Fig 1: Analysis of the glucose transporters GLUT1-3 in the primary uveal melanomas (UMs) by immunohistochemistry. (A) Representative images from the immunostainings for GLUT1-3. Signal detection was performed using the horseradish peroxidase-green substrate, which generates a blue-green reaction product. The nuclei were counterstained with nuclear fast red. The strength of the immunohistochemical reaction within a circumscribed area was graded using a scale of 0–3 (0: negative. 1: weak. 2: intermediate. 3: strong). The mean intensity of the entire tumor area was determined by a semiquantitative approach as described in the Methods section. Images were acquired at an original magnification of 200×. Arrows indicate the pigmented regions. Scale bar = 25 µm. (B) Immunoblots with GLUT1-3 peptides or full-length recombinant human GLUT3 protein (f), demonstrating the specificity of the antibodies used. The total protein amount in the wells was detected by the stain-free imaging of gels after ultraviolet-activation. The white bands (arrows) correspond to the bromophenol blue dye in the loading buffer, which has not migrated out of the gels. kDa: Kilodalton. (C) Quantification of the GLUT1, GLUT2, and GLUT3 intensities in the primary UMs with regard to the monosomy-3 status. The sum of the GLUT1-3 intensities was also presented as “total”. p-values were determined by the Mann-Whitney U test. (D) Ratios of GLUT1, GLUT2, and GLUT3 to the total GLUT1-3 levels in the primary UMs with respect to the monosomy-3 status. p-values were analyzed by the Mann-Whitney U test. The significant p-values (<0.05) in (C,D) were stated in red.
Fig 2: Expression of the glucose transporters GLUT1, GLUT2, and GLUT3 in the irradiated uveal melanoma (UM) samples. (A) Representative images from the immunohistochemistry for GLUT1-3. The disomy-3 tumor originates from a male patient (60 years), who remained metastasis-free during the follow-up, whereas the monosomy-3 tumor was obtained from another male patient (52 years), who has developed liver metastases. Irradiation was performed two months prior to the acquisition of both tumors. Images were collected at an original magnification of 200×. Arrows indicate varying degrees of pigmentation. Scale bar = 25 µm. (B) Immuno-Fluorescent in situ hybridization (Immuno-FISH) analysis, demonstrating the copy number of chromosome-3 (red) in the UM cells that were expressing the melanoma marker Melan-A (green). The nuclei were counterstained in blue with DAPI. Arrows indicate several cells with monosomy-3. The percentage (%) of monosomy-3 cells and the chromosome index of these tumors were presented underneath the respective images (please see the Methods for the detailed description of the calculation of these parameters, n: number). Scale bar = 10 µm. Quantification of the intensity and ratio of (C) GLUT1, (D) GLUT2, and (E) GLUT3 in the irradiated primary UMs with regard to the monosomy-3 status. (F) The sum of the GLUT1-3 intensities was presented as “total.” p-values were determined by the unpaired, two-sided t-test. The significant p-values (<0.05) were highlighted in red. a.u.: Arbitrary units.
Fig 3: Expression of the glucose transporters GLUT1, GLUT2, and GLUT3 in the non-irradiated uveal melanoma (UM) samples. (A) Representative images from the immunostainings for GLUT1-3. The disomy-3 tumor was obtained from a male patient (69 years), who did not develop metastases during the follow-up, whereas the monosomy-3 tumor was removed from a female patient (39 years), who presented with bone metastases that have subsequently spread to the brain. Images were acquired at an original magnification of 200×. The black arrows indicate the pigmentation whereas the white arrows with a dark contour point out the staining of the plasma membrane. Scale bar = 25 µm. (B) Immuno-Fluorescent in situ hybridization (Immuno-FISH) was performed to detect the copy number of chromosome-3 (red) in the UM cells that were positive for the melanoma marker Melan-A (green). The nuclei were counterstained in blue with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI). The chromosome 3 status of the tumors was evaluated by determining the percentage (%) of cells with monosomy-3 and the chromosome 3-index as described in the Methods section. The values obtained for these parameters in the previously mentioned tumors are stated underneath the respective image panels (n: number). Arrows indicate several examples of cells with monosomy-3. Scale bar = 10 µm. Quantification of the intensity and ratio of (C) GLUT1, (D) GLUT2, and (E) GLUT3 in the primary UMs with regard to the monosomy-3 status. (F) The sum of the GLUT1-3 intensities was also presented as the “total.” p-values were determined by the unpaired, two-sided t-test. The significant p-values (<0.05) were indicated in red. a.u.: Arbitrary units.
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