Fig 5. YB-1-silenced SH-SY5Y cells exhibited reduced tumorigenicity and delayed tumor formation in vivo.

(A) SH-SY5Y, SH-shCON and SH-shYB-1 cells were seeded sparsely on culture dishes and allowed to form colonies for 2 weeks. Colonies consisting of more than 50 cells were counted and colony forming ratio was calculated. (B) 10⁶ SH-SY5Y, SH-shYB-1 or SH-shCON cells were inoculated subcutaneously into an 8-week-old BALB/c nude mouse (n = 6 each group), and tumor volumes were measured externally with a caliper every week and calculated as previously described. (C, D) By end of week 8, the mice were sacrificed, and the tumors were excised, photographed and weighed. (E) Tumor tissues were fixed, paraffin embedded, sectioned and stained with hematoxylin and eosin (H&E) for histological examination. (F) Tumor sections were permeabilized, enzyme inactivated, and incubated sequentially with TUNEL reaction solution, Converter-POD and DAB, followed by counterstaining with hematoxylin. Fragmented DNA was labeled and turned brownish under an optical microscope. (G) Tumor tissues were lysed and total proteins were extracted. Expression levels of YB-1 and Cyclin-D1 in the tumor cells were examined by Western blot analysis. E-G show representative images from all the experimental mice. Values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, *P<0.05; ***P<0.001.