Figure 4
iASPP supported cell proliferation and tumor growth. (a) The morphology of cells presented at spheroid formation after 14 days. Scale bars: 750 μm. (b) The number of spheriods larger than 50 μm was counted under a dissecting microscope. The data represented the mean spheroid formation rate±S.E.M. of cells from three independent experiments. (c–e) Tumors formed from H1975 cells in xenografts of NOD/SCID mice were measured, separated and weighed up. (f) The protein expression levels of iASPP and conversion of LC3-I to LC3-II in mixture of six tumors for each group were determined by western blotting. GAPDH was used as a loading control. (g) Immunohistochemical staining of PCNA protein for measurement of proliferation and immunofluorescence staining for measurement of apoptosis by TUNEL assay in tumor xenografts. (h) Quantification of PCNA-positive or TUNEL-positive cells as the mean±S.E.M. percentage of positive cells per field from six random microscopic fields. (i–k) Tumors formed from A549 cells in xenografts of NOD/SCID mice were measured, separated and weighed up. Tumor volume was calculated using the formula: V=length × width2/2. The curve in c and i represented the tumor volumes±S.E.M. of six mice for each group on different days. The data in (e and k) represented the mean tumor weight±S.E.M. for each group. (l–o) The expression of pluripotency markers, including OCT4, NANOG and SOX2 were performed by qPCR and western blotting. Western blot quantification estimated as relative ratio of each protein to GAPDH were shown under individual blots
