Figure 4. Sufficient glutamine catabolites are required to maintain or enhance GFAT1 expression during glucose starvation.
A. Growing WT and SIN1−/− MEFs were resuspended in culture medium with (+) or without (−) glucose for the indicated number of hours. Cells were lysed in CHAPs buffer and cytosolic extracts were fractionated by SDS-PAGE and immunoblotted for the indicated antibodies. See also Figure S3C, S5B–C.
B. WT or SIN1−/− MEFs were grown as in A. qRT-PCR was performed on Gfat1, with tubulin as internal control. Gfat1 mRNA levels relative to basal levels from WT MEFs (1 hr in complete media), are averaged and plotted (n=3). Error bars indicate SD.
C. HeLa cells were grown and processed as in A. See also Figure S3D, S5A, S5D.
D. Growing WT or SIN1−/− MEFs were resuspended in complete media or media lacking both glucose and glutamine for the indicated time (hr). Cytosolic extracts were fractionated by SDS-PAGE and immunoblotted for the indicated proteins. See also Figure S3E, S5E.
E. Growing HeLa cells were resuspended in complete media (+) or media lacking either glucose or glutamine or both for the indicated hours. Cytosolic extracts were isolated using NE-PER kit. See also Figure S3F.
F–G. Growing HeLa cells were resuspended in complete media with (+) or without (−) glucose at the indicated hours. At the last hour of starvation (either after 1 or 17 hr), glutamine (Q) was re-added at the indicated concentrations (mM). Cell extracts were analyzed for protein levels by immunoblotting (F) or mRNA levels by qRT-PCR (G). For G, error bars represent SD; n=3; * indicates p<0.05.
H. Growing HeLa cells were resuspended in media with (+) or without (−) glucose and BPTES (25μM) for the indicated time. Cells were lysed in CHAPS-based detergent. See also Figure S5F.