Figure 6. NCAN is essential for the malignancy of tumor spheres derived from TH-MYCN mice.

(A) RT-qPCR revealed the efficient knockdown of NCAN mRNA by two independent shRNAs in the tumor sphere line #6673 derived from TH-MYCN mice. ^*p < 0.05, ^**p < 0.01. (B) Western blot for mouse endogenous NCAN showing the efficient knockdown by shRNAs in the tumor sphere line #6673 derived from TH-MYCN mice. (C) Schematic of NCAN knockdown in mouse tumor sphere cells. (D) Representative images of tumor spheres after the knockdown of NCAN. Day 4: Four days after infection with shRNA-expressing lentivirus. Day 8: Just before the quantification of spheres. Scale bars: 100 μm. (E) Quantification of the sphere numbers and sizes shown in panel C. ^**p < 0.01. (F) RT-qPCR analysis of stemness marker genes in each tumor sphere. ^**p < 0.01. (G) Here, 1 × 10⁵ tumor sphere cells expressing each shRNA were subcutaneously inoculated into KSN/Slc nude mice. The tumor forming ratio is indicated (n = 6 for shRNA-NT, n = 8 for shRNA#59 and shRNA#60).