Figure 3: Loss of AF6 promotes tumour growth and metastasis via Snail.

(a) Real-time PCR analysis of the mRNA expression of the indicated genes in Bxpc-3 or Bxpc-3 shAF6 cells. Data represent the mean±s.d. (n=3) from three separate experiments. *P<0.05, **P<0.01, Student’s t-test. (b) Western blot analysis of AF6 and Snail expression in Bxpc-3, HPAC, Capan-1, Capan-2 and Miapaca-2 cells; the protein levels of AF6 and Snail were inversely correlated. (c) Western blot analysis of AF6 and Snail expression in Bxpc-3 and HPAC cells stably transfected with control (CT) or either of two AF6 shRNAs, and in Capan-1 and Capan-2 cells stably transfected with control (CT) or AF6 (AF6)-encoding vectors. (d) Phase-contrast images of Bxpc-3 control (CT), shAF6 and shAF6 shSnail cells grown as 2D, 3D or 3D invasion cultures (Day 10), respectively. Black arrows indicate invasive structures. Scale bars, 100 μm (2D); 50 μm (3D and 3D invasion). (e) Western blot analysis of AF6 and Snail protein levels in cells used in d. (f) The sizes of the spheroids were measured. Data represent the mean±s.d. (n=30) from three independent experiments. ***P<0.001, Student’s t-test. (g,h) Four-week-old nude mice were orthotopically transplanted with 1 × 10⁶ cells per animal (n=7); tumours were harvested, photographed and weighed at autopsy (Day 60). Results are presented as mean±s.d. (n=7) of calculated tumour weight (g). *P<0.05, **P<0.01, Student’s t-test. (h) Liver tissues were photographed, fixed and stained with haematoxylin and eosin (H&E); black arrows indicate the liver metastatic lesions. The number of metastatic lesions in each specimen was counted. Results are presented as mean±s.d. (n=6). **P<0.01, Student’s t-test. Scale bar: 100 μm.