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Itemised List of Supplemental Material 1. Supplemental Materials And Itemised list of Supplemental Material 1. Supplemental Materials and Methods 2. Supplemental References 3. Supplemental Table S1. Summary of pathological abnormalities observed with variable penetrance in L597VBraf-expressing mice. 4. Supplemental Figures S1-7. Fig. S1: Multiplex PCR genotyping of Braf+/LSL-L597V mouse ear samples Fig. S2: H&E-stained sections of benign growths Fig. S3: PCR to detect Cre-mediated recombination in MEFs Fig. S4: Characterisation of the Erk pathway in MEFs: Dusp 6 knockdown and p90RSK phosphorylation Fig. S5: PCR to detection Cre-mediated recombination in lung Fig. S6: Expression of D-type cyclins in primary MEFs. Fig. S7: Heterodimer formation between L597VBRAF and CRAF in HEK293T cells. 5. Supplemental Table S2. Summary of microarray data 6. Supplemental Table S3. Genes whose expression is significantly altered in V600EBraf, G12DKras and L597VBraf/G12DKras MEFs. 7. Supplemental Table S4. Genes whose expression is altered in L597VBraf/G12DKras MEFs but not G12DKras or V600EBraf MEFs Supplemental Materials and Methods Transfection of HEK293T cells HEK293T cells were cultured in DMEM with 10% FCS and penicillin/streptomycin and transfected with lipofectamine 2000 using conditions recommended by the manufacturer (Invitrogen). Cells were either mock transfected or transfected with myc-tagged expression vectors for human WTBRAF or L597VBRAF. 48 hours after transfection, cells were harvested and protein lysates generated by previously reported methods (Huser et al. 2001). BRAF was immunoprecipitated using the BRAF antibody (Santa Cruz Inc. #SC-5284). Immunoblotting Protein lysates were prepared by previously published methods (Huser et al. 2001). Antibodies used were as follows: Cyclin d1 (Cell Signaling Tech. #2922), Cyclin d2 (Santa Cruz Inc. #SC-593), Cyclin d3 (Cell Signaling Tech. #2936), Gapdh (Millipore #MAB374), CRAF (BD Biosciences #610153), BRAF (Santa Cruz Inc. #SC-5284), MYC (Cell Signaling Tech. #2276), Gapdh (Millipore #MAB374), Erk2 (Santa Cruz Inc. #SC-1647), Dusp6 (Abcam #AB76310) and phospho-Ser380 p90RSK (Cell Signaling Tech. 9341S). siRNA knockdown ON-TARGETplus SMARTpool Dusp6 siRNA (Dharmacon) was used and transfected using lipofectamine 2000 as described in Materials and Methods of the main text. Supplemental References Ritt, D.A., Monson, D.M., Specht, S.I., Morrison, D.K. (2010) Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling. Mol. Cell. Biol. 30: 806-819. Supplemental Table S1. Summary of pathological abnormalities observed with variable penetrance in L597VBraf-expressing mice. Pathology Braf+/Lox-L597V Braf+/+ a)Splenomegaly 17/31 (55%) 0/26 (0%) Stroke 8/31 (26%) 0/26 (0%) b)Growth in internal organs 3/31 (16%) 0/26 (0%) c)Eye abnormalities 4/31 (13%) 0/26 (0%) Uterine prolapse 4/31 (13%) 0/26 (0%) d)Skin lesions 2/31 (6%) 0/26 (0%) Overgrown incisors 4/31 (13%) 0/26 (0%) a) Increased spleen weight by ~4-fold was observed in these mice b) These included: a small intestinal adenoma in one animal at 79 weeks, hyperplasia of the lymphoid tissue in one animal at 73 weeks and hyperplasia of the smooth muscle of the uterus in one animal at 23 weeks. c) Eye abnormalities included cataracts and watery eyes. d) Skin lesions were histologically confirmed as benign skin papillomas in two mice at 29 and 48 weeks of age. Pathologies arising in mice of 6-80 weeks of age with the indicated genotypes are included in the table: all of these mice arose from Braf+/LSL-L597V x CMV-Cre+/o intercrosses and recombination efficiencies were as those indicated in Supplemental Figure 1. + + + - - - CMV-Cre + + - - + - LSL-L597V BrafLox-L597V BrafWT BrafLSL-L597V Supplemental Figure S1. Multiplex PCR genotyping of Braf+/LSL-L597V mouse ear samples with and without the CMV-Cre transgene, utilizing three primers shown in Figure 1A. A B C Supplemental Figure S2. H&E-stained sections of benign growths arising in aged Braf+/Lox-L597V mice. A. Small intestinal adenomatous polyp B. Skin papilloma C. Hyperplasia of lymphoid tissue. D. Hyperplasia of smooth muscle of the uterus. Scale bars = 200 µm β-gal 0 hour 24 hours 48 hours 72 hours 96 hours . WT LV VE WT LV VE WT LV VE WT LV VE WT LV VE WT LV VE BrafLox BrafWT BrafLSL Supplemental Figure S3. PCR to detect Cre-mediated recombination of BrafLSL-L597V and BrafLSL-V600E alleles in MEFs. Braf+/+, Braf+/LSL-L597V or BrafLSL-V600E MEFs were treated with AdCre for 0-96 hours or Adβgal for 96 hours, and genomic DNA was isolated. DNA was PCR genotyped using the three primers (A-C) indicated in Fig. 1A and described in Methods. Cre-mediated recombination is indicated by the appearance of the BrafLox band and the disappearance of the BrafLSL band. A high level of recombination was achieved within 24 hours, and recombination was virtually complete within 72 hours for both alleles. A - Scr D P-Mek P-Erk Dusp6 Erk2 B 0 24 48 72 96 hr + AdCre WT LV VE WT LV VE WT LV VE WT LV VE WT LV VE P-Mek P-Erk P-p90Rsk Erk2 Supplemental Figure S4. Characterisation of the Erk pathway in MEFs: Dusp6 knockdown and p90RSK phosphorylation. A, Dusp6 siRNA knockdown in V600EBraf MEFs. To investigate control of the Erk pathway by Dusp6 in V600EBraf cells, immortalised VE MEFs were either untransfected (-) or transfected with Scrambled (Scr) or Dusp6 (D) siRNA. Protein lysates were western blotted and analysed with antibodies for phosphoMek, phosphoErk, Dusp6 and Erk2. As expected, phosphoErk levels were observed to increase significantly following Dusp6 knockdown while P-Mek levels were slightly reduced, possibly as a result of increased negative feedback regulation of the V600EBRAF/MEK pathway by ERK as previously documented (Ritt et al 2010). B, Analysis of the phosphorylation of the Erk target p90RSK. Protein lysates from Braf+/+, Braf+/LSL-L597V and Braf+/LSL-V600E MEFs treated with AdCre for 0-96 hours were western blotted and analysed with antibodies for phosphoMek, phosphoErk and phospho-p90RSK (Ser380). Erk phosphorylates and activates the C-terminal kinase domain of p90RSK which in turn autophosphorylates Ser380 and so phosphorylation of Ser380 of RSK is Erk- dependent. RSK phosphorylation was observed to correspond to levels of Mek/Erk activation with higher levels being observed in the VE cells compared to LV and WT cells at 48 and 72 hours following Cre-induced recombination. Levels of phosphoRSK were more similar at the 96 hour time point when phosphoErk levels were more similar between the LV and VE cells. A WT LV G12D VE G12D/LV Braf BrafLox BrafWT KrasLox-G12D Kras KrasWT WT VE LV G12D G12D/LV B BrafLox Braf BrafWT KrasLox-G12D Kras KrasWT Supplemental Figure S5. PCR to detection Cre-mediated recombination of BrafLSL- L597V, BrafLSL-V600E and KrasLSL-G12D alleles in lung and MEFs. A, Genomic DNAs taken from lungs of Braf+/+ (WT), Braf+/LSL-L597V (LV), Kras+/LSL-G12D (GD), Kras+/LSL-G12D ;Braf+/LSL-L597V (G12D/LV) or Braf+/LSL-V600E (VE) mice treated with AdCre were PCR genotyped using the two primers (A,C) indicated in Figure 1A and described in Methods for Braf (top panel) and the two primers described in Methods for Kras (lower panel). B, Genomic DNAs taken from MEFs of Braf+/+ (WT), Braf+/LSL-L597V (LV), Kras+/LSL-G12D (G12D), Kras+/LSL-G12D;Braf+/LSL-L597V (G12D/LV) or Braf+/LSL-V600E (VE) mice treated with AdCre were PCR genotyped using the two primers (A,C) indicated in Figure 1A and described in Methods for Braf (top panel) and the two primers described in Methods for Kras (lower panel). G12D/ WT VE LV G12D LV Cyclin d1 Cyclin d2 Cyclin d3 Gapdh Supplemental Figure S6. Expression of D-type cyclins in primary MEFs. Protein lysates from pimary MEFs from WT, LV, VE, G12D and G12D/LV MEFs were prepared 96 hours after infection with AdCre and western blots were analysed with the indicated antibodies. While Cyclin D1 and Cyclin D2 levels were increased to similar levels in the VE, G12D and G12D samples compared to WT and LV samples, Cyclin D3 levels were higher in the G12D MEFs. - WT LV CRAF IP: BRAF BRAF CRAF WCL BRAF MYC Supplemental Figure S7. L597VBRAF forms a heterodimer with CRAF following overexpression in HEK293T cells. HEK293T cells were either mock-transfected (-) or transfected with vectors expressing myc-tagged WTBRAF or L597VBRAF. Protein lysates were immunoprecipitated for BRAF and immunoprecipitates analysed for BRAF and CRAF. Whole cell lysates (WCL) were also analysed with antibodies for BRAF, CRAF or the myc-tag. Supplemental Table S3. Genes whose expression is significantly altered in V600EBraf, G12DKras and L597VBraf/G12DKras MEFs compared to Braf+/+ MEFs Fold change Fold change Fold change Gene Name Gene Description Entrez ID (V600E over WT) (G12D over WT) (LV/G12D over WT) Gap43 growth associated protein 43 14432 16.60 11.70 10.20 Etv1 ets variant gene 1 14009 15.60 10.90 11.40 Etv1 /// Gm5454 ets variant gene 1 /// predicted gene 5454 14009 /// 432800 13.80 10.80 9.62 Olfr1318 /// Olfr1317 olfactory receptor 1318 /// olfactory receptor 1317 258022 /// 258440 12.70 9.95 6.93 Sh3bgrl2 SH3 domain binding glutamic acid-rich protein like 2 212531 9.87 2.88 4.37 Ramp3 receptor (calcitonin) activity modifying protein 3 56089 9.75 8.14 6.74 Pde3b phosphodiesterase 3B, cGMP-inhibited 18576 9.23 18.90 9.27 Pdgfra platelet derived growth factor receptor, alpha polypeptide 18595 8.50 6.28 6.11 Lrrtm2 leucine rich repeat transmembrane neuronal 2 107065 8.36 7.70 9.43 Tmeff2 transmembrane protein with EGF-like and two follistatin-like domains 2 56363 7.19 4.45 3.98 Gpm6b glycoprotein m6b 14758 6.67 6.88 7.99
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