Fig 2: T3 represses STMN1 expression in HepG2 cell lines.(A) THR protein expression levels in HepG2 control and THR overexpressing cell lines. (B) qRT-PCR of STMN1 mRNA expression levels in HepG2-THRA cells (**P < 0.01, ***P < 0.001, n = 3). (C) Northern blots of HepG2-THRA cell RNAs. (*P < 0.03, n = 2). (D) Immunoblots of HepG2-THRA, HepG2-THRB and HepG2-Neo cell lysates (*P < 0.03, n = 3). Error bars, s.e.m. Electrophoretic gels and blots in each experiment were conducted under the same experimental conditions, and images were cropped.

Fig 3: Thyroid hormone receptor represses STMN1 in HepG2 cell at the transcription level directly.(A) Schematic representation of the STMN1 promoter (+1, translation start site). A serial deletion fragments of STMN1 5'-flanking DNA were cloned into pGL3 (construct I~V, XI) or pGL3tk-luc (construct VI~X) reporter plasmids, as indicated. The T3 repression folds were presented as mean values ± s.e.m. (n = 3). (B) HepG2-THRA was incubated for 12–24 h with 0 to 10 µg/ml cycloheximide (CHX) in the absence or presence of T3, after which total RNA was isolated and sequentially analyzed by Northern blot. (n = 3, Error bars, s.e.m.). (C) ChIP assay of STMN1 5'-flanking region (-101~+1). The promoter region of GAPDH was used as the negative control. Electrophoretic gels and blots in each experiment were conducted under the same experimental conditions, and images were cropped.

Fig 4: Overexpression of STMN1 attenuates T3-suppressed cell growth.(A) Fluorescent image of stable expressing STMN1-GFP fusion protein in HepG2-THRA cell. (B) Cell growth in the absent or present of T3 for 4 days were analyzed. The relative folds of T3-suppressed growth of each individual clone were normalized by 0 nM T3. (C) The STMN1 overexpressing and control cells were analyzed by flow cytometry. Percent of cell cycle distributions were presented in (D). (***P < 0.001; **P < 0.01; *P < 0.05 n = 3). Error bars, s.e.m. (E) Immunoblots of STMN1-EGFP and EGFP stable expressing cells in the absent or present of 4 days T3 treatment. Gel electrophoresis was conducted under the same experimental conditions, and images of blots were cropped. Uncropped blot images are shown in Supplementary Fig. S3B.