Fig. 3: RNF121 is necessary for neuroblastoma development in TH-MYCN mice.

mRNA expression level of a RNF121^WT, and, b a MYC gene signature²³ in ganglia tissue and tumours from wild-type (WT) and TH-MYCN^+/+ mice, obtained at different postnatal ages (1, 2, and 6 weeks of age; n  =  4 per group). Gene expression values are represented as log2-transformed microarray probe intensities of RNA levels. c The Pearson correlation coefficient (r) and p value for RNF121^WT and the MYC signature expression scores for the tissue samples in (a) and (b). d Schematic of targeted RNF121 conditional allele: Two sgRNAs (yellow) were complexed with Cas9 nuclease to generate double stranded breaks in the RNF121 locus. A targeting vector featuring exon 3 (blue) of the RNF121 gene flanked by loxP sites was used as a template for homologous recombination. The resulting RNF121 conditional mice was crossed to Cre deleter mice to delete exon 3, leading to the generation of RNF121 knockout mice. e PCR products of mouse tail genomic DNA using primers which distinguished RNF121^WT (540 bp in lanes 2–5) and the successfully floxed RNF121^+/− allele (269 bp in lanes 6–7). Positive control for RNF121 hemizygous mutant allele (+ ve): DNA from RNF121 targeted embryonic stem cell clone. f Immunohistochemical staining for RNF121^WT protein in coeliac ganglia from 20-week-old RNF121^WT or RNF121^+/− mice using an anti-mouse RNF121 polyclonal antibody and a control IgG antibody. g Tumour-free survival assessed using Kaplan–Meier analyses of TH-MYCN^+/+ mice back-crossed with either RNF121^WT (n = 25, black line) or floxed RNF121^+/− knockout (n = 21, red line) mice (**p < 0.01). h A graphical representation showing RNF121 protein expression levels in tumour tissues from TH-MYCN^+/+ mice crossed with either RNF121^WT or floxed RNF121^+/− knockout mice, across 5 independent western Blots (each with 3 different tumour lysates per group).