hTSHR(2)

Fig.1 Detection of TSHR expression in mammary gland by RT-PCR.

RT-PCR analysis confirmed specific human TSHR expression in HO hTSHR mice(female, 10 weeks old), while WT mice expressed only endogenous mouse TSHR. No cross-amplification was observed, confirming the specificity of the humanized TSHR transgene and the absence of endogenous mouse TSHR expression in HO mice. 

Fig.2 Detection of human TSHR expression in thyroid in hTSHR(2) mice by WB. Protein lysates from thyroid tissues of WT and HO hTSHR mice were analyzed using an anti-human TSHR antibody. Vinculin was used as a loading control. Positive control (PC) was Hela cells expressing human TSHR. A specific band at approximately 70 kDa corresponding to human TSHR was detected in HO mice and PC, but not in WT mice. Western blot analysis confirmed human TSHR protein expression in thyroid tissues of HO hTSHR mice.

Abbr: WT, wild-type; HO, homozygous; PC, positive control; hTSHR, human thyroid-stimulating hormone receptor.

Fig.3 Representative FACS plots showing human TSHR expression in thyroid cells from HO hTSHR and WT C57BL/6 mice. Single-cell suspensions from mouse thyroid tissue (9 weeks, male). Figures were analyzed. Live cells were first gated from the CD45⁻ population , followed by gating on CD31⁻ EPCAM⁻ cells (not shown) to exclude endothelial cells. hTSHR⁺ cells were identified from the TER119⁻ population. In HO hTSHR(2) mice, strong hTSHR expression was detected (70 %), confirming successful transgene expression in thyroid cells. The anti-hTSHR antibody also recognized endogenous mouse TSHR, as evidenced by low but positive signals in WT mice (40%).

Fig.4 Body weight changes following T3 pretreatment and single-dose M22 stimulation in WT and HO hTSHR(2) mice. WT C57BL/6 and HO hTSHR (2) mice (female, 8 weeks old, n = 8/group) were pretreated with L-T3 (5 µg/day, i.p.) for 4 days, followed by a single dose of M22 (2 µg, i.p.) or PBS control on day 0. (A) Body weight measured daily from day-3 to day 1 across experimental groups. (B) Body weight change calculated as the difference.  Neither M22 stimulation nor T3 pretreatment caused significant changes in body weight across all groups. These data indicate that acute M22 stimulation under T3-suppressed conditions does not affect body weight in this short-term experimental setting.

Fig.5 Serum T4 levels before and after single-dose M22 stimulation. WT C57BL/6 and HO hTSHR mice (female, 8 weeks old, n = 8/group) were pretreated with L-T3 (5 µg/day, i.p.) for 4 days, followed by a single dose of M22 (2 µg, i.p.) or PBS control on day 0. Blood was collected on day 0 (before M22) and day 1 (24 h post-injection). Serum T4 levels were measured by ELISA. A single dose of M22 induced a significant increase in serum T4 levels within 24 hours in both WT and HO hTSHR mice, demonstrating that M22 cross-reacts with endogenous mouse and humanized TSHR and effectively stimulates thyroid hormone production.

Fig.6 TG mRNA expression in thyroid tissues following M22 stimulation. WT C57BL/6 and HO hTSHR mice (female, 8 weeks old, n = 8/group) were pretreated with L-T3 (5 µg/day, i.p.) for 4 days, followed by a single dose of M22 (2 µg, i.p.) or PBS control. Thyroid tissues were collected 24h post-M22 injection, and TG mRNA expression was measured by qPCR. Data are shown as relative expression of M22 treated group normalized to T3 only group.

M22 stimulation significantly increased TG expression in HO hTSHR mice compared to T3-only controls. This suggests that the humanized TSHR confers specific transcriptional responsiveness to M22, enabling upregulation of TG expression under T3-suppressed conditions.