Pt202 y204 erk1 2

X-370 were synthesized with a purity >99% by Xcovery (West Palm Beach, FL, USA) and AZ628, PLX4032, AZD6244, BX-912, MK-2206, Rapamycin, AZD8055 (Selleck, Houston, TX), BI-D1870 (Enzo Life Sciences, Farmingdale, NY) were dissolved in DMSO at 10 mM and stored at −20°C. Antibodies against Akt, Akt-pS473, Akt-pT308, S6k1, S6k1-pT389, Erk1/2, Erk1/2-pT202/Y204, p110δ and MEK1/2-pS217/S221 were from Cell Signaling (Cell Signaling, Danvers, MA). Antibodies against Gab1, CD19, MEK1 and PDK1 were from Eptomics (Hangzhou, China). Antibodies against β-Actin and GAPDH were from Sigma-Aldrich (Sigma-Aldrich, MO) and KangChen Bio-tech (Shanghai, China) respectively. IGF-1, MCP-1 and M-CSF were from R&D systems (Minneapolis, MN) and LPA was from Sigma-Aldrich (St. Louis, MO). Raji and SU-DHL-6 cells were obtained from ATCC (Manassas, VA). Raji-R cells were kindly provided by Yajun Guo (Shanghai Jiao Tong University, Shanghai, China) and MEF cells were generous gift from Jean Zhao (Dana-Farber Cancer Institute, Boston, MA). Rh30 cells were obtained from Dr. P.J. Houghton (St. Jude Children's Research Hospital, Memphis, TN, USA).

Intestinal tissue was harvested, flushed with PBS solution and fixed as “Swiss-roll” sections in PFA for 24 h at 4 °C. For tumour scoring, intestines were fixed in Methacarn (60% methanol, 30% chloroform and 10% glacial acetic acid). Tissue was automatedly processed through the Tissue-TeK VIP infiltration Processor (Sakura) for paraffin embedding and cut into 5 µm sections with the microtome (Leica). Standard IHC techniques were conducted during this study. Antibodies used were as follows: BrdU, 1:500 (Bioss, bs-0489H), β-catenin, 1:50 (BD Biosciences, 610154), cleaved Caspase 3, 1:800 (R&D), Lyz1 (DAKO, A009), Muc2 (Genetex, GTX100664), EGFR^pY1068, 1:25 (Cell Signalling, 3777S), EGFR^pY1068, 1:400 (Abcam, ab40815) and ERK1/^2pT202/Y204, 1:100 (Cell Signalling, 4370S). At least 3 different mice of each genotype were used as biological replicates in every IHC experiment. Scoring of the staining was done blinded for evaluation and representative images were selected. Images were digitalised using the Nanozoomer Digital slide scanner (Hamamatsu) and analysed with the viewer software NDP.view2 (Hamamatsu). Scoring of tumour sections was automatedly performed using the QuPath software (qupath.github.io), whilst proliferation scoring of normal intestine was carried out manually.

Lungs were collected at a treatment endpoint predefined as 27 weeks of age. For tissue preparation, lungs were fixed in 10% phosphate-buffered formaldehyde for 24–48 hrs, dehydrated by incubation in ethanol followed by xylene (70% ethanol, 3 hr; 95% ethanol, 2 hr; 100% ethanol, 2 hr; ethanol-xylene, 1hr; xylene, 3hr) then embedded in paraffin. Paraffin blocks were cut into 5 μm sections, mounted on microscope slides, and stored at room temperature until used. Prepared specimens were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO) and analyzed by immunohistochemistry (IHC) or immunofluorescence using standard protocols. Antibodies used in IHC were to Ki-67 (DAKO, Carpinteria, CA), cleaved caspase 3 (#9664L, Cell Signaling, Beverly, MA), pS⁸²⁴-KAP1 (ab70369, Abcam, Cambridge, MA), pT²⁰²/Y²⁰⁴-ERK1/2 (#4996S Cell Signaling, Beverly, MA) and HSP90 (NB120-2928, Novus Biologicals, Littleton, CO). For immunofluorescence, we used antibody to vimentin (ab92547, Abcam, Cambridge, MA) as primary reagent and secondary Alexafluor 568 tagged donkey anti-rabbit antibody (Life Technologies, Eugene, OR), and counterstained with a 2 μmol/L 4′, 6-diamidino-2-phenylindole (DAPI) (#1652731, Life Technologies, Eugene, OR) solution to visualize DNA.