Figure 6.

Repression of HCRT gene expression in hiONs by hyperglycemia in vitro. (A-J) HCRT gene expression in hyperglycemic condition. hiONs established by treatment with ManNFAc for 20 d were used (see Fig. S3A). HCRT expression was measured during hyperglycemia in vitro for 12 d by RT-qPCR (A). Values were normalized to ACTB expression. Means ± SD (n = 3). Relative values were expressed, with the expression of day 0 equal to 1.0. HCRT expression in hiONs under euglycemic or hyperglycemic conditions for 8 d (days 0–8) (B) or 4 d (days 8–12) (C). On days 0, 8, and 12, the cells were subjected to the TUNEL assay (D). Treatments with ManNAc (10 μM) and ManNFAc (1 μM) in hyperglycemic conditions were effective in maintaining HCRT gene expression during the sensitive phase (E) and in the reactivation during the refractory phase (F). The ManNAc derivatives (5S-ManNAc, 5S-ManNAcF, ManNCOOMe, and ManNCOOEt) were also effective during both phases, but the pharmacological spectrum was different between the phases (G and H). Treatments with EX-527 and BADGP, in hyperglycemic conditions were effective in maintaining HCRT gene expression during the sensitive phase (I). Treatments with TSA (200 nM), but not Zebularine (100 μM), EX-527 and BADGP, were effective in the reactivation of HCRT expression during the refractory phase (J). (K) Summary of pharmacological activities of ManNAc and its derivatives.