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. 2018 Apr 16;39(9):1836–1848. doi: 10.1177/0271678X18769770

Figure 3.

Figure 3.

Knockdown or knockout of Thorase blocks preconditioning and increases sensitivity to neuronal injury. (a and b) Immunoblot of Thorase expression in primary cortical cultures transfected with lentivirus expressing DsRed shRNA (LV.shRed), Thorase-shRNA2 (LV.sh2) or Thorase-shRNA3 (LV.sh3) and exposure to 15 min OGD preconditioning (PC) (a) or 50 µM NMDA/10 µM glycine (PC) (b). No viral infected neurons were used as control. (c) Cell viability assessed by PI/Hoechst staining in primary cortical cultures transfected with DsRed shRNA (LV.shRed), Thorase-shRNA2 (LV.sh2) or Thorase-shRNA3 (LV.sh3) and exposed to 15 min OGD followed 24 h later to 90 min OGD. Scale bar, 25 μM. (d) Percentages of neuron death in (c) were quantified and expressed as the mean ± SEM from three independent experiments performed in triplicate (n = 9). *p < 0.05 by ANOVA with Tukey–Kramer post hoc test compared to LV.shRed viral control. (e) Cell viability assessed by PI/Hoechst staining in primary cortical cultures transfected with DsRed shRNA (LV.shRed), Thorase-shRNA2 (LV.sh2) or Thorase-shRNA3 (LV.sh3) and exposed to 50 μM NMDA and 24 h later to 500 µM NMDA. Scale bar, 25 μM. (f) Percentages of neuron death in (e) were quantified and expressed as mean ± SEM from three independent experiments performed in triplicate (n = 9). **p < 0.01 by ANOVA with Tukey–Kramer post hoc test compared to LV.shRed viral control. (g) Cell viability assessed by PI/Hoechst staining in primary cultured Thorase KO and WT neurons exposed to 15 min OGD followed by 90-min OGD 24 h later. Scale bar, 25 μM. (h) Percentages of neuron death in (g) were quantified and expressed as mean ± SEM from three independent experiments performed in triplicate (n = 9). *p < 0.05, **p < 0.01 by ANOVA with Tukey–Kramer post hoc test compared to WT control. (i) Cell viability assessed by PI/Hoechst staining in primary cultured Thorase KO and WT neurons treated with 50 µM NMDA followed 24 h later by 500 μM NMDA. Scale bar, 25 μM. (j) Percentages of neuron death in (i) were quantified and expressed as the mean ± SEM from three independent experiments performed in triplicate (n = 9). *p < 0.05, **p < 0.01 by ANOVA with Tukey–Kramer post hoc test compared to WT control. (k) Cell viability in primary cultured Thorase KO and WT neurons exposed to 45 min or 60 min OGD. Scale bar, 25 μM. (l) Quantification of cell viability in (k). Experiments were performed three independent experiments performed in triplicate (n = 9). The data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA with Tukey–Kramer post hoc test compared to WT control. (m) Cell viability in primary cultured Thorase KO and WT neurons treated with 200 μM or 400 μM NMDA. Scale bar, 25 μM. (n) Quantification of cell viability in (m). Experiments were performed three independent experiments performed in triplicate (n = 9). The data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA with Tukey–Kramer post hoc test compared to WT control.