Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host (2021)
Figure 3

Figure 3. Inhibition of O-GlcNAcylation restricts the activation of apoptotic and pro-inflammatory responses caused by Stx2a through directly influencing the phosphorylation status of Akt and p65

A. Representative western blot showing changes of overall O-GlcNAcylation (upper panel) or phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of OSMI-1 (10 µM, final).

B. Quantification of the relative band intensities for O-GlcNAcylation (RL-2) or each of the phosphorylated- and total-protein bands in (A). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with lysates prepared from cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a.

C. Representative western blot showing changes in OGT expression (top panel), overall O-GlcNAcylation (second panel), or phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of OGT knockdown.

D. Quantification of the relative band intensities between each of phosphorylated- and total-protein bands in (C). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in siScr controls were compared with those of lysates from cells maintained in the absence of Stx2a, and OGT knockdown was compared with that of siScr controls in the presence of Stx2a. (n.s. = not significant).

E. LDH cytotoxicity assay performed during 9 h culture period following Stx2a (10 ng/mL) treatment of THP-1 cells in the presence or absence of OGT knockdown (n = 3 biological replicates). The effects of OGT siRNA (siOGT) were compared with those of scrambled siRNA (siScr) controls at each time-point.

F. ELISAs were used to analyze the inhibitory effect of siOGT on cytokine/chemokine production from THP-1 cells exposed to Stx2a (10 ng/mL) for 9 h (n = 3 biological replicates). The effects of OGT knockdown were compared with those of siScr controls in the presence of Stx2a.

G. Representative western blot showing changes of overall O-GlcNAcylation (upper panel), phosphorylation status of each target protein (middle panels) or pro-caspase-3 cleavage (lower panel) in THP-1 cells treated with Stx2a (10 ng/mL) for 9 h in the presence or absence of overexpression of Akt (wild-type or T305A/T312A).

H. Quantification of the relative band intensities of each of the phosphorylated- and total-protein bands or cleaved caspase-3 in (G). Data are normalized against β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with lysates prepared from cells maintained in the absence of Stx2a, and overexpression of mutant Akt (T305A/T312A) was compared with that of Akt_wild-type in the presence of Stx2a. (n.d. = not detected; C-Casp-3 = Cleaved Caspase-3; P-Casp-3 = Pro-Caspase-3).

I. LDH cytotoxicity assay performed at 9 h following Stx2a (10 ng/mL) treatment of THP-1 cells in the presence or absence of overexpression of Akt (wild-type or T305A/T312A (n = 3 biological replicates). The effects of Stx2a were compared with those of culture medium from cells transfected with Akt (wild-type or T305A/T312A), and overexpression of mutant Akt (T305A/T312A) was compared with that of Akt_wild-type in the presence of Stx2a.

Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.