Journal: NAR Cancer

Article Title: G-quadruplex ligand RHPS4 compromises cellular radioresistance by inhibiting the mitochondrial adaptive response induced by ionizing irradiation

doi: 10.1093/narcan/zcaf033

Figure Lengend Snippet: Increase in mitochondrial mass and mitochondrial membrane potential induced by ionizing radiation. U2OS cells were irradiated by γ-irradiation (6 Gy) and compared with non-irradiated (NI) cells. ( A ) Mitochondrial genes ND1, ND4, COX2, COX3, CYB, TRNA1, and TRNA2 were quantified by qPCR 96 h after irradiation. Statistical analysis was performed for five independent experiments with one-way analysis of variance (ANOVA) (**<0.005). ( B ) Confocal microscopy images of U2OS cells stained with anti-DNA (yellow), anti-TFAM (cyan), and TOM20 (magenta). Nuclear DNA was stained with DAPI (blue). The scale bar is 10 μm and 5 μm for insets. ( C ) Number of nucleoids and ( D ) the area of mtDNA per cell was quantified from >1000 cells from four independent experiments. ( E ) Confocal microscopy images of U2OS living cells 96 h after irradiation. Mitochondria were stained with MitoTracker Green (magenta) and TMRE (cyan); the scale bar is 10 μm. ( F ) TMRE and MitoTracker Green probes were used to measure mitochondrial membrane protential and mitochondrial mass, respectively, in U2OS cell by cytometry 3 days after irradiation (6 Gy). ( G ) Representative quantification of cytometry results for single-cell MitoTracker Green, TMRE, and the ratio of TMRE to MitoTracker Green (MTG). A total of 10 000 cells were analysed for each condition. One representative experiment out of three is shown. Statistical analyses were performed with GraphPad using Mann–Whitney test (*** P ≤ 0.001).

Article Snippet: Taqman probes against ND1 (Hs02596873_s1), ND4 (Hs02596876_g1), COX2 (Hs02596865_g1), COX3 (Hs02596866_g1), CYTB (Hs02596867_s1), tRNA1 (Hs02596871_s1), tRNA2 (Hs02596871_s1), and RNaseP (4401631) were purchased from Thermo Fisher Scientific.

Techniques: Membrane, Irradiation, Confocal Microscopy, Staining, Cytometry, MANN-WHITNEY

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: Reduced levels of mt-tRNA Pro aminoacylation and OXPHOS complex proteins in dPARS2-deficient flies. (A) Northern blot analysis of mitochondrial tRNA Pro aminoacylation in total RNA samples from control and elav -Gal4-driven dPARS2 knockdown fly heads. Upper bands represent the charged tRNAs and lower bands represent the uncharged tRNAs. (B) Western blot analysis of mtDNA-encoded OXPHOS complex subunits in protein extracts from control and elav -Gal4-driven dPARS2 knockdown fly heads. Antibodies against individual subunits of OXPHOS complexes (MT-ND1, complex I; MT-CO2, complex IV) were used. Porin was used as a loading control. (C) Quantification of the Western blots shown in B. MT-ND1, N = 3; MT-CO2, N = 4. ∗p < 0.05, ∗∗p < 0.01. (D) Western blot analysis of nuclear-encoded OXPHOS complex subunits in protein extracts from control and elav -Gal4-driven dPARS2 knockdown fly heads. Antibodies against individual subunits of OXPHOS complexes (NDUFS3 and NDUFS1, complex I; SDHB, complex II; UQCRFS1, complex III; ATP5A, complex V) were used. Porin was used as a loading control. (E) Quantification of the Western blots shown in D. NDUFS1 and UQCRFS1, N = 3; NDUFS3, SDHB and ATP5A, N = 4. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns, not significant. (F) Northern blot analysis of mitochondrial tRNA Pro aminoacylation in total RNA samples from control and Da -Gal4-driven dPARS2 knockdown larvae. (G) Western blot analysis of mtDNA-encoded OXPHOS complex subunits in protein extracts from control and Da -Gal4-driven dPARS2 knockdown larvae. (H) Quantification of the Western blots shown in G. N = 3. ∗∗∗∗p < 0.0001. (I) Western blot analysis of nuclear-encoded OXPHOS complex subunits in protein extracts from control and Da -Gal4-driven dPARS2 knockdown larvae. (J) Quantification of the Western blots shown in I. NDUFS3, UQCRFS1 and ATP5A, N = 3; NDUFS1 and SDHB, N = 4. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant.

Article Snippet: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

Techniques: Northern Blot, Control, Knockdown, Western Blot

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: Defective assembly of OXPHOS complexes in dPARS2-deficient flies. (A) BN-PAGE followed by Western blot analysis of isolated mitochondria from control and elav -Gal4-driven dPARS2 knockdown fly heads. Antibodies against individual subunits of OXPHOS complexes (NDUFS3, complex I; SDHB, complex II; UQRCFS1, complex III; MT-CO2, complex IV; ATP5A, complex V) were used. Porin was used as a loading control. (B) Quantification of the Western blots shown in A. complex II, complex III and complex V, N = 3; complex IV, N = 4; complex I, N = 5. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C) BN-PAGE followed by Western blot analysis of isolated mitochondria from control and Da -Gal4-driven dPARS2 knockdown larvae. (D) Quantification of the Western blots shown in C. complex II and complex III, N = 3; complex I and complex V, N = 4; complex IV, N = 5. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

Techniques: Western Blot, Isolation, Control, Knockdown

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: Primary antibodies used were anti-MT-ND1 (Abcam, AB181848-1001), anti-MT- CO2 (Proteintech, 55070-1-AP), anti-MT-ATP8 (Proteintech, 26723-1-AP),anti-NDUFS1 (Proteintech, 12444-1-AP), anti-NDUFS3 (Abcam, ab14711), anti-UQCRFS1 (Abcam, ab14746), anti-ATP5A (Abcam, ab14748), anti-SDHB (Proteintech, 10620-1-AP), anti-Porin/VDAC (Abcam, ab14734), anti-P-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 2103), anti-P-PERK (ABclonal, AP0886), anti-PERK (ABclonal, A27664 ), anti-P-GCN2 (Abcam, ab75836), anti-GCN2 (ABclonal, A2307), anti-LDH (ThermoFisher, PA5-26531), anti-PARS2 (ABclonal, A16512), anti-His (yeasen, 30405ES50), anti-ATF4 (Abcam, ab1371), anti-Alpha actin (Proteintech, 23660-1-AP) and anti-Alpha tubulin (Proteintech, 66031-1-Ig).

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

Journal: bioRxiv

Article Title: Oxa1L-Mediated Co-translational Protein Insertion Maintains Mitochondrial Function in Parkinson’s Disease Models

doi: 10.64898/2025.12.22.696118

Figure Lengend Snippet: A Immunoblot blot analysis of Oxa1L and mitochondrial OXPHOS complex subunits in control and MPP⁺-treated SH-SY5Y cells. Mitochondrial genome-encoded subunits (MT-CYTB, MT-CO1, MT-CO2, MT-ATP8) and nuclear genome-encoded subunits (NDUFB8, SDHB, ATP5A1) are indicated. B Immunoblot analysis of OXPHOS subunits in scramble and Oxa1L knockdown (shOxa1L) cells following MPP⁺ treatment, showing selective reduction of mitochondrial genome–encoded proteins upon Oxa1L deficiency. C-D Cryo-EM density map (C) and corresponding atomic model (D) of the Oxa1L-mitoribosome complex, revealing Oxa1L positioned adjacent to the ribosomal exit tunnel. E AlphaFold3-predicted structure of monomeric human Oxa1L, highlighting its five transmembrane helices. F Sequence alignment of Oxa1L transmembrane regions across representative species, illustrating strong evolutionary conservation. Multiple sequence alignment was performed using WebLogo. G AF3-predicted model of Oxa1L-uL24m-bL29m complex. The black box indicates the focused area shown in (H, I). H, I Enlarged views of interaction interfaces between Oxa1L and ribosomal proteins uL24m (H) and bL29m (I), identifying conserved matrix-exposed regions implicated in co-translational membrane insertion.

Article Snippet: Membranes were probed with primary antibodies against DYKDDDDK (FLAG) (Flag tag, proteintech, 1:5000), GAPDH (proteintech, 1:5000), Oxa1L (proteintech, 1:5000), MT-CYTB (proteintech, 1:2000), MT-CO1 (Cell Signaling Technology, 1:1000), MT-CO2 (proteintech, 1:2000), MT-ATP8 (abclonal, 1:1000), NDUFB8 (Cell Signaling Technology, 1:1000), SDHA (abclonal, 1:1000), SDHB (Cell Signaling Technology, 1:1000), and ATP5A1 (abclonal, 1:10000).

Techniques: Western Blot, Control, Knockdown, Cryo-EM Sample Prep, Sequencing, Membrane