Figure 1: Induction of LASY expression and Lipoic acid (LA) levels in HSMM treated with compound 1
A: Induction of LASY mRNA levels in HSMM treated with compound 1. Cells were grown and differentiated in 96 well plates and treated with either DMSO, 50, 100 or 250 μM of compound 1 in treatment medium (HSMM basal medium +1 % fetal bovine serum (FBS)). Treatments were done for 18 hours at 37°C/5 % CO2. Cells were lysed in lysis buffer and RNA extracted using Absolutely RNA 96 well RNA isolation kit (Stratagene, Inc., La Jolla, CA). Real time PCR quantification was done using the SYBR green real time PCR kit (Stratagene). Ct values obtained from LASY primers were corrected for Ct values from Actin primers. Fold changes compared to DMSO control are shown. Results shown are averages of three independent experiments, and represent means ± standard deviation (SD). P values were calculated using one way ANOVA with post-hoc testing. *P=0.0000027 for compound-1 100 μM versus DMSO control; P=0.0000003 for compound 1 250 μM versus DMSO control
B: Induction of LASY promoter by compound 1. HSMM were grown and differentiated in 24 well plates. A pGL3 construct containing the 200 base pair LASY promoter region (pGL3-200) was transfected into the differentiated cells for 24 hours, followed by treatment with either DMSO or 250 μM of compound 1 in treatment medium (HSMM basal medium +1 % fetal bovine serum (FBS)). A pGL3 control vector was used as transfection control. Luciferase expression was quantitated in terms of luminescence. All values were normalized for protein content. Values obtained for pGL3-200 were subtracted from values obtained for the pGL3 control vector to correct for background luminescence. Results shown are averages of three independent experiments and represent means ± SD. P values were calculated using a two-sample, paired t-test. *P=0.007 for compound 1 versus DMSO control
C: Induction of PDC-associated LA levels in HSMM treated with compound 1. Cells were grown and differentiated in 24 well plates and treated as described above for RNA. Cells were lysed in T-PER protein lysis buffer (Pierce, Inc.) containing protease inhibitors. Protein was quantitated using a BCA kit (Pierce). LA ELISA was used to quantitate LA amounts in samples according to the protocol described in Supplementary methods. LA concentration associated with PDC (μg/ml) is shown. Results shown are averages of three independent experiments, and represent means ± SD. P values were calculated using one way ANOVA with post-hoc testing. *P=0.01 for compound-1 100 μM versus DMSO control; P=0.0001 for compound 1 250 μM versus DMSO control