Morton et al. 10.1073/pnas.0701158104. |
Fig. 6. Examples of histology of pancreatic lesions observed in immunocompromised mice orthotopically transplanted with RCAS-Shh-infected PDECs. (A) Normal pancreatic duct. (B and C) Ductal lesions induced in immunocompromised mice orthotopically transplanted with RCAS-Shh-infected PDECs. Note papillary growth and changes in epithelium to columnar phenotype with expanded cytoplasm (D), mitosis (E), and loss of polarity (F). Detection of proliferation associated marker Ki67 in (G) a normal pancreatic duct and (H) an RCAS-Shh-induced ductal lesion. (I) Pdx1 expression in an RCAS-Shh-induced ductal lesion.
Fig. 7. Characterization of pancreatic tumors and cell lines induced by K-Ras and Shh. (A) Immunoblot analysis of Shh expression (by myc epitope tag) and Ras expression (by Flag epitope tag) in tumors induced by orthotopic transplant of RCAS-K-Ras-infected PDECs (R1-R6) or RCAS-Shh- and RCAS-K-Ras-infected PDECs (RS1-RS7). Tumors induced by transplant of Ink4a/Arf null PDECs are designated R1-R3 and RS1-RS3. Tumors induced by transplant of Trp-53, Ink4a/Arf null PDECs are designated R4-R6 and RS4-RS7. (B) Immunocytochemical detection of Keratin 8, Shh, K-Ras, and Pdx1 in tumor cell lines established from tumors induced by orthotopic transplant of RCAS-K-Ras-infected PDECs or RCAS-Shh- and RCAS-K-Rasinfected PDECs.
Table 2. Antibodies for immunoblotting
Primary antibody | Dilution | Manufacturer |
Mouse anti-myc (9E10) | 1:1,000 | Developmental Studies Hybridoma Bank, University of Iowa |
Rabbit anti-Cyclin D1 | 1:200 | Santa Cruz |
Rabbit anti-Cyclin A | 1:200 | Santa Cruz |
Rabbit anti-Cyclin E | 1:200 | Santa Cruz |
Mouse anti-p21 | 1:1,000 | Santa Cruz |
Rabbit anti-phospho Akt (Ser473) | 1:1,000 | Cell Signaling |
Rabbit anti-Akt | 1:1,000 | Cell Signaling |
Rabbit anti-phospho ERK1/2 | 1:1,000 | Cell Signaling |
Rabbit anti-ERK1/2 | 1:1,000 | Cell Signaling |
Rabbit anti-caspase 3 | 1:1,000 | Cell Signaling |
Rabbit anti-cleaved caspase 3 | 1:1,000 | Cell Signaling |
Hamster anti-Bcl2 | 1:200 | BD Pharmingen |
Rabbit anti-Bcl-xL | 1:1,000 | BD Pharmingen |
Rabbit anti-Bad (Y208) | 1:1,000 | Abcam |
Mouse anti-Flag | 1:1,000 | Sigma |
Anti-phospho mTOR | 1:1,000 | Cell Signaling |
Anti-mTOR | 1:1,000 | Cell Signaling |
Rabbit anti-b-actin | 1:5,000 | Santa Cruz |
Table 3. Antibodies for immunostaining
Primary antibody | Dilution | Manufacturer |
Rabbit anti-Ki67 | 1:1,000 | Novocastro |
Rabbit anti-mouse Pdx1 | 1:5,000 | Gift of Chris Wright |
Rat anti-keratin 8 (Troma1) | 1:50 | Developmental Studies Hybridoma Bank, University of Iowa |
Mouse anti-Myc (9E10) | 1:1,000 | Developmental Studies Hybridoma Bank, University of Iowa |
Mouse anti-Flag | 1:2,500 (2 h at 37°C) | Sigma |
Table 4. RT-PCR primers
Transcript | Primers | Conditions |
Gli1 | F: 5'-TGGGATGAAGAAGCAGTTGGG-3' | Annealing temp: 58°C |
R: 5'-TTGATGAAAGCCACCAGGGAG-3' | 35 cycles | |
Gli2 | F: 5'-TCAGCCCAGCCTTCACTTTTCC-3' | Annealing temp: 55°C |
R: 5'-TCTTGACCTTGCTCCGCTTATG-3' | 30 cycles | |
Smo | F: 5'-TGCGAACAGACAACCCCAAG-3' | Annealing temp: 60°C |
R: 5'-CAATGCTGCCCACGAAGAAAC-3' | 35 cycles | |
Ptch | F: 5'-ctcgcttacaaactcctggtgc-3' | Annealing temp: 58°C |
R: 5'-gccgttgaggtagaaagggaac-3' | 35 cycles | |
Cyclin D1 | F: 5'-GGATGTCCACACACGCATTCAG-3' | Annealing temp: 58°C |
R: 5'-TGTCCCCAATCTCCTTGTCCAG-3' | 35 cycles | |
Cyclin A | F: 5'-CATCTGTCCTGGATTGGGTCAC-3' | Annealing temp: 58°C |
R: 5'-TTCGGAAAGAGTGTCAGCCTCC-3' | 27 cycles | |
Cyclin E | F: 5'-CTTATGGTGTCCTCGCTGCTTC-3' | Annealing temp: 58°C |
R: 5'-TCGCTGCTCTGCCTTCTTACTG-3' | 30 cycles | |
p21 | F: 5'-TCCTTGCCACTTCTTACCTGG-3' | Annealing temp: 55°C |
R: 5'-cggttgagtcctaactgccatc-3' | 35 cycles | |
p27 | F: 5'-GGGAAGGCTGGAATCACTTGAG-3' | Annealing temp: 58°C |
R: 5'-AAAACGAACCTCTGGGAAATGG-3' | 30 cycles | |
Bcl-2 | F: 5'-GGCTTCACACCCAAATCTCACC-3' | Annealing temp: 58°C |
R: 5'-ACTTCCTAAACCCTGCTTCCCC-3' | 25 cycles | |
Bcl-xL | F: 5'-ACTGTGCGTGGAAAGCGTAGAC-3' | Annealing temp: 58°C |
R: 5-TGAAGAGAGAGTTGTGGTGGGG-3' | 30 cycles | |
b -actin | F: 5'-GGCTGTATTCCCCTCCATCG-3' | Annealing temp: 58°C |
R: 5'-AGATGGGCACAGTGTGGGTG-3' | 25 cycles |
SI Methods
Construction of RCAS-Kras Vector
A 3X-FLAG epitope was added to the amino-terminal end of a cDNA-encoding K-RasG12D by PCR and the resulting PCR product cloned into the PCR 2.1 vector. A sequence-verified clone was digested with EcoRI and cloned into the EcoRI site of the pIRES2-EGFP vector (Invitrogen, Carlsbad, CA). A DNA fragment encompassing Kras and EGFP was excised with BglII and Not1 and cloned into BamHI- and NotI-digested pENTR1A (Invitrogen). The insert was then transferred into the vector RCAS-DV (1) by Gateway recombination cloning.
1. Loftus SK, Larson DM, Watkins-Chow D, Church DM, Pavan WJ (2001) DNA Res 8:221-226.