Fig 2: (A) Western blot analysis for the expression of GLUT3 in control and human glioma U251 cells treated with siRNA-SP1 (10 and 20 pmol), shRNA-GLUT3 (10 and 20 pmol) and SP1-inhibitor (300 nM). Notably, GLUT3 expression is decreased following treatment with siRNA-SP1, shRNA-GLUT3 and SP1-inhibitor. (B) GLUT3 relative mRNA level. GLUT3 mRNA expression was detected using quantitative polymerase chain reaction in control and U251 cells treated with GLUT3 shRNA (5, 10 and 20 pmol), SP1 siRNA (10 and 20 pmol) and SP1-inhibitor (300 nM). (C) Glucose uptake in U251 cells. Uptake of-2-DG in control and U251 cells treated with GLUT3 shRNA (5, 10 and 20 pmol), SP1 siRNA (10 and 20 pmol) and SP1-inhibitor (300 nM). Data are the means of three independent experiments performed in triplicate. Error bars indicate the standard deviation from the mean; *P<0.05;**P<0.01 vs. control. GLUT3, glucose transporter 3; control, untreated U251 cells; siRNA, small interfering RNA; SP1, specific protein 1; shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SP1-inhibitor, mithramycin A; mRNA, messenger RNA; 2-DG, [3H]-2-deoxyglucose; pm, picomole.

Fig 3: (A) Assay for promoter responsiveness to SP1. Human glioma U251 cells were co-transfected with the indicated luciferase reporter plasmids, and control or siRNA-SP1. Luciferase activity was analyzed 24 h later. (B) Effect of SP1 binding to GLUT3 chromatin. Extracts from U251 cells were used for chromatin immunoprecipitation followed by quantitative polymerase chain reaction. Data represent the means of three experiments. Error bars indicate the standard deviation from the mean; *P<0.05;**P<0.01 vs. control. GLUT3, glucose transporter 3; pGL3-GLUT3-WT, wild-type reporter; pGL3-GLUT3-MUT, mutated reporter; pGL3-Basic, control; SP1, specific protein 1; siRNA, small interfering RNA; anti-SP1, antibody to SP1

Fig 4: (A) Glut3-IR in cytotypes of normal and cryptic gonad of dogs. (a,g) A large group of Leydig cells positive to Glut3 in NT and CT (asterisk) were found; (b,h) Glut3-IR were described in the apical and basal portions of the cytoplasm of Sertoli cells (arrowhead) in NT and CT; (c,d) few spermatogonia (c) and resting or pre-leptotene spermatocytes (d) positive to Glut3 were found along the basal membrane of the testicular tubule of NT (arrow and double arrow, respectively); (e,f) round or immature (e) and elongated or mature (f) spermatids were localized toward the tubular lumen (hashtag) in TN. Bar: 50 µm. (B) Western blot analysis of Glut3 protein expression level in the canine NT, CLT, and CT. Samples from rat testis (RT) were blotted as positive controls. In all the examined tissues, Glut3 appears as a band at ~ 56 kDa. A significative increase in Glut3 expression is observed in CT respect to NT and CLT (densitometric analysis). Data are expressed as mean ± SD (n = 5 samples/group), *p < 0.01.

Fig 5: AMP-activated protein kinase signaling and GLUT3 trafficking were manipulated in an in vitro trophoblastic GDM model. HTR8/SVneo cells were treated with 11.1 mM D-glucose + 18.9 mM mannitol (osmotic control), 30 mM glucose (high glucose) or 11.1 mM D-glucose (control) and were then subjected to Western blotting analyses of (A) the p-AMPK and AMPK levels (n = 6), (B) the p-ACC and ACC levels (n = 6), (C) the Ki67, p-Akt and Akt levels (n = 6), (D) the total GLUT3 and HIF-1a protein levels (n = 4) and (E) the membranous GLUT3 levels (n = 4). PM GLUT3 was normalized to GAPDH. (F) Representative fluorescence images from each group after incubation with 2-NBDG for 30 min are shown. Bar: 400 µm. The fluorescence intensity of 2-NBDG taken up by HTR8/SVneo cells was measured by flow cytometry at 488/530 nm (n = 4). The intensity of each experiment was calculated using flow cytometry software, and the intensity was normalized to the cell number. (G) p-AMPK and AMPK levels in HTR8/SVneo cells treated with vehicle or 100 µM AICAR for 24 h in the presence of a high or normal concentration of glucose (n = 6). (H) Subcellular GLUT3 distribution after the treatments (n = 4). PM GLUT3 was normalized to ß-actin. (I) Total GLUT3 protein levels (n = 6). (J) Representative fluorescence images of 2-NBDG obtained from each group after 24 h of treatment with vehicle or AICAR. Bar: 400 µm. The fluorescence intensity of 2-NBDG was measured at 535/485 nm by flow cytometry (n = 4). The analysis was in line with that presented in panel (F). The data that are presented as the means ± SEMs are annotated in normal font, and ordinary ANOVA test was used among multiple groups. The data presented as the medians (IQRs) are shown in bold font, and Kruskal-Wallis test was used among multiple groups. *p < 0.05, **p < 0.01, and ***p < 0.001.