Fig. 7.

Effect of keratin depletion on basal and SPC-induced random migration of pancreatic cancer cells. (A) K8 and/or K18 were depleted in Panc-1 cells using specific siRNAs (siK8, siK18). Relative K8 mRNA (upper panel) and K18 mRNA levels (lower panel) were determined in Panc-1 cells by qRT-PCR using the ICyclerIQ system from Bio-Rad. Graphs depict percentage mRNA expression as compared with control. Figures shows representative data obtained in triplets of at least three independent experiments. (B) Top panel shows Panc-1 cells transfected with specific K8 siRNA (siK8) and/or K18 siRNA (siK18). After 72 hours, keratins were extracted and expression analyzed by western blotting using a pan-CK antibody. Bottom panel is densitometric evaluation of keratin protein levels. Data represent the means ± s.e.m. of three independent experiments. (C) Panc-1 cells were transfected with scrambled siRNA (siCon) or siK8. After 48 hours, cells were incubated with 10 μM SPC (+) or solvent (−) and subjected to a random migration that was analyzed by time-lapse video microscopy. Cells were tracked and the velocity was calculated using the ImageJ program. Data represent the fold increase in migration above control and are the means ± s.e.m. of three independent experiments. (D) Panc-1 cells were transfected with scrambled siRNA (siCon) or siK18. 24 hours after knockdown, cells were incubated with solvent or 10 μM SPC and subjected to random migration. Migration was analyzed as described above. Data represent the fold increase in migration above control and are the means ± s.e.m. of three independent experiments. (E) Panc-1 cells were incubated with 10 μM U0126, 10 μM SPC and 5 ng/ml TGF-β as indicated. Random migration was determined by time-lapse video microscopy as described above. Data represent the fold increase in migration above control and are the means ± s.e.m. of four independent experiments. (F) Panc-1 cells were incubated with 10 μM U0126, 10 μM SPC and 5 ng/ml TGF-β as indicated. TGF-β was used as a positive control (Rahimi and Leof, 2007). Migration through size-limited pores was determined using a Boyden chamber assay as described previously (Beil et al., 2003). Data represent the fold increase in migration above control and are the means ± s.e.m. of three independent experiments. (G) Panc-1 cells were transfected with K8(WT),K18(WT), K8(SE),K18(WT) or K8(SA),K18(WT) and subsequently incubated with 10 μM SPC (+). Random migration was determined as above. Data represent the fold increase in migration above control and are the means ± s.e.m. of four independent experiments. In all experiments described in 7C-G significant differences were tested using the Student's t-test. *P<0.05; **P<0.01; ***P<0.001.