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Supplementary Figures S1 Experimental Procedures Immunoblotting Cells were washed twice with cold PBS and lysed in lysis buffer (containing 125mM HEPES, pH7.5, 750mM NaCl, 5% Igepal CA-630, 50mM MgCl2, 5mM EDTA and 10% glycerol) supplemented with proteinase and phosphatase inhibitors (Roche). Protein concentration was determined using the BCA kit (Thermo Scientific). Western blots were conducted on 20 μg protein separated by 4-12% Bis-Tris SDS-PAGE gels (Invitrogen), transferred to PVDF membranes, and immunoblotted after blocking with 5% skim milk with the corresponding primary antibodies (listed below) in 5% BSA (SIGMA). This was followed by incubation for 1h with secondary antibodies conjugated with goat anti-rabbit HRP-conjugated antibody (1:5,000; Santa Cruz sc-2054) or goat anti-mouse HRP-conjugated antibody (1:5,000; Santa Cruz sc-2005). Bound antibodies were detected by chemiluminescence with the ECL detection system (GE Healthcare Biosciences). In Figures 2C, 3C and 4C we show the same representative Merlin and Actin blots as aliquots of the same shNF2 clone M2-transduced C643 cell lysate were used for multiple different blots. Supplementary Figure 11 shows the individual blots and the original films corresponding to Figures 2C, 3C, and 4A. Aliquots of the same shNF2 clone M4-transduced C643 cell lysates were used for all blots in Figure 2C and 3C. RAS-GTP or RAC1-GTP immunoprecipitation was conducted using the RAS or RAC1 activation assay kits, respectively, from Millipore, according to the manufacturer's protocol and subjected to Western blotting with the indicated antibodies. Cells were treated during 72h with dox in media with 1% of FBS. Subcellular fractions were prepared using the Subcellular Protein Fractionation Kit for cultured cells following the manufacturer’s instructions (Thermo Fisher Scientific). Fractions were subjected to Western blotting with the indicated antibodies. Copy number alterations, chr 22, IMPACT Copy number alterations, chr 22, CGH-array A. CRKL MAPK1 SMARCB1 CHEK2 NF2 EP300 B. CRKL MAPK1 SMARCB1 CHEK2 NF2 EP300 JF_thy_005_PDTC -0.86 JF_thy_005_PDTC -0.45 JF_thy_019_PDTC -0.80 JF_thy_019_PDTC -0.45 JF_thy_020_PDTC -0.74 JF_thy_020_PDTC -0.37 JF_thy_021_PDTC -0.63 JF_thy_021_PDTC -0.36 JF_thy_009_PDTC -0.55 JF_thy_009_PDTC -0.32 JF_thy_018_PDTC -0.52 JF_thy_018_PDTC -0.31 JF_thy_007_PDTC -0.39 JF_thy_007_PDTC -0.25 JF_thy_034_ATC -0.17 JF_thy_034_ATC -0.13 JF_thy_008_ATC -0.17 JF_thy_008_ATC 9 NF2 NF2 9 loss 9 NF2 NF2 9 loss -0.10 37 frozen tumors frozen 37 37 frozen tumors frozen 37 C. D. NF2 CNA values CRKL MAPK1 SMARCB1 CHEK2 NF2 EP300 log ratio log ratio Tumor ID Tumor type Preservation Driver alteration JJ_thy_027_PDTC -0.73 IMPACT CGH-array JJ_thy_053_PDTC -0.53 JJ_thy_007_PDTC -0.47 JF_thy_005 PDTC frozen NRAS Q61R -0.86 0.45 JJ_thy_004_PDTC -0.43 JJ_thy_026_PDTC -0.42 JF_thy_019 PDTC frozen NRAS Q61R -0.80 -0.45 JJ_thy_017_PDTC -0.36 JF_thy_020 PDTC frozen unknownRET/PTC -0.64 -0.37 JJ_thy_001_PDTC -0.28 7 NF2 NF2 7 loss JF_thy_021 PDTC frozen HRAS Q61R -0.63 -0.36 JF_thy_009 PDTC frozen unknownRET/PTC -0.55 -0.32 JF_thy_018 PDTC frozen unknown -0.52 -0.31 JF_thy_007 PDTC frozen unknownRET/PTC -0.39 -0.25 JF_thy_034 ATC frozen unknown -0.17 -0.13 JF_thy_008 ATC frozen NRAS Q61R -0.17 -0.10 JJ_thy_027 PDTC FFPE NRAS Q61R -0.73 N/A JJ_thy_053 PDTC FFPE NRAS Q61R -0.53 N/A JJ_thy_007 PDTC FFPE NRAS Q61R -0.47 N/A JJ_thy_004 PDTC FFPE NRAS Q61R -0.43 N/A 46 FFPE tumors 46 JJ_thy_026 PDTC FFPE unknown -0.42 N/A JJ_thy_017 PDTC FFPE HRAS Q61R -0.36 N/A JJ_thy_001 PDTC FFPE unknown -0.28 N/A Supplementary Figure S1: Loss of heterozygosity of Ch22q genes in poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC). Copy number alterations (CNA) were investigated in frozen (A,B) or formalin-fixed paraffin embedded (FFPE) (C) tissue samples. Frozen tissues were studied with two different approaches: A) CNA- IMPACT, an exon-capture, next generation sequencing panel of 341 cancer genes, 6 of which mapped to 22q (CRKL, MAPK1, SMARCB1, CHEK2, NF2, EP300), from which copy number information was derived by comparing sequencing read counts to those of a diploid control. B) SNP-CGH, using an Agilent 500K SNP array. Ch22q loss was present in 9/37 tumors, all of which had NF2 LOH. This was confirmed with both strategies, including the relative quantification of allelic loss in the individual tumors (D), thus confirming the ability of CNA-IMPACT to detect copy number losses. In most cases the Ch22q arm was lost in its entirety, with the exception of one PDTC that lost only the distal arm of the chromosome, encompassing CHEK2, NF2 and EP300. C) CNA of 46 FFPE tumors by IMPACT, 7 of which had Ch22q and NF2 loss. One tumor had LOH of the distal arm only. D) NF2 CNA log ratios in tumors analyzed by IMPACT or CGH. 9/16 tumors with NF2 LOH had RAS mutations. CGH CGH - rray a copy number copy NF2 2 level level copy - 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -2 -4 number -6 -8 logratio [log2(CN/2)] -10 -12 NF2 exon # IMPACT exon IMPACT level level exon 2 exon 3 exon 4 - aligned reads IMPACT exon IMPACT cell cell line NF2 normal normal NF2 Supplementary Figure S2: Focal NF2-exon 4 homozygous deletion in KHM-5M anaplastic thyroid cancer cells. Top panel: Focal deletion within NF2 gene as detected by array-CGH. Middle panels: NF2 exon-level copy number analysis by IMPACT, shows selective copy number loss of exon 4 of NF2. Bottom panel: Exon 4 read counts in a cell line with wild-type merlin, demonstrating adequate sequencing coverage at that site. A B Supplementary Figure S3: FISH for NF2 in anaplastic thyroid cancers. FISH analysis for NF2 was performed by hybridizing a tissue microarray consisting of 16 different ATC (anaplastic) specimens with NF2 probe (BAC clone RP11-551L12, 22q12.2, red) and BCR probe (22q11.2, green), as control. Deletion of NF2 was defined as tumors with > 25% of cells with a single NF2 hybridization signal (a minimum of 200 cells were scored for each tumor). This threshold was selected because of the high admixture of stromal cells, particularly tumor associated macrophages, in these cancers (Ryder et al., 2008; Caillou et al., 2011). A) A total of 10/16 ATCs had NF2 LOH or homozygous deletion, with the remainder being polyploid at this site (note that the FISH analysis alone is not sufficient to determine loss of NF2 locus in a polyploid population) B) Representative FISH image to show nuclei with either LOH (yellow arrows) or homozygous loss (red arrows) of NF2 locus in an ATC sample. A MERLIN protein BRAF RAS WT PTEN B MERLIN protein C D 1 - 8505c Cal62 TCO KMH5 Hth74 BCPAP Hth83 TTA1 Mutation Hom Exon Del Decreased mRNA Stability Decreased mRNA Supplementary Figure S4: NF2/Merlin defects in thyroid cancer cell lines. A) Western blots of thyroid cancer cell lines for merlin. The driver mutation of each line is indicated. Cell lines were defined as merlin normal (black), low (grey) or null (white) based on protein abundance. B) Merlin mRNA by quantitative RT-PCR in thyroid cancer lines categorized based on merlin protein levels. C) Decreased merlin mRNA half-life in HTh74 cells. Merlin mRNA levels were measured by quantitative RT-PCR at various times after addition of actinomycin D. D) Events associated with merlin loss in thyroid cancer cell lines. A B C643 (HRASG13R) Cal62 (KRASG12R) C * ** ** C643(HRASG13R) D Supplementary Figure S5: Merlin inhibits RAC1-PAK activity: A) Top: RAC1-GTP levels in Cal62 (KRASG12R) cells treated with dox for 72h. Bottom: Westerns of input lysates for the indicated proteins are shown below. pMEK-S298 is a PAK substrate. B) Western blots of: Left: Cal62 cells and Hth83 cells following dox-induction of merlin. Right: C643 cells transfected with five NF2 siRNAs. C) Growth of Cal62 cells expressing control (pEBG) or a dominant negative PAK (dnPAK) vector treated with or without dox for 4 days. ***p ≤ E x 10-3; ** p ≤ E x 10-2 ; n=3, two independent experiments). Bars represent mean +/- SD. D) Left: Growth effects of FRAX597 in C643 (HRASG13R, NF2-WT) cells stably expressing scrambled or shNF2.M2. Cells were counted at 6days. *p < 4 x E-2; **p < 1 x E -2. Right: Western blots of C643 cells expressing scrambled or shNF2.M2 after incubation with FRAX 597 for 48h. Supplementary Figure S6: Merlin effects on EGFR signaling and growth of RAS mutant thyroid cancer cell lines. Left: Western blots of Cal62 (KRASG12R) cells treated with dox for 72h in 1% serum, and then with EGF (5nM) for 10 min as shown. Right: hEGF (5ng/ml) effects on growth of Cal62 cells treated with or without dox in 1% serum (n=3). HrasG12V+/- wt HrasG12V+/- HrasG12V+/+ Nf2 1 2 3 4 1 2 3 1 2 3 1 2 3 4 5 M WT Supplementary Figure S7: PCR of genomic DNA from mouse thyroid tissues of the indicated genotypes with primers that distinguish wild-type from mutant Hras alleles. M: 668 bp; WT: 622 bp. A TEAD expression in TCGA-PTC B Hth83(HRASQ61R) C643(HRASG13R) seq - RNA expression C Hth83(HRASQ61R) D Cal62 (KRASG12R) G13R E C643(HRAS ) Supplementary Figure S8: A) Data represent TEAD isoform mRNA levels derived from RNASeq of PTCs reported by the TCGA program (Cancer Genome Atlas Research Network., 2014). B) ChIP-PCR with antibodies to YAP and TEAD1 in Hth83 cells treated with or without dox for 72h and in C643 cells transfected with plko.1 or shNF2 (*p < 1 x E-2) C) Left: Verteporfin (VP) dose- dependent decrease of YAP, pYAP and TEAD at 72h in Hth83 cells.
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