Supplementary Figure 1

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1 Supplementary Figure 1 60 HRAS G12V 40 * Percent BRDU Positive NS Percent BRDU Positive NS dox dox doxkt5720 -doxkt5720 Treatment TSHKT dox dox doxkt5720 -doxkt5720 Treatment TSHKT5720 Supplementary Figure 1: The PKA antagonist KT5720 inhibits TSH but not oncoprotein-induced DNA synthesis in rat PCCL3 cells. PCCL3 cells conditionally expressing the indicated oncoprotein in a dox-inducible manner were grown in Coon s/h4 serum and then switched to Coon s/h3 serum (without TSH) for 3 days. Cells were then treated with 10 µg/ml BrdU for 24 h in the absence or presence of 1 µg/ml of doxycycline or 10 miu/ml TSH with or without 5 µm KT5720 for 24 h. BrdU incorporation was analyzed by FACS. Data represent mean SD of 3 wells. *p<0.05; p<0.009; NS: Not significant, p > 0.05.

2 Supplementary Figure 2 A Braf terk Dox KT5720 B PCCL3 PCCL3 RET/PTC3 Braf RET/PTC3 rs6 ERK Dox - - camp rs6 ERK Dox - - camp Supplementary Figure 2 Effects of camp on ribosomal S6 phosphorylation in PCCL3 cells expressing oncogenic BRAF or RET/PTC3. A,B) PCCL3 cells conditionally expressing Braf V600E (left) or RET/PTC3 (right) were incubated in Coon s/h3 serum for 2 days. A) Cells were then incubated in Coon s - serum with or without dox for 2 days. KT5720 (10 µm) was added 6 hours prior to preparing protein lysates. B) Cells were then incubated in Coon s - serum with or without dox for 4 days. camp was added 60 min prior to preparing protein lysates. Western blotting was performed for the indicated proteins.

3 BHT101 ps6k T389 prs6 S240/244 p4ebp1 T37/46 prsk 380 trsk terk Rapa 20nM 2h AZD nM 2h FMK 5 µm 2h Supplementary Figure 3 Role for RSK in ribosomal S6 phosphorylation in a human thyroid cancer cell line harboring oncogenic BRAF. Cells were incubated in medium with 10% FBS, and then with DMSO, rapamycin (20 nm), AZD6244 (500 nm) or RSK inhibitor (FMK, 5 μm) alone or in combination for 2 h. Cells lysates were Western blotted with the indicated antibodies.

4 8Br-cAMP KT5720 (µm) ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 KT5720 (µm) ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 8Br-cAMP - - rapa - AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 8Br-cAMP - - rapa - U ps6k T389 prs6 S240/242 p4ebp1 T37/ Supplementary Figure 4 Densitometry of Western blots shown in Figure 1. Bars represent the ratio of the indicated phosphoprotein relative to the loading control.

5 Dox rapa U0126 AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 Dox rapa U AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 Dox rapa U0126 AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 Supplementary Figure 5 Densitometry of Western blots shown in Figure 2B. Bars represent the ratio of the indicated phosphoprotein relative to the loading control α-tubulin.

6 8 Br camp - - ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 8 Br camp - - ps6k T389 prs6 S240/242 p4ebp1 T37/46 pakt S473 Supplementary Figure 6 Densitometry of Western blots shown in Figure 3B. Bars represent the ratio of the indicated phosphoprotein relative to the loading control α-tubulin.

7 rapa U AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pmek pakt S473 rapa U AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pmek pakt S473 rapa U AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pmek pakt S473 rapa U AKTi ps6k T389 prs6 S240/242 p4ebp1 T37/46 pmek pakt S473 Supplementary Figure 7 Densitometry of Western blots shown in Figure 5B. Bars represent the ratio of the indicated phosphoprotein relative to the loading control α-tubulin.

8 Fig. 6A ERK norm alized expression AZD6244 AZD8055 BHT101 KTC1 pakt S473 Hth104 ps6k T389 ERK norm alized expression AZD6244 AZD8055 BHT101 KTC1 pakt S473 Hth104 ps6k T389 Fig. 6D β -actin Norm alized Expression AZD AZD pakt S473 * β -a c tin N o rm a liz e d Expression AZD AZD pakt S Supplementary Figure 8 Densitometry of Western blots shown in Figure 6A and C. (upper) Bars represent the ratio of the indicated phosphoprotein relative to total ERK1 for Figures 6A. (lower) Bars represent ratio of the indicated phosphoprotein relative to β-actin for Figure 6C. *p<0.05, p<0.006.

9 Supplementary Table 1: Cell lines used in study. Cell Source Mutation Line 1 identified 2 WRO G. Juilliard, University of California Los none BHT101 OCUT2 KTC1 FRO Angeles German Collection of Microorganisms and Cell Cultures ( Naoyoshi Onoda, Osaka City University German Collection of Microorganisms and Cell Cultures ( Junichi Kurebayashi, Kawasaki Medical School G. Juilliard, University of California Los Angeles PI3KCA H104 7R KRAS G12R none TPC1 Sissy M. Jhiang, Ohio State RET/PTC1 Hth104 C c FTC133 Nils-Erik Heldin, Uppsala University Hospital, Sweden Nils-Erik Heldin, Uppsala University Hospital, Sweden Nils-Erik Heldin, Uppsala University Hospital, Sweden Nils-Erik Heldin, Uppsala University Hospital, Sweden German Collection of Microorganisms and Cell Cultures ( German Collection of Microorganisms and Cell Cultures ( HRAS Q61R HRAS G13R PTEN 1 All cell line were subjected to STR analysis and found to have a unique STR profile (i.e. no match to cell lines in ATCC, DSMZ, or EACAC databases) (1). 2 Cell lines were screened for point mutations in BRAF, RAS, AKT, and PI3KCA as well as for RET, ntrk, PPARγ rearrangements as described in Ricarta-Fihlo et al., (2). References 1. Schweppe RE, Klopper JP, Korch C, Pugazhenthi U, Benezra M, Knauf JA, et al. DNA Profiling Analysis Of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting In Cell Line Redundancy And Misidentification J Clin Endocrinol Metab 2008; 15, Ricarte-Filho JC, Ryder M, Chitale DA, Rivera M, Heguy A, Ladanyi M, et al. Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1 Cancer Res 2009; 69,

10 Supplementary Table 2: Different PCCL3 media used in study. Medium Contents Coon s modified F12 supplemented with TSH (10 miu/ml), insulin (10 Coon s/h4serum mg/ml), apotransferrin (5 mg/ml), hydrocortisone (10 nmol/l) and fetal calf serum (5%) Coon s/h3serum Coon s modified F12 supplemented as above except TSH omitted. Coon s modified F12 supplemented with TSH (10 miu/ml), insulin (10 Coon s/h4-serum mg/ml), apotransferrin (5 mg/ml), hydrocortisone (10 nmol/l) and bovine serum album (0.5%) Coon s/h3-serum Coon s modified F12 supplemented as above except TSH omitted. Coon s modified F12 supplemented apotransferrin (5 mg/ml), Coon s/h2-serum hydrocortisone (10 nmol/l) and bovine serum album (0.5%)