Knockdown of GLUT1 decreases de uptake and phototoxicity of PcGal16.
Western blotting analysis and quantification of GLUT1 protein levels in HT-1376 and UM-UC-3 cells (panel A), in HT-1376 cells (panel E) or UM-UC-3 cells (panel F) after uptake with PcGal16 in darkness and after PDT. β-actin was blotted as loading control. Quantitative analysis of GLUT1 (normalized to β-actin) expressed as a ratio of the levels found in HT-1376 cells (panel A). *(p<0.05) significantly different from HT-1376 cells. Quantitative analysis of GLUT1 (normalized to β-actin) expressed as a ratio of the levels found in untreated HT-1376 or UM-UC-3 cells (panel E, F). Data represents mean ± S.D. of five independent experiments. *(p<0.05) significantly different from untreated HT-1376 cells. Knockdown of GLUT1 in HT-1376 bladder cancer cells as determined by Western blotting 24 and 48 h post-transfection (panel B). Quantitative analysis of GLUT1 (normalized to β-actin) expressed as a ratio of the levels found in non-transfected control cells. Data represents mean ± S.D. of five independent experiments. *(p<0.05), ***(p<0.0001) significantly different from non-transfected control cells. $(p<0.05), $$$(p<0.0001) significantly different from cells treated with scrambled siRNA. Intracellular uptake of PcGal16 by HT-1376 bladder cancer cells transfected with GLUT1 siRNA (panel C). The cells were incubated with PcGal16 24 h post-transfection. Data are the mean ± S.D. of at least three independent experiments performed in triplicates. *(p<0.05) significantly different from non-transfected control cells. Photocytotoxic effects after PcGal16-PDT in UM-UC-3 cells transfected with galectin-1 siRNA (panel D). Phototoxicity was evaluated 72 h after PDT. Data are the mean ± S.D. of at least three independent experiments performed in triplicates. *(p<0.05), ***(p<0.0001) significantly different from control cells. *(p<0.05), significantly different from PDT with PcGal16 in non-transfected cells. Representative fluorescence images (panel G) of GLUT1 protein (green) in HT-1376 and UM-UC-3 cells before and after incubation with PcGal16 (red), with DAPI staining the nucleus (blue). Scale bars 20 µm.
