(A) Endogenous D3 activity in SK-N-AS neurons, rat neonatal cardiomyocytes, NCLP6E hepatocytes, choriocarcinoma cells (JEG-3 cells), endometrial cells (ECC-1 cells), and AG04526 fibroblasts exposed to normoxia (21% O₂) versus hypoxia (1% O₂) for 24 h. Values are mean ± SEM of 2 or 3 cell plates; mean of 3 experiments is shown for each cell type. *P < 0.005. (B) Northern blot analysis of total RNA obtained from SK-N-AS or NCLP6E cells exposed to hypoxia versus normoxia for 24 h. Lanes were run on the same gel but were noncontiguous. (C) D3 activity and Northern blotting in NCLP6E cells exposed to continuous normoxia (condition A), continuous hypoxia (condition B), or transient hypoxia for 24 h followed by normoxia (condition C). Representative experiment with mean ± SEM of 2 cell plates is shown; this experiment was reproduced.

(A) D3 activity and Western blot analysis of HIF-1α protein in NCLP6E hepatocytes 24 h after exposure to the indicated hypoxia mimetics. Hepatocytes that were exposed to hypoxia (without hypoxia mimetics) are included in the HIF-1α Western blot as a positive control. (B) D3 activity and Western blot analysis of HIF-1α protein in SK-N-AS neurons 6 or 24 h after exposure to DFO or CoCl₂. Values are mean ± SEM of 3 cell plates. *P < 0.05; **P < 0.005. (C) Western blot analysis of HIF-1α protein in cells that increase endogenous D3 expression in response to hypoxia (JEG-3, SK-N-AS neurons, and NCLP6E hepatocytes) and in cells with undetectable endogenous D3 activity (HepG2, HEK-293 cells, and MSTO cells). (D) ChIP analysis of the DIO3 or the SCN3A promoter in DFO-stimulated SK-N-AS cells using antibodies directed against endogenous HIF-1α. Representative experiments are shown and were reproduced.

Oxygen consumption (A and C) and extracellular acidification (B and D) in SK-N-AS neurons and NCLP6E hepatocytes that were propagated in media with (A and B) or without (C and D) a euthyroid concentration of T3 and exposed to iopanoic acid (Iop) versus vehicle control. Oxygen consumption rate (pmol/min/mg protein) and extracellular acidification rate (mpH/min/mg protein) are expressed as relative fold change from vehicle control. Values are mean ± SEM of 10 cell plates. *P = 0.07; **P < 0.05; ***P < 0.005.

Thickening of the RV wall (A) and increased RV weight (B) were observed as CHF developed after monocrotaline administration. Values are mean ± SEM of 11–16 animals per group. (C–F) Western blot analysis of HIF-1α (C) and HIF-2α protein (D), D3 activity (E), and quantitative real-time PCR of D3 mRNA (F) in tissue prepared from the RV and LV of rats administered monocrotaline (CHF) versus saline control (CON). The HIF-2α Western blot in D depicts RV samples from control and CHF animals, with extracts of primary microvascular endothelial cells obtained from human foreskin and cultured under hypoxic conditions (1% O₂) serving as a positive control (+). Values are mean ± SEM of 3–9 animals per group. (F) D3 mRNA is expressed as the RV/LV ratio and shown as the relative fold change from the saline control group. *P < 0.05 versus control; **P < 0.05 versus LV; ***P < 0.005 versus control. (G) Myocardial reporter activity after in vivo cardiomyocyte transfection of the pLuc-TRE T3-responsive Firefly luciferase reporter, normalized to the expression of the pRen-C transfection control. Values are mean ± SEM of 5–29 animals per group. LV and RV reporter activity is shown in CHF versus control animals. In control animals with systemic hypothyroidism (Hypo), euthyroidism (Eu), or thyrotoxicosis from T3 treatment (Hyper), reporter levels from pooled LV/RV homogenates are shown. *P < 0.05 versus euthyroidism or hypothyroidism, ANOVA; **P < 0.05 versus control and LV.