Fig 1: RA sustained the intracellular level of Asc. a The protein levels of Glut1 and Glut3 were determined by immunoblotting on day 6 during reprogramming with AscPNa. b-c The bind sites of RARA::RARG were identified on the promoters of Glut1 and Glut3 by Pscan software. Promoter assay confirmed the abilities of RA to affect the functions of the promoters of Glut1 and Glut3 in MEFs. d The intracellular level of Asc was determined on day 6 during reprogramming with N2B27 medium (24 h after replacing medium with fresh medium containing AscPNa). e-j MEFs were cultured with N2B27 medium containing 0.16 mM AscPNa or equivalent concentration of DHAA (E&H). Different concentrations of RA were also used. The intracellular levels of Asc was determined at different time points during the treatment with AscPNa (F&I) or DHAA (f), and the area under curve (intracellular levels of Asc multiple time length) were summarized in (g) and (j) respectively. k-n Different concentrations of RA were used to treat MEFs cultured in N2B27 medium in the presence or absence of 0.16 mM AscPNa (k). The expression of Glut1 (l), Glut3 (m), and Svct2 (n) was determined by qPCR. The comparisons were preformed between RA-treated groups and corresponding control (DMSO) groups or between indicated groups with one-way ANOVA except with two-way ANOVA in (c, f & j). Experiments were repeated for at least five times (n = 5). Standard deviations were provided
Fig 2: Model describing how RIP140 inhibits tumorigenesis by affecting glycolysis through the blockade of GLUT3 expression. RIP140 and p53 cooperate to inhibit the expression of GLUT3 induced by HIF-a. Glycolysis-dependent proliferation of breast cancer cells is reduced, due to a decrease in glycolysis. The prognostic value of RIP140 is associated with good survival in patients with low GLUT3, high p53 and low HIF-a (left panel). In patients with high HIF-a, low p53 and high GLUT3, RIP140 and p53 do not inhibit the transcriptional activity of HIF-a; GLUT3 is highly expressed and glycolysis is enhanced. In this sub-group, RIP140 expression level is not correlated with good survival (right panel). Double thick blue lines represent cytoplasmic membranes, double thin ones that of nucleus. The grey square represents GLUT3 gene, the grey ovoid forms represent GLUT3 protein. The orange circle represents glucose.
Fig 3: (A) Western Blot analysis of GLUT1 and ß-actin proteins. (B) Western Blot analysis of GLUT3 and ß-actin proteins. Represented Western Blots correspond to 10 µg total protein of MKN-28 cells grown on 24 well plates and incubated for 24 h with fresh RPMI medium supplemented with 5.5 mM fructose and 0.75 nM AuNP@PEG1, 0.63 nM AuNP@PEG3, 1.38 nM AuNP@PEG1 + 3, 30 nM AuNP@GLUT1, 20 nM AuNP@GLUT3, or a mixture of AuNP@GLUT1 + 3. After this 24 h period, cells were incubated for an additional 24 h with fresh medium supplemented according to the first incubation. (C) GLUT1 relative intensity values normalized to corresponding intensity of ß-actin protein and to the corresponding AuNP@PEG control sample. (D) GLUT3 relative intensity values normalized to corresponding intensity of ß-actin protein and to the corresponding AuNP@PEG control sample. Error bars represents SEM of at least three independent experiments. The grey line on the border of blots represent the place where images were cropped. Full length blot images can be found on Supplementary Fig. S2. *p-value < 0.5, **p-value < 0.005, ***p-value < 0.0005.
Fig 4: GLUT1 (A) and GLUT3 (B) relative expression in MKN-28 cells incubated for 24 h (black bars) or 24 h + 24 h (grey bars) with fresh RPMI medium supplemented with 5.5 mM fructose and 30 nM AuNP@GLUT1, 20 nM AuNP@GLUT3, or a mixture of AuNP@GLUT1 + 3. After 24 h cells were collected or incubated for an additional 24 h with fresh medium supplemented according to the first incubation. Gene expression was calculated through 2-??Ct, using as internal reference GAPDH gene, and normalized to respective control samples consisting in MKN-28 cells treated with RPMI medium supplemented with 5.5 mM fructose and 0.75 nM AuNP@PEG (control of AuNP@GLUT1), 0.63 nM AuNP@PEG (control of AuNP@GLUT3), or 1.38 nM AuNP@PEG (control of AuNP@GLUT1 + 3), and collected at the same time point. Error bars represent the SEM of at least three independent experiments. *p-value < 0.5, **p-value < 0.005, ***p-value < 0.0005.
Fig 5: Downregulation of GLUT3 inhibits STYK1/NOK-mediated cell migration. (A) NIH-3T3 stable cells were cultured under different treatment conditions for the Transwell migration assay. A light microscope was used to count the number of stained cells in random fields within each Transwell insert after 24 h (scale bar, 50 µm). (B) Quantitative analysis of cell migration. Each value is presented as the mean ± SD of three independent experiments. Following statistical analysis, results were considered to be significant if ***P<0.001. ns, no significance; GLUT, glucose transporter; siCtrl, control siRNA; siGLUT3, GLUT3 siRNA; STYK1, serine threonine tyrosine kinase 1; NOK, novel oncogene with kinase domain.
Supplier Page from Abcam for Anti-Glucose Transporter GLUT3 antibody [EPR10508(N)] - N-terminal