Bovine tsh

Pre-infusion (time 0) blood samples were collected in chilled,
EDTA-treated vacutainer sample tubes and kept on ice until they could be
centrifuged. Pre-infusion (time 0) adipose and muscle tissue biopsies were
collected by first cleaning a small region in the flank of the animal near the
hind flipper with alternating wipes of isopropyl alcohol and betadine. A small
(<1.5 cm) incision was made using a sterile scalpel, and a blubber and
muscle biopsy (ca. 50–200 mg) was collected with a sterile biopsy punch
needle (Henry Schein). The biopsy samples were rinsed with cold, sterile saline,
placed in cryogenic vials, immediately frozen by immersion in liquid nitrogen,
and stored at −80°C until later analyses. Following initial
sample collection, animals (n=10 early, n=10 late) were infused with 8 IU of
bovine TSH (Sigma) (n=6) or with saline (n=4). Following the infusion, blood
samples were collected at 15, 30, 60, 120, and 1440 mins. Additionally, adipose
and muscle biopsies were collected at 60, 120, and 1440 mins. Blood samples were
centrifuged for 15 min at 3000g, and the plasma
was transferred to cryo-vials, immediately frozen by immersion in liquid
nitrogen, and stored at −80°C (Viscarra et al., 2011 (link); Viscarra et
al., 2013; Viscarra et al.,
2010; Viscarra et al.,
2012).

We measured the stimulating activity of TSHR antibodies in mouse sera by testing their ability to induce cAMP production in cells expressing the TSHR. A stable cell line of CHO-HA-TSHR luciferase cells was generously provided by Drs. Rauf Latif and Terry Davies [30 ]. Cells were grown in Ham’s F12 medium (Gibco, Gaithersburg, MD) supplied with 10% FBS (Sigma-Aldrich, St. Louis, MO), 1× penicillin-streptomycin (Corning, NY) and 100 μg/ml of hygromycin (Invitrogen, Carlsbad, CA). CHO-HA-TSHR luciferase cells (500,000 cells/ml) were seeded in a 96-well plate and incubated for 24 h at 37 °C. Bovine TSH (Sigma-Aldrich, catalog no. T8931) at concentrations of 1 μU/ml to 10,000 μU/ml was added to generate a dose response curve. Sera from BALB/c-DR3 mice immunized with AdTSHR (n = 14) or AdLacZ (n = 12) were diluted 1:2 in Ham’s F12 medium. 50 μl of increasing concentrations of TSH and diluted serum samples were added to cells and incubated for 5 h at 37 °C. After 5 h incubation, 50 μl of Bright-Glo reagent (Promega, WI, catalog no. E2610) were added to each well. Followed by 2–3 min gentle shaking, luciferase activity was measured using a BMG ELISA reader (BMG Labtech, Cary, NC).

Thyroid tumors were collected aseptically from donor mice (BVE^Cyp24a1-wt and BVE^Cyp24a1-wll) using blunt dissection, then mechanically dissociated by mincing and passing through a 40μm/mesh sterile screen, and suspended in DMEM/F12 growth medium (10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin). The cells were further dissociated by incubation in growth medium containing 100 U/mL type I collagenase (Sigma-Aldrich) and 1.0 U/mL dispase I (Roche Diagnostics) at 37°C in a rocking water bath for 60 minutes. The cell suspension was washed twice with growth media and resuspended in a 10mm culture dish with DMEM/F12 growth medium containing 4 mU/mL bovine TSH (Sigma-Aldrich) to establish BVE^Cyp24a1-wt and BVE^Cyp24a1-null cell lines. The established cell lines were propagated in DMEM/Ham’s F12 growth medium. Two cell lines were established each from two separate BVE^Cyp24a1-wt or BVE^Cyp24a1-null primary thyroid tumors in 2015: BVE^Cyp24a1-wt-1, BVE^Cyp24a1-wt-2, BVE^Cyp24a1-null-1, and BVE^Cyp24a1-null-2. The thyroid origin of these cell lines was confirmed by genotyping in the lab as described above, which was performed 1 month before using the cell lines.

A PTC tumor from a 4-month-old TPO-BRAF^V600E mouse was collected aseptically using blunt dissection and mechanically dissociated by mincing and passaged through a 40-μM mesh sterile screen, and suspended in DMEM/F12 growth medium (10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin). Cells were further dissociated by incubation in the growth medium containing 100 U/ml type I collagenase (Sigma-Aldrich) and 1.0U/ml dispase I (Roche Diagnostics, Indianapolis, IN, USA) at 37 °C rocking water bath for 60 min. The cell suspension was washed twice and resuspended in 10 mm culture dish with DMEM/F12 growth medium containing 2 mU/ml bovine TSH (Sigma-Aldrich) to establish a BVE cell line (BRAF^V600E-induced tumor cell line). The genetic background was confirmed by genotyping.