Notch2

Immunoblot and immunofluorescence assays were performed as previously described [29 (link)]. Antibodies employed to perform these studies were as follows: centrin (20H5 kindly provided by Dr. Salisbury’s laboratory at the Mayo Clinic); ERα and pericentrin (Santa Cruz Biotechnology, Dallas, TX, USA); AURKA (Cell Signaling Technology, Danvers, MA, USA); CD44, CD24, NOTCH1, NOTCH2, and NOTCH3 (Abcam, Cambridge, MA, USA); and β-actin and α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA).

Proteins in tissues and cells were extracted. The loading quantity of the sample was adjusted to 30 µg by deionized water. Proteins were conducted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto membranes, which were blocked with 4% skim milk powder at 4 °C overnight and incubated with primary antibodies NOTCH2 (1:1000) and GAPDH (1:2000, both from Abcam) for 1 h. Afterward, the membranes were incubated with relative secondary antibodies (Abcam) for 1 h, soaked in enhanced chemiluminescent reagent (Pierce Manufacturing Inc., WI, USA) for 1 min, and covered with plastic wrap, then observed through X-ray. GAPDH was taken as the internal reference and the relative protein expression was calculated.

Cells were lysed and the protein concentration was determined using BCA assay (Beyotime Biotechnology, China). The protein were subjected to polyacrylamide gel electrophoresis. Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with blocking buffer [0.1% Tween-20 in 5% skimmed milk (Gibco, USA)] for 1.5 h. The membrane was incubated with a primary antibody [Nanog, Cell Signaling Technology (CST), #3580; β-catenin, CST, #9582; Hsp90B1, CST, #20292; Notch2, abcam, ab#8927] at 4°C overnight. Next, the membrane was washed with 0.1% Tween-20 Tris-buffered saline. Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1.5 h at room temperature. The bound antibodies were detected using a chemiluminescence detection kit ECL (Millipore, USA). β-actin was used as an internal control.

Protein extraction was performed using RIPA lysis buffer (Wuhan Boster, China). Protein samples were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with 5% skimmed milk for 90 min at room temperature. The PVDF membrane was incubated with the primary antibody at 4 °C overnight on a shaker. The membrane was then incubated with secondary antibody at room temperature for 1 h. The membrane was placed into a chemiluminescence machine to visualize the protein bands after washing. ImageJ software was used to analyze band densities. Tubulin was used as a loading control. All antibodies were purchased from Abcam, UK; the antibody dilutions used were as follows: MK 1:1000; Notch2, 1:1000; HES1, 1:500.

Western immunoblotting analysis was carried out for CSCs, CSCs/DDP treated with 80 μM DDP and CSCs/DDP treatment with 80 μM DDP and lumiflavin 80 μM for 72 h, as described previously (24 (link), 25 ). Cellular proteins were extracted with RIPA buffer [150 mM NaCl, 50 mM Tris (pH 8.0), 10% glycerol, 2% Triton X-100, 1 mM ethylene diamine tetra acetic acid disodium, and a protein inhibitor mixture (Beyotime Biotechnology)]. For immunoblotting, solubilized proteins were loaded on a gel and resolved by 8% – 15% SDS-PAGE. The proteins were then transferred to PVDF membranes and incubated at 4°C overnight with primary antibodies Jagged1 (Jag1,Abcam,1:1000), Jagged2 (Jag2, Abcam,1:1000), Delta-like canonical notch ligand 1 (Dll1, Abcam,1:500), Delta-like canonical notch ligand 3 (Dll3, Cell Signaling Technology,1:1000), Delta-like canonical notch ligand 4 (Dll4, Cell Signaling Technology,1:1000), Notch1 (Abcam,1:1000), Notch2 (Abcam,1:5000), Notch3(Santa Cruz, 1:500), Notch4 (Santa Cruz, 1:500) or NICD (Cell Signaling Technology, 1:1000). Membranes were washed and incubated with anti-rabbit/anti-mouse antibodies (MultiSciences Biotech, 1:5000) for 1 h at room temperature. Chemiluminescent images of the blots were finally captured using a ChemiDoc System (Bio-Rad, USA). ImageJ software was used to calculate relative protein expression.

Total protein was separated from the cell lysates with the use of SDS-PAGE, which was further transferred to PVDF membranes (Thermo Fisher Scientific). Subsequently, involved primary antibodies were added onto membranes for incubation overnight at 4 °C. After PBS rinsing for 3 times, secondary antibodies (1/2000) were added for 1-h incubation. The primary antibodies against Oct4 (1/1000), Nanog (1/1000), SOX2 (1/1000), NOTCH2 (1/2000), HES1 (1/1000), HES6 (1/1000), and β-actin (1/5000) were purchased from Abcam. Chemiluminescence system (GE Healthcare, Chicago, USA) was applied for protein detection. The assay was conducted for three times.

Tibias were fixed in 4% paraformaldehyde at 4 °C for 3 days. After fixation, bones were decalcified and dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin. The entire tibia was then sectioned longitudinally at 3 μm per section. To measure BM cell proliferation, sections from the center of the femur were stained with Ki67, Notch2, p63 (Abcam), and Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Histologic staining was performed with hematoxylin and eosin.