Fig. 2

Protein synthesis inhibition drives MondoA transcriptional activity. TXNIP mRNA levels following 16-h CHX treatments of a MondoA+/+ and MondoA−/− MEFs, and b MondoA−/− MEFs expressing empty vector or MondoA. c Immunofluorescence was used to assess the subcellular localization of MondoA in HeLa cells treated with CHX for 16 h. Cells were scored for localization of MondoA (cytoplasmic > nuclear or cytoplasmic ≤ nuclear). d Chromatin immunoprecipitation was used to determine the enrichment of MondoA on the TXNIP promoter in HeLa cells treated with CHX for 16 h. e HeLa cells transfected with the indicated reporter luciferase constructs were treated with CHX for 16 h. The ChoREmut TXNIP promoter lacks the double CACGAG carbohydrate responsive element located directly upstream of luciferase. The media was replaced with regular medium for 1 h to wash out CHX, allowing translation of accumulated luciferase mRNA. f To ensure that TXNIP levels were at a minimum, HeLa cells were starved of glucose for 6 h prior to treatment with CHX. We then measured TXNIP mRNA levels in cells growing in DMEM +10% FBS or glucose-free DMEM +10% FBS following 16-h CHX treatments. g TXNIP mRNA levels in HeLa cells growing in glucose-free DMEM +10% FBS or in glucose-free DMEM +10% dialyzed FBS following 16-h treatments. h TXNIP mRNA levels in HeLa cells growing in glucose-free DMEM +10% dialyzed FBS with the indicated amount of glucose following 16-h treatments with CHX. TXNIP mRNA levels were determined using RT-qPCR