Anti ki67

The tumor formed in the mouse model was used for the IHC staining study. The staining assay was performed according to the standard protocol on five-micrometer formalin-fixed paraffin sections. After staining, a pathologist and an investigator blind to the study design scored the staining intensities according to an immunoreactive score (IRS) system [14 (link)]. The primary antibodies for IHC are anti-Nrf2 (ABclonal, 1 : 200), anti-Ki-67 (Santa Cruz, 1 : 200), anti-Notch1 (Cell Signaling, 1 : 100), anti-c-Myc (Santa Cruz, 1: 100), and anti-Slug (Santa Cruz, 1 : 100).

Cells were washed and then lysed in ice with Complete Tablet buffer (Roche) supplemented with 2 mmol/L sodium orthovanadate, and 1:1000 phenylmethanesulfonylfluoride (PMSF; Sigma‐Aldrich). A quantity of 35 μg of protein from each lysate was loaded on 15 or 5% SDS‐PAGE gels and transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy). They were incubated overnight at 4°C with specific primary antibody: anti‐ferritin (1:250, Santa‐Cruz), anti‐VDR (D‐6) (1:200, Santa‐Cruz), anti‐p53 (1:500, Santa‐Cruz), anti‐cMyc (1:200, Millipore S.p.A., Milan, Italy), anti‐Ki67 (1:500, Santa‐Cruz), anti‐Phospho‐p44/42 MAPK (Thr202/Tyr204, 1:1000 Cell‐Signaling). Protein expression was normalized and verified through ß‐actin detection (1:5000; Sigma, Milan, Italy).

Immunofluorescence was performed as described in a previous study [67 (link)]. Osteosarcoma cells were seeded at an initial confluence of 30% and were treated with the NR modulators at 10 μM for 2 days. The expression of the proliferation marker KI-67 in the osteosarcoma cells was measured by immunofluorescence staining. The area of positive fluorescence in 5 randomly selected fields in each group was calculated by using ImageJ 1.51j8 (NIH, USA). The relative KI-67-positive area was normalized to the costained DAPI-positive area. Osteosarcoma cells were seeded at an initial confluence of 15% and were treated with the NR modulators at 10 μM for 5 days. The expression of the apoptotic marker Cleaved-Caspase 3 in the osteosarcoma cells was measured by immunofluorescence staining. The antibodies that were used were anti-KI-67 (1:500, Santa Cruz Biotechnology, #sc-23900, USA) and anti-Cleaved-Caspase 3 (1:500, Cell Signaling Technology, #9661, USA) antibodies.
Osteosarcoma cells were treated with the NR modulators for 2 days and harvested for cell cycle analysis. In brief, the cells were fixed in precooled 70% ethanol for 30 min at 4 °C and stained in the dark for 30 min with a solution containing 50 μg/ml propidium iodide (PI). Cells in the different phases of the cell cycle were then identified by using a FACScan flow cytometer (BD Biosciences, USA).

The immunohistochemical staining procedure was performed as previously described [12 (link)]. The antibodies used were as follows: anti-PRDM4 (1:200 dilution, #ab126939, Abcam, Cambridge, MA, USA), anti-Ki67 (1:400 dilution, #sc-23900, Santa Cruz, Biotechnology Inc., Santa Cruz, California, USA), anti-E-cadherin (1:150 dilution, #sc-8426, Santa Cruz, CA, USA), anti-vimentin (1:200 dilution, #sc-6260, Santa Cruz, CA, USA), and anti-β-catenin (1:150 dilution, # sc-7963, Santa Cruz, CA, USA).