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 LEGENDS
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.