Fig 1.
2NBDG uptake is unaffected by Slc2a1 deletion in 5TGM1 cells.
(A) GLUT1 staining of gRNA-transduced cells. Representative histogram (left) showing GLUT1 expression on 5TGM1 cells transduced with a control gRNA (black) and a Slc2a1-targeting gRNA (blue). Quantification of percent GLUT1 positive cells (right) in control and Slc2a1 gRNA-transduced cells across 9 independent experiments. Each circle represents a single group from a single experiment. ***p<0.0001 by Brown-Forsyth and Welch one-way ANOVA multiple comparisons test. (B) 14C-glucose uptake in gRNA-transduced cells. Lysates from cells transduced with gRNAs as in (A) were cultured in 14C-glucose containing media for 30 minutes and 14C signal was quantified in the cell pellet. Background counts indicated by the dotted line. Representative graph of three independent experiments. *p<0.05 by Brown-Forsyth and Welch one-way ANOVA multiple comparisons test. (C) Flow cytometric analysis of 2NBDG uptake in control gRNA- (black) and Slc2a1 gRNA-transduced (colored) 5TGM1-Cas9 cells. Cells were cultured in media containing 2NBDG for 60 minutes. Histogram representative of three independent experiments. (D) 2NBDG uptake in control gRNA (black) and Slc2a1 gRNA-transduced (colored) 5TGM1-Cas9 cells. Mean fluorescence intensity (MFI) +/- SEM shown for 0-, 15-, 30-, 45-, and 60-minutes post culturing with 2NBDG. Pooled data from three independent experiments. No significant differences were observed with ordinary two-way ANOVA with Dunnett’s multiple comparison test.
Fig 2.
Plasma cells take up 2NBDG and retain it in the cytosol.
(A) 2NBDG is retained in the cytosol of cells. 5TGM1 cells previously transduced with a Cas9-T2A-mCherry lentivirus were treated with 2NBDG, stained for surface CD138 and Hoeschst 33342, and analyzed by imaging flow cytometry. Representative images of cells treated with 2NBDG (top left) relative to untreated cells (bottom left) at 60x magnification are shown. Mean similarity morphology indices for 2NBDG and other stains/dyes were quantified and shown (right). (B) Cas9-mCherry expressing 5TGM1 cells were transduced with control gRNA or Slc2a1 gRNA lentiviruses. Representative images of each gRNA group (left) at 60x magnification are shown with overlays for 2NBDG/CD138 and 2NBDG/mCherry. Mean similarity morphology indices were quantified (right). For both panels, each dot indicates the mean value for a group in a single experiment. No significant differences were observed with ordinary two-way ANOVA with Dunnett’s multiple comparison test.
Fig 3.
Plasma cells specifically transport 2NBDG.
(A) Mice were injected with 100μg of either 2NBDG or 1NBDF and assessed for NB fluorescence in splenic and bone marrow plasma cells. Representative flow cytometry plots (left) on splenic (top row) and bone marrow (bottom row) CD138+ plasma cells showing gated percent NB-positive cells. Quantification of NB-positive plasma cell percentages (left) in the spleen and bone marrow and groups from each mouse are connected by a line. Data from three independent experiments with n = 8 mice in both groups. ****p<0.0001 by ordinary two-way ANOVA with post hoc Šídák’s multiple comparison test. (B) Freshly isolated ex vivo plasma cells (CD138+ B220+/-, orange), B cells (CD138- B220+, purple), and total spleen cells (green) as well as cultured 5TGM1 cells (black) were examined for 2NBDG uptake by flow cytometry. Pooled data from three independent experiments shown as mean+/-SEM for mentioned time points.
Fig 4.
The kinetics of 2NBDG uptake is unaffected by GLUT1 inhibition or deletion.
(A) 5TGM1 cells were treated with DMSO, 30μM Cytochalasin B (CytoB), 10μM BAY-876, or 100μM WZB-117 for 30 minutes in complete media followed by 60μM of 2NBDG for another 30 minutes. Mean +/- SEM of 2NBDG MFI are shown as bar graphs. Pooled data from three independent experiments. No significance was observed by one-way ANOVA. (B) 5TGM1 cells were treated with 300nM Carbenoxolone (CBX) and assayed for 2NBDG uptake as in (A). Mean +/- SEM of 2NBDG MFI are shown as bar graphs. Pooled data from three independent experiments. No significance observed by the paired t-test. (C) Control gRNA- or Slc2a1 gRNA-transduced 5TGM1-Cas9 cells were administered 2NBDG at a final concentration of 60μM and intensity monitored immediately after addition by flow cytometry. Mean +/- SEM is shown for each group for the mentioned time points. Pooled data from three independent experiments.
Fig 5.
2NBDG uptake takes place independently of sugar transporters.
(A) Transcripts per million kilobase (TPM) values of sugar transporters in ex vivo bone marrow plasma cells (orange) and 5TGM1 cells (black). Mean values +/- SEM are shown. (B) Quantification of gene modifications in gRNA-transduced cultures. Exons of indicated genes were PCR amplified and sequenced to assay for in-frame and frame shift mutations. Mean values +/- SEM shown for each of the genes and modifications within it. Pooled data from three experiments. (C) 2NBDG uptake in sugar transporter deleted cultures. 5TGM1-Cas9 cells were transduced with control gRNA (black) or gRNAs targeting Slc2a3 (pink), Slc2a5 (blue), Slc2a6 (violet), Slc2a8 (purple), and Slc50a1 (cyan). MFIs across three independent experiments are quantified and displayed with SEM. No significant differences observed with Brown-Forsyth and Welch one-way ANOVA multiple comparisons test. (D) Frequency of 2NBDG-negative cells for groups in (C). 2NBDG- gate drawn based on control cells cultured without 2NBDG in complete media. Mean values +/- SEM are shown. No significant differences observed with Brown-Forsyth and Welch one-way ANOVA multiple comparisons test.
Fig 6.
Combined deletion of sugar transporters does not affect 2NBDG uptake.
(A) Quantification of in-frame and frameshift mutations in Slc2a1, Slc2a3, Slc2a5, Slc2a6, and Slc2a8 genes of the Slc2a1/3/5/6/8 deleted cultures. Pooled data from three experiments. (B) 2NBDG uptake in Slc2a1/3/5/6/8 cultures. Representative histogram (left) showing control gRNA- (black) and the five gRNA-transduced cultures (green). Pooled MFI +/- SEM for both groups across three independent experiments are depicted (right). No significance observed with the paired t-test. (C) Frequencies of 2NBDG-negative cells in cultures described in (B). 2NBDG- gating carried out as in Fig 5D. Mean values +/- SEM are shown for both groups. No significance observed with the paired t-test.
Fig 7.
2NBDG uptake in cells is independent of competing glucose.
(A) 5TGM1 cells were resuspended in either glucose-free RPMI or RPMI with D-glucose at the mentioned final concentrations. 2NBDG was added at a final concentration of 60μM and intensity monitored immediately after addition by flow cytometry. (B-D) CD138+ enriched spleen cells were resuspended in glucose-free RPMI or RPMI with D-glucose at the specified concentrations. 2NBDG was added and intensity monitored immediately by flow cytometry. 2NBDG intensity in (B) plasma cells (CD138+ B220+/-), (C) B cells (CD138- B220+), and (D) unenriched total spleen cells are shown for the mentioned time points. Mean +/- SEM shown for each group for the indicated time points. Pooled data from three independent experiments each.
Fig 8.
2NBDG is not imported through nucleoside or nucleoside-sugar transporters.
(A) TPM values of nucleoside and nucleoside-sugar transporters in ex vivo bone marrow (orange) plasma cells and 5TGM1 cells (black). Mean values +/- SEM are shown. (B) Estimation of indels in nucleoside and nucleoside-sugar transporter deleted cultures done as in Fig 5B. Mean values +/- SEM shown for each of the genes and modifications within it. Pooled data from three independent experiments. (C) 2NBDG uptake in deleted cultures. 5TGM1-Cas9 cells transduced with gRNAs targeting indicated nucleoside or nucleoside-sugar transporters (colored) or with a control gRNA (black). 2NBDG MFIs across three independent experiments are quantified and displayed with SEM. (D) Frequencies of 2NBDG negative cells in cultures described in (C). Pooled data from three experiments. No statistical significance observed with the Brown-Forsyth and Welch one-way ANOVA multiple comparisons test.
Fig 9.
2NBDG transport is a low affinity process.
(A) 2NBDG was added to 5TGM1 cells resuspended in 1xPBS to the specified final concentrations and uptake measured by flow cytometry. (B) 5TGM1 cells were resuspended in either DMEM or Sodium-free MEM and measured for 2NBDG import by flow cytometry. Mean +/- SEM shown for each group for the indicated time points. Pooled data from three independent experiments each.