Myc d84c12

After the mice were sacrificed, the hearts and tumors were collected and fixed in 4% paraformaldehyde for 24 h and subsequently embedded in paraffin. Tissue sections (5 μm) were stained with H&E. IHC staining on tumor tissues harvested from treated mice was performed using an automated immunostainer (Leica, Bond-III, Leica, Buffalo Grove, IL) with the primary polyclonal antibodies against Ki67 and mouse CD31 (Abcam, Cambridge, MA). The stained tissue sections were analyzed using the Aperio ScanScope Image Analysis System (Aperio, Vista, CA). TCF7 (C63D9) and MYC (D84C12) antibodies were obtained from Cell Signaling Technology. Ki67 antibodies were obtained from Dako (MIB-1; Carpinteria, CA, USA).

Retinal culture, transduction, and immunostaining were as described (21 (link)). Immunostaining antibodies were: Ki67: 550609 (BD), 1:200; MDM2: SC-965 (Santa Cruz), 1:150; MYCN: SC56729 (Santa Cruz) 1:100; MYC: D84C12 (Cell Signaling Technology) 1:800; RXRG: SC-555 (Santa Cruz) 1:1000, SC-514134 (Santa Cruz) 1:200. MDM2 expression was quantitated using Trainable Weka Segmentation plugin available in Fiji (27 (link)) (https://imagej.net/Trainable_Weka_Segmentation) trained to recognize and segment nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). Trained data was applied to confocal images of fluorescent antibody stained sections to obtain probability maps followed by watershed and segmentation of DAPI+ nuclei. Mean gray values of fluorochromes were measured from single cells using Fiji. Sample sizes were chosen based on availability. All segmented nuclei were included. Cell culture groups were compared using two‐tailed, unpaired, non-parametric Mann-Whitney U test. Transduced retinae groups were compared using one‐way analysis of variance (ANOVA) Kruskal-Wallis test followed by Dunn’s multiple comparisons test. GFP^Lo and GFP^Hi cells were distinguished based on mean GFP pixel density relative to 7% of the maximum in MYCN- and MYCC-transduced tissues and cell lines.

Protein extracts were obtained and subjected to western blot analysis as described previously (17 (link)). For MYC and β-actin immunodetection, the following specific primary antibodies were used: MYC (D84C12), β-actin (13E5) (both Cell Signaling Technology, Danvers, Massachusetts, USA). Secondary antibody: anti-rabbit (7074; Cell Signaling Technology). Chemiluminescence was detected with Clarity™ Western ECL Blotting Substrate (Bio-Rad, Hercules, USA). Signals were quantified with ImageJ Software (www.imagej.net) and referred to the respective controls, i.e., β-actin levels in individual samples. Protein extracts from cell lines NALM6 and MEC-1 were used as positive controls.