Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: Genetic suppression of GCN2 reverses developmental delay and seizure phenotypes in dPARS2-deficient flies. (A) Western blot analysis of P-GCN2, GCN2 and P-PERK in protein extracts from control and elav- Gal4-driven dPARS2 knockdown fly heads. α-tubulin was used as a loading control. (B) Quantification of the Western blots shown in A. P-GCN2, N = 3; GCN2 and P-PERK, N = 4. ∗∗p < 0.01, ns, not significant. (C) Western blot analysis of P-eIF2α in protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown fly heads. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 3. ∗p < 0.05, ∗∗p < 0.01. (E) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown fly heads. Flies were fed with puromycin. α-actin was used as the loading control. (F) Quantification of the Western blots shown in E. N = 3, ∗p < 0.05, ∗∗∗p < 0.001. (G) Images of control, elav -Gal4-driven dPARS2 knockdown and elav -Gal4-driven dPARS2 and GCN2 double knockdown flies at different developmental stages. Scale bars: 500 μm. (H) Graph showing pupariation rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown larvae. N = 3, n = 28–30. (I) Graph showing eclosion rate of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown, and elav -Gal4-driven GCN2 knockdown pupae. N = 3, n = 28–30. (J) Graph showing percentage of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies displaying Bang-sensitive paralytic phenotypes. N = 3, n = 10 sample. ∗∗∗∗p < 0.0001. (K) Graph showing the recovery time of control, elav -Gal4-driven dPARS2 knockdown, elav -Gal4-driven dPARS2 and GCN2 double knockdown and elav -Gal4-driven GCN2 knockdown flies from paralysis. n = 30. ∗∗∗∗p < 0.0001.

Article Snippet: Mouse anti-Alpha tubulin , Proteintech , 66031-1-Ig.

Techniques: Western Blot, Control, Knockdown, Staining

Journal: Redox Biology

Article Title: Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency

doi: 10.1016/j.redox.2025.103966

Figure Lengend Snippet: PARS2 V95I mutation causes mitochondrial dysfunction and ISR activation in human cells (A) Western blot analysis of ectopically expressed PARS2 proteins. Lysates from HEK-293T cells transfected with plasmids encoding His-tagged wild-type (WT) or the indicated PARS2 variants were immunoblotted with an anti-His antibody. α-actin was used as a loading control. (B) Quantification of the Western blots shown in A. N = 5, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Western blot analysis of endogenous PARS2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (D) Quantification of the Western blots shown in C. N = 4, ∗∗∗p < 0.001. (E) Western blot analysis of mtDNA-encoded CO2 and ATP8 and nuclear-DNA encoded NDUFS1, NDUFS3, UQCRFS1 and ATP5A in protein extracts from the wild-type controls and the PARS2 V95I cells. VDAC was used as a loading control. (F) Quantification of the Western blots shown in E. MT-CO2, MT-ATP8, NDUFS1, NDUFS3, and ATP5A, N = 4; UQCRFS1, N = 7. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant. (G) CI, CII and CIV in-gel activity analysis of isolated mitochondria from the wild-type controls and the PARS2 V95I cells. (H) Western blot analysis of P-eIF2α and eIF2α in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (I) Quantification of the Western blots shown in H. N = 5, ∗∗∗∗p < 0.0001. (J) Western blot analysis with anti-puromycin antibody and ponceau staining on protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as the loading control. (K) Quantification of the Western blots shown in J. N = 4. ∗∗∗∗p < 0.0001. (L) Western blot analysis of ATF4 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-actin was used as a loading control. (M) Quantification of the Western blots shown in L. N = 5, ∗∗∗p < 0.001. (N) Western blot analysis of P-GCN2 and GCN2 in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (O) Quantification of the Western blots shown in N. N = 4, ∗∗p < 0.01. (P) Western blot analysis of P-PERK and PERK in protein extracts from the wild-type controls and the PARS2 V95I cells. α-tubulin was used as a loading control. (Q) Quantification of the Western blots shown in P. N = 5, ns, not significant.

Article Snippet: Mouse anti-Alpha tubulin , Proteintech , 66031-1-Ig.

Techniques: Mutagenesis, Activation Assay, Western Blot, Transfection, Control, Activity Assay, Isolation, Staining