Fig. 5

1,25(OH)₂D₃ induces proteasomal degradation of TXNIP in MCF-7 cells. a The reduction in TXNIP protein levels by 1,25(OH)₂D₃ (100 nM) in MCF-7 cells is rescued by MG-132 (5 μM) or 2-deoxyglucose (10 mM), but not leupeptin (20 μM). The various molecules were added to the conditioned medium of DMSO- and 1,25(OH)₂D₃-treated (66 h) MCF-7 cells, for an additional 6 h. b, c ITCH mRNA and protein expression is not markedly influenced by 1,25(OH)₂D₃. Relative expression was calculated using the ∆∆Ct method with vinculin as the housekeeping gene. Error bars ± SD; n > 3. d Overall protein ubiquitination in MCF-7 cells was not changed by 1,25(OH)₂D₃ treatment. e Co-immunoprecipitation studies illustrate that the TXNIP-ITCH interaction is not altered by 1,25(OH)₂D₃ treatment of MCF-7 cells. f Negative regulation of TXNIP protein expression by 1,25(OH)₂D₃ is observed in MCF-7 cells with knocked-down AMPKα1 levels. g The non-calcemic 1,25(OH)₂D₃ analogue, calcipotriol (100 nM; 72 h) induces similar effects on TXNIP expression as 1,25(OH)₂D₃. The cell permeable Ca²⁺ chelator BAPTA-AM (20 μM) does not hamper 1,25(OH)₂D₃’s effects on TXNIP expression. BAPTA-AM was added to the conditioned medium of DMSO- and 1,25(OH)₂D₃-treated MCF-7 cells, 2 h prior to the end of the treatment period (72 h)