hydrochloride - CAS 25316-40-9-Calbiochem, Merck, Germany). Hydroxyapatite powder was purchased from FLUIDINOVA, Portugal (micro hydroxyapatite powder, 10 ​μm and nano hydroxyapatite paste, <50 ​nm particle size). RPMI 1640 Medium and GlutaMAX™ Supplement were purchased from Thermo scientific, U.S.A. Heat inactivated fetal bovine serum (FBS) and MTT reagent was purchased from Sigma Aldrich, Germany. All the chemicals used in immunohistochemistry staining were purchased from Abcam: Citrate Buffer pH 6.0 (ab93678), Hydrogen Peroxide Blocking Reagent (ab64216), Protein Block (ab64226), Recombinant Anti-Ki67 antibody (ab16667), Antibody Diluent (ab64211), Goat Anti-Rabbit IgG H&L (HRP) (ab205718), DAB Substrate Kit (ab64238) and Hematoxylin Solution (Mayer's, Modified) (ab220365). TUNEL Assay Kit (ab206386) was also purchased from Abcam. ATPlite Luminescence Assay (6016943) was bought from PerkinElmer. Endocytosis inhibitors (Amiloride, LY2940002, MβCD, Dynasore) were purchased from Thermo Fisher. Hochst 33342, GFP-LAMP1, Mitotracker and Tom20 were all purchased from Thermo Fisher. 143B human osteosarcoma cells were purchased from American Type Culture Collection (ATCC). Athymic nude mice (Fox1^nu/nu) were procured from Janvier Labs (France).