Dexamethasone and phloretin were purchased from Sigma-Aldrich (St. Louis, MO) Cells were routinely cultured in RPMI1640/10%FBS/5 mM glucose. For chronic hyperglycemia conditions, cells were chronically grown in RPMI 1640/10% FBS containing 20 mM glucose. For dexamethasone response cells were cultured in either 5 or 20 m chronically

and dexamethasone (25 uM) added to media for 24 hours prior to harvest. Glucose uptake inhibition studies were accomplished by adding phloretin (200 uM) to media and cells harvested after 24 hours. TXNIP RT-PCR, ROS assay and TRX activity All experiments BIBW2992 mw were run in triplicate for analysis. Cells were harvested and each sample split into three aliquots for RNA isolation, ROS and TRX activity analysis. Total RNA was isolated using Aquapure RNA isolation kit (Bio-Rad, Hercules, CA) and first strand c-DNA synthesis by iScript c-DNA amplification kit (Bio-Rad) according to manufacture’s protocol. Primers and PCR conditions were as previously described [5]. We have previously shown that increased RNA correlates with level of TXNIP protein [5]. ROS were detected

by 5-6-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) and measured for mean fluorescence intensity by flow cytometry as previously described [5]. TRX-activity was assessed by the insulin disulfide assay as previously described [5]. Fold-change (> 1 versus < 1 fold increase/decrease, 1 = no change) was obtained for each cell line. Cell lines which showed response LXH254 concentration (NCIH929, ARH77, U266B1) were further grouped and compared to non-responsive MC/CAR cell line. Dexamethasone IC50 calculation IC 50 were calculated by the method of Chou and Talalay using Calcusyn software (Biosoft, Cambrigdge UK) Statistical analysis Differences between treatments were Methamphetamine evaluated by ANOVA or student’s t-test and accepting as significant differences if p < 0.05. Results Differences in TXNIP-ROS-TRX axis-response to hyperglycemia in MM cells We assessed the TXNIP RNA level, ROS production and TRX activity in response to isolated hyperglycemia. The function of TXNIP as a modulator of the redox system

through the binding of the TRX active cysteine residues has been elucidated [7, 8]. Furthermore, the promoter region of the TXNIP gene buy H 89 contains carbohydrate responsive elements (ChoRE) conferring the responsiveness of the gene directly to glucose [9, 10]. We have also recently shown that there is strong correlation between TXNIP RNA and TXNIP protein level to justify our decision to assess only RNA levels in the cells [5]. Hyperglycemia [20 mM versus 5 mM glucose] significantly affected the fold-change of increased levels of TXNIP RNA level (mean 1.37 ± 0.17) and ROS level (mean 1.70 ± 0.25) in NCIH9292, ARH77 and U266B1 cells (Figure 1A). As expected TRX activity concurrently declined an average of 0.77 ± 0.12 in the same cell lines (Figure 1C).