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Results: 7

1.
Figure 2

Figure 2. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

A) OGT overexpression dose-dependently improves post-hypoxic cardiac myocyte survival. LDH release was spectrophotometrically determined from the media following hypoxia-reoxygenation (n=6/group). B) OGT overexpression attenuates post-hypoxic injury according to propidium iodide staining. *p<0.05 vs. 0 MOI AdOGT or 100 MOI AdGFP.

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.
2.
Figure 6

Figure 6. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

Assessment of sensitivity to loss of mitochondrial membrane potential in NRCMs following inhibition of OGT (TT04) during hypoxia. TMRM fluorescence was used to indicate mitochondrial membrane potential (n=6/group) beginning at post-hypoxic reoxygenation and continuing through the first hour of reoxygenation. A) In post-hypoxic cardiomyocytes, OGT overexpression improved the recovery of mitochondrial membrane potential compared to Hypoxia + AdGFP. Quantification of the change in relative fluorescent intensity (left) and representative montages (right) are shown. B) In post-hypoxic myocytes, OGT inhibition (via TT04) repressed the recovery of of mitochondrial membrane potential compared to Hypoxia + Vehicle. Quantification of the change in relative fluorescent intensity (left) and representative montages (right) are shown.

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.
3.
Figure 4

Figure 4. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

Effects of OGT gene knockdown on post-hypoxic NRCM survival (n=6/group). A) NRCMs treated with fluorescently tagged scrambled (Scr) control RNAi showing a transfection efficiency of more than 90%. B) Knockdown of OGT significantly reduces OGT mRNA levels according to qRT-PCR in OGT siRNA-treated compared with scrambled control siRNA-treated NRCMs. C) Knockdown of OGT significantly reduces OGT protein levels according to immunoblotting in OGT siRNA-treated compared with scrambled control siRNAtreated NRCMs. D) Whole cell lysates from OGT siRNA-treated NRCMs showed a significant decease in O-GlcNAc levels compared with those from scrambled control siRNA-treated NRCMs according to western blotting. E) NRCMs treated with OGT siRNA were more sensitive to hypoxia-induced injury (according to LDH release) compared with NRCMs treated with scrambled control siRNA. *p< 0.05 vs Scr siRNA.

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.
4.
Figure 5

Figure 5. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

Effects of OGT deletion on survival following hypoxia in cardiac myocytes. NMCMs were infected with AdCre 72 hours prior to protein harvest or H/R. A) Representative genotyping PCR results showing loxP-flanked or WT OGT gene. Cardiac myocytes from homozygous litters were exposed to AdCre or no virus. There was a significant (p<0.05) increase in Cre-recombinase expression (panel B) and a significant (p<0.05) decrease in OGT levels (panel C) with 50 MOI compared with 0 MOI AdCre (n=4/group). D) O-GlcNAc levels were markedly decreased at 50 MOI compared with 0 MOI AdCre (n=4/group). E) LDH release was significantly (p<0.05) elevated at 50 MOI compared with 0 MOI AdCre following hypoxia-reoxygenation (n=4/group). *p<0.05 vs. 0 MOI AdCre

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.
5.
Figure 1

Figure 1. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

Assessment of the effects of OGT overexpression (using AdOGT) on NRCM survival following hypoxia. Myocytes were infected with AdOGT 48 hours prior to protein harvest or hypoxia-reoxygenation. A) Summary densitometric analysis (n=3/group) and immunoblot of OGT (using SQ17) show a dose dependent increase in OGT protein with increasing MOI of AdOGT. A significant (p<0.05) increase is seen at 20 and 100 MOI compared with 0 MOI AdOGT. B) Densitometric analysis (n=6/group) and representative immunoblot of the functional readout of OGT, O-GlcNAc. A dose dependent increase in O-GlcNAc levels (n=6/group) is seen with the RL2 antibody, while a trend is evident with the CTD antibody. As expected, multiple immunopositive bands appear because the O-GlcNAc modification occurs on numerous proteins throughout the cell. C) OGT overexpression (n=6/group) dose-dependently augmented O-GlcNAc levels according to O-GlcNAc-TAMRA. *p<0.05 vs. 0 MOI AdOGT.

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.
6.
Figure 3

Figure 3. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

Evaluation of effects of OGT inhibition on cardiac myocyte survival post-hypoxia. NRCMs were treated with the OGT inhibitor, TT04, prior to protein harvest or H/R (n=6/group). A) Densitometric analysis and representative immunoblots for O-GlcNAc levels following TT04 treatment show a dose dependent decrease in O-GlcNAc levels. Multiple bands occur because O-GlcNAc is a post-translational modification occurring on a plethora of intracellular proteins. B) OGT inhibition dose-dependently exacerbates post-hypoxic cardiac myocyte survival. LDH release was spectrophotometrically determined from the media following hypoxia-reoxygenation. C) OGT inhibition via TT04 worsens post-hypoxic injury reflected by the significant increase in PI positivity release. *p<0.05 vs. 0 µmol/L TT04.

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.
7.
Figure 7

Figure 7. From: Noncanonical Glycosyltransferase Modulates Post-hypoxic Cardiac Myocyte Death and Mitochondrial Permeability Transition.

OGT inhibition (via TT04) reduces O-GlcNAc levels on at least one mitochondrial protein (VDAC). A–C) Co-immunoprecipitation for O-GlcNAc-modified-VDAC showed a reduction in O-GlcNAc-modified-VDAC in adult cardiac mitochondria from TT04-treated mice compared with Vehicle-treated mice, despite no difference in total VDAC levels from both groups (n=4/group; *p<0.05 vs. Vehicle). D) Immunoprecipitation for VDAC and enzymatic labeling of O-GlcNAc-modified proteins confirmed the reduction in O-GlcNAc-modified-VDAC in cardiac mitochondria from TT04-treated compared with Vehicle-treated mice. There was no difference in protein loading according to SYPRO ruby staining. E) Sensitivity to mPTP formation according to calcium-induced mitochondrial swelling. Adult mouse cardiac mitochondria were subjected to time control or challenged with 100 µmol/L CaCl2. Change in absorbance (at 520 nm) was measured over time. Cardiac mitochondria from TT04-treated mice were significantly more sensitive to calcium-induced swelling compared with those from Vehicle-treated mice (n=4/group). p< 0.05 vs. Vehicle.

Gladys A. Ngoh, et al. J Mol Cell Cardiol. ;45(2):313-325.

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