Orbital adipose tissue was obtained with informed consent and relevant local ethical approval. The research adhered to the tenets of the Declaration of Helsinki.
Six tissue samples were collected, 3 from GO patients (undergoing decompressive surgery) and 3 from individuals free of GO and thyroid autoimmunity (undergoing blepharoplasty with fat pad excision).
Preadipocytes were obtained from explants of the adipose tissues, cultured in DMEM/F12 10% FCS (complete medium). Adipogenesis was induced in confluent cells by replacing with medium having reduced FCS and containing a range of hormones and PPARγ agonists (differentiation medium), for 10 days, as previously described (11 (link)).
Activating mutant TSHR, L629F and M453T, and the WT were introduced using retroviral vectors, previously produced in our laboratory (12 (link)). Geneticin selection resulted in mixed populations stably expressing the various TSHR, all as previously described (12 (link)).
Six tissue samples were collected, 3 from GO patients (undergoing decompressive surgery) and 3 from individuals free of GO and thyroid autoimmunity (undergoing blepharoplasty with fat pad excision).
Preadipocytes were obtained from explants of the adipose tissues, cultured in DMEM/F12 10% FCS (complete medium). Adipogenesis was induced in confluent cells by replacing with medium having reduced FCS and containing a range of hormones and PPARγ agonists (differentiation medium), for 10 days, as previously described (11 (link)).
Activating mutant TSHR, L629F and M453T, and the WT were introduced using retroviral vectors, previously produced in our laboratory (12 (link)). Geneticin selection resulted in mixed populations stably expressing the various TSHR, all as previously described (12 (link)).
