Supplement Methods

Supplement Methods


Thyroid Ultrasonography:

Anesthesia was achieved using isofluorane (1.5%) in oxygen. Mice were then placed in dorsal recumbencyon a heated stage (37◦C) and anesthesia was maintained during the procedure. Neck hair was removed using a depilatory cream. Ultrasound images of the neck were acquired with the Vevo 660 high-frequency ultrasound system (VisualSonics, Toronto, Ontario, Canada) using a 40 mHz probe as per the manufacturer’s recommended methods.

Protein Isolation and Western Blot:

Frozen tissues were homogenized in M-PER buffer (Fisher Scientific, Pittsburg, PA) containing 0.3 M okadaic acid and 1 g/ml each of aprotinin, pepstatin, leupeptin, and 20 mM of 4-amidino-phenyl methane-sulfonyl fluoride (APMSF) (EMD Chemicals, Inc., Gibbstown, NJ). Protein was extracted by centrifuging the homogenates at 16,000 g for 15 min at 4°C. Supernatant was collected and protein concentrations were measured using a BCA protein assay reagent kit (Fisher Scientific). Twenty-thirty g of total lysate were mixed with final 0.05M DTT and SDS buffer (Invitrogen Co., Carlsbad, CA), and boiled for 5 min. The reduced and denaturized lysate was loaded into 4-12% SDS-PAGE, separated by electrophoresis and then transferred to nitrocellulose membranes. Immunoblotting was performed as previously described (36, 37). Proteins were detected using ECL solution (GE Healthcare, Piscataway, NJ) and X-Ray film (ISC Bioexpress, Kaysville, UT). Relative quantitation of proteins was determined by scanning and band densitometry using Image Gauge software (Fuji Photo Film Co, Ltd, Tokyo, Japan).


Supplement Figure 1: Thyroid ultrasonography was performed on wild type (WT), PVPV-Akt1WT and PVPV-Akt1KO mice at 9 months of age. Representative images are shown. The white arrow denotes the trachea. In comparison to the WT mice, the PVPV-Akt1WT mice display bilateral goiter that is partially attenuated by the absence of Akt 1 in the PVPV-Akt1KO mice.

Supplemental Figure 2: Panel A:Representative pituitary histology (H&E) is demonstrated from 9 month old wild type (WT) mice, PVPV-Akt1WT and PVPV-Akt1KO mice. In comparison to the pituitaries from the WT litter mate control mice, the PVPV-AktWT mice demonstrated variable hyperplasia of basophils containing abundant cytoplasm. The results were similar in the PVPV/Akt1KO mice. Panel B: Representative pituitary immunohistochemical staining for Akt1, 2, and 3 demonstrate loss of Akt1 expression with similar levels of Akt2 and 3 in the PVPV-Akt1WT and PVPV-Akt1KO mice. Methods are described in the primary manuscript.

Supplemental Figure 3: Western blot of proteins from thyroid tissues from 9 month old PVPV-Akt1KO and PVPV-Akt1WT mice are compared with age-matched litter mate control Akt 1 KO and wild type (WT)mice. Panel A: Akt isoform immunoblot demonstrates loss of Akt1 (upper band, arrow) in PVPV-Akt1KO versusPVPV-Akt1WT mice. Akt 2 and 3 proteins were not increased in the PV/PV-Akt1KO thyroid cancers despite the reduction in Akt1 levels. Protein from thyroid tissue from age-matched WT and Akt1 KO mice are included as positive and negative controls, respectively. Panel B: pAkt (ser473) levels are reduced in thyroid tissue from PVPV-Akt1KO vs PVPV-Akt1WT mice. pAkt levels were similar in the thyroid glands ofWT vs Akt1 KO mice.