Free fatty acids, however, did result in a significant rescue. to compose Physique 4a and 4c. The active and precursor SREBP bands shown in Physique 4a and 4c were taken from the same samples, but with a different exposure. This was done in order to have and accurate detection of both the active and the precursor form. Panels (a-d) show composition of panel a of Physique 4 (SREBP1 data), panels (e-h) show composition of panel c of physique 4 (SREBP2 data).(TIF) pone.0106913.s003.tif (1.4M) GUID:?C1DEE3BB-2794-4BD7-A863-228496F360F2 Physique S4: Lipid-reduced (LR) growth conditions do not change the expression and nuclear translocation of ChREBP. (a) ChREBP expression at protein level was analyzed by western blot analysis in HepG2, PC3M, HOP62 and T24 cells, cultured for 72 hours in normal (N) or LR growth conditions. Human liver was used as a positive control for ChREBP detection. Beta-actin was used as a loading control. (b) ChREBP translocation to the nucleus was determined by western blot analysis of cytosolic and nuclear fractions of HepG2, PC3M and T24 cells, cultured for 72 hours in normal (N) or LR growth conditions. Total cellular extract of human liver was used as a positive control for ChREBP detection. Alpha-tubulin was and lamin A/C were used as a loading controls.(TIF) pone.0106913.s004.tif (841K) GUID:?9824A21A-7A43-4746-BFC5-8E502F7CE41F Physique S5: Addition of very-low density lipoproteins (VLDL), free fatty acids and cholesterol to lipid reduced (LR) growth conditions reverses the increased expression of SREBP1 and SREBP2 in T24 cell line. Gene expression levels of SREBP-1a, SREBP-1c and SREBP-2 were analyzed by qPCR analysis in T24 cells cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL (a), different fatty acid mixtures (b) and different concentrations cholesterol (c). VLDL was added at a concentration of 607 g triglycerides/ml serum (corresponding to the concentration triglycerides in normal FBS). Fatty acid (FA) mixtures were as follows, FA Mix 1: 20 M linoleic (182), 20 M Pipemidic acid -linolenic (183), 5 Pipemidic acid M arachidonic (204), 5 M docosahexaenoic acid (226), FA Mix 2: 10 M 182, 15 M 183, 10 M 204, 15 M 226 and FA Mix 3: 20 M 182, 20 M 183, 5 M 204, 5 M 226, 30 M oleic acid, 30 M palmitic acid. Different cholesterol (Ch) concentrations are as indicated in the figures (25 M, 50 M or 100 M). Data are normalized to 18S and represented as mean S.D. (triplicate per experiment and n?=?3). *Significantly different (*p0,05; **p0,01; ***p0,001) from normal Pipemidic acid medium control. #Significantly different (#p0,05; ##p0,01; ###p0,001) from LR medium control.(TIF) pone.0106913.s005.tif (126K) GUID:?F0693DB0-C9BD-4384-ACA1-6717BA6C7321 Physique S6: Addition of triglycerides (TG) in combination with recombinant lipoprotein lipase (LPL) to lipid-reduced (LR) growth conditions reverses the increased activation of the lipogenic pathway. Pipemidic acid (a) T24 cells were cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of different lipid mixtures. Composition of lipid mixtures were as follows: Mix a: 20 M linoleic (182) and 20 M -linolenic acid (183) and Mix b: 44 g/ml glyceryltrilinoleate and 44 g/ml glyceryltrilinolenate. LPL was added at a concentration of 10 g/ml. Mouse Monoclonal to Rabbit IgG During the last 4 hours of culturing 14C-acetate was added and the incorporation of radioactivity in cellular lipids was normalized to sample DNA content. Representative experiment is usually shown, experiment was repeated two times. Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. *Significantly different (**p0,01; ***p0,001) from normal medium Pipemidic acid control. #Significantly different (###p0,001) from LR control.(TIF) pone.0106913.s006.tif (21K) GUID:?6A613A3E-1793-4925-B09B-EE3F0841CA05 Table.