Figure 2

Analysis of mRNA expression of glucose transporters and glutamine metabolizing enzymes by qRT-PCR and ASCT2 translocation in MCF-7 cells. (A) mRNA expression analysis of glucose transporters (GLUT) 1, 6, 8, 10, 11, 12 and 13 related to the housekeeping gene RPL37A. Data are presented as a mean of n = 2–5 ± SEM. Unpaired t test with Welch’s correction. (B) mRNA expression analysis of the transporters alanine-serine-cysteine transporter 2 (ASCT2), glutamate/cystine antiporter (xCT), mitochondrial aspartate-glutamate-carrier (Aralar), glutamate carrier (GluR1), glutamate dehydrogenase (GLUD) and L-type amino acid transporter 1 (Lat1) related to the housekeeping gene RPL37A. Data are presented as a mean of n = 3–4 ± SEM. Unpaired t test with Welch’s correction. (C) mRNA expression analysis of the GSH synthesis key enzymes glutaminase (GLS) and GLS2, glutathione reductase (GSR), glutathione synthase (GSS), glutamate-cysteine-ligase (GCLC) and the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) related to the housekeeping gene RPL37A. Data are presented as a mean of n = 3–5 ± SEM. Unpaired t test with Welch’s correction. (D) Glycosphingolipid-enriched microdomains (GEMs) were isolated by sucrose density gradient centrifugation and GEM containing fractions 2 and 3 were identified by cholesterol and caveolin-1 content (data not shown,⁴). The ASCT2 protein concentration of the fractions was determined by Western blot analysis and related to Ponceau S staining. One representative blot of three is displayed. Data are presented as a mean of n = 3 ± SEM. Unpaired t test with Welch’s correction. (E) Immunohistochemistry of formaldehyde-fixed paraffin-embedded (FFPE) human breast cancer tissue surrounded by non-cancerous tissue. Sections were incubated with an anti-ASCT2 antibody and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain DNA. Images were recorded by Axio ObserverZ.1 microscope (Carl Zeiss AG, Oberkochen, Germany). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.