American Society of Hematology

Figure 2

$Figure 2. Expression of constitutively plasma membrane-localized GLUT4 is necessary for glucose consumption, lactate production, growth, and viability of myeloma cells. (A) Cells were transduced with control (C), nontargeted shRNA or GLUT4-targeted shRNA and incubated 3 (L363) or 4 (JJN3, KMS11) days before protein extraction, and analysis of GLUT4 protein expression was performed. Representative blot is shown. (B) Cells from panel A were cultured in 5mM glucose-containing medium for 5 hours. Glucose consumption rates and lactate production rates were determined and normalized to control shRNA-expressing cells. (C-E) Cells from panel A were analyzed for viability and proliferation. Viable cell densities are expressed as fold change relative to the day 0 reading of control shRNA-expressing cells. (F-H) Cells from panels C through E were evaluated for viability via trypan blue exclusion. Values are normalized to control shRNA-expressing cells. (I) GLUT4 localization in CD138+ primary myeloma cells, myeloma cell lines, and NBLs was assessed via confocal immunofluorescence microscopy. Arrows indicate regions of cell surface GLUT4 immunoreactivity. Black boxes represent normal controls. Representative images are shown (n = 1 for primary samples). (J) KMS11 cells, L363 cells, and normal PBMCs were lysed for extraction of plasma membrane-associated proteins or total cellular protein content. GLUT4 immunoblot analysis was performed on the resulting fractions. Na+/K+ ATPase and GAPDH serve as loading controls. (K) Densitometric quantification of band intensities in panel G are displayed, normalized first to corresponding loading controls and subsequently to KMS11 cells. (B-H,K) Data are mean ± SEM. With exception noted in panel I, n ≥ 3 for data in panels A through K. *P < .05. **P < .01. ***P < .005.$

Expression of constitutively plasma membrane-localized GLUT4 is necessary for glucose consumption, lactate production, growth, and viability of myeloma cells. (A) Cells were transduced with control (C), nontargeted shRNA or GLUT4-targeted shRNA and incubated 3 (L363) or 4 (JJN3, KMS11) days before protein extraction, and analysis of GLUT4 protein expression was performed. Representative blot is shown. (B) Cells from panel A were cultured in 5mM glucose-containing medium for 5 hours. Glucose consumption rates and lactate production rates were determined and normalized to control shRNA-expressing cells. (C-E) Cells from panel A were analyzed for viability and proliferation. Viable cell densities are expressed as fold change relative to the day 0 reading of control shRNA-expressing cells. (F-H) Cells from panels C through E were evaluated for viability via trypan blue exclusion. Values are normalized to control shRNA-expressing cells. (I) GLUT4 localization in CD138⁺ primary myeloma cells, myeloma cell lines, and NBLs was assessed via confocal immunofluorescence microscopy. Arrows indicate regions of cell surface GLUT4 immunoreactivity. Black boxes represent normal controls. Representative images are shown (n = 1 for primary samples). (J) KMS11 cells, L363 cells, and normal PBMCs were lysed for extraction of plasma membrane-associated proteins or total cellular protein content. GLUT4 immunoblot analysis was performed on the resulting fractions. Na⁺/K⁺ ATPase and GAPDH serve as loading controls. (K) Densitometric quantification of band intensities in panel G are displayed, normalized first to corresponding loading controls and subsequently to KMS11 cells. (B-H,K) Data are mean ± SEM. With exception noted in panel I, n ≥ 3 for data in panels A through K. *P < .05. **P < .01. ***P < .005.

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