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. Author manuscript; available in PMC: 2016 Aug 8.
Published in final edited form as: Nat Cell Biol. 2014 Dec 1;17(1):20–30. doi: 10.1038/ncb3072

Fig. 6. Ambra1 affects tumorigenesis.

Fig. 6

a) MEFs Ambra1+/+ and Ambra1gt/gt have been immortalized through RasV12/E1A oncogene infection. Subsequently, cells were subcutaneously injected into athymic mice and the insurgence and the size of masses were analysed. Data are presented as means±s.d. and significance is *P<0.05, **P<0.005 (n=3 independent experiments). b) Protein extracts have been obtained from +/+ and gt/gt derived tumours (in a). The extracts have been analysed by western blot, with anti-P c-MycS62, total c-Myc and Tubulin. c) Ki-67 staining on tumours derived from +/+ and gt/gt MEFs (shown in a). Scale bar, 50μm. d) Analysis of the frequence of spontaneous tumours in Ambra1+/gt mice between 12 and 17 months of age. e) H&E and TTF-1 (scale bars, 100μm and 50μm, respectively) immunofluorescence of close sections from the same lung mouse (Ambra1+/+ or Ambra1+/gt) are shown. PLA: Papillary Lung Adenocarcinoma; NT: Normal Tissue. f) Trichromic staining on Ambra1+/gt lung tumours. Scale bars: 2mm, 50μm, 100μm. g) Ki-67 staining on lung of Ambra1+/gt mice. H&E and IHC, second row: tumour tissue (scale bars, 100μm and 50μm). IHC, first row: healthy tissue (Scale bars, 50μm and 5μm). h) Western blot analysis of AMBRA1 and P C-MYCS62 in human adenocarcinoma cell lines. On the right panel, A549 cells have been reconstituted for AMBRA1. The levels of P C-MYCS62 have been analysed by western blot. Bronc. E.: Bronchiolar epithelium. The vertical line represents a splice mark of samples from the same gel. i) Graph showing the correlation between AMBRA1 and P C-MYCS62 protein levels in lung cell lines. The densitometric analysis of the western blots reported in (h) is reported in the graph. Data are presented as means±s.d. and significance is *P<0.05; **P<0.005 (n=4 independent experiments). j) A549 cells, overexpressing AMBRA1 or βGal as control, by retroviral infection, were analysed by MTS assay at different time points. Data are presented as means±s.d. and significance is **P<0.005 (n=3 independent experiments). k) The tumorigenicity of the cells used in j was assessed by a colony formation assay in soft agar. Data are presented as means±s.d. and significance is **P<0.005 (n=4 independent experiments).