Journal: Cell reports

Article Title: Mitochondrial protein OPA1 is required for the expansion of effector CD8 T cells

doi: 10.1016/j.celrep.2025.115610

Figure Lengend Snippet: 500 WT (CD45.1/2) and 500 Opa1 −/− (CD45.1/1) OT1s were transferred into B6 recipients, followed by a challenge of either poly(I:C), αCD40, and Ova (vaccine); 10 6 plaque-forming units of SIINFEKL-expressing vaccinia virus (VV.siin); 10 6 colony-forming units (CFUs) of the attenuated ActA-deficient Ova-expressing L. monocytogenes ( L. monocytogenes .Ova.ActA); or 2,000 CFUs of virulent Ova-expressing L. monocytogenes ( L. monocytogenes .Ova.Vir). Mice were euthanized 7 days following infection or vaccination and analyzed via flow cytometry. Cell populations were identified and quantified based on the gating scheme shown in . (A) Representative flow plots of WT and Opa1 −/− OT1s for each challenge. (B) Total splenic OT1s were quantified 7 days after challenge. (C) Representative flow plot for KLRG1 and CD127 for splenocytes harvested. (D–G) Splenic short-lived effector cells (SLECs; KLRG1 + CD127 − ) or memory precursor effector cells (MPECs; KLRG1 − CD127 + ) were quantified by (D and E) percentages or (F and G) total cell counts of SLECs and MPECs. (H and I) WT: Opa1 −/− ratio of SLECs and MPECs recovered from each mouse. (A–I) Two to three replicate experiments using B6 donor and recipient mice. (J and K) Following transfer of 500 WT and 500 Opa1 −/− OT1s, OT1s were quantified in the blood 7, 14, and 63 days following L. monocytogenes .Ova.ActA infection. On day 64, mice were rechallenged with 5 × 10 6 CFUs of L. monocytogenes .Ova.ActA. (L) Four days after the secondary challenge, blood was collected to calculate the fold expansion of WT and Opa1 −/− OT1s, calculated based on pre- and post-challenge WT and Opa1 −/− percentages of all CD8s in the blood. (J–L) One experiment using B6 donors and recipients (shown) and one experiment using mixed-background donor and recipient mice. Data shown are means ± SEM; n ≥ 4 mice per group, representative of 2–3 experiments. Significance was defined by two-tailed paired Student’s t tests, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: anti-mouse OPA1 , Cell Signaling , D6U6N.

Techniques: Expressing, Virus, Infection, Flow Cytometry, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial protein OPA1 is required for the expansion of effector CD8 T cells

doi: 10.1016/j.celrep.2025.115610

Figure Lengend Snippet: 500 WT and 500 Opa1 −/− OT1s were transferred into B6 recipients, followed by either L. monocytogenes .Ova.ActA or vaccine challenge. Twelve hours prior to euthanasia (day 7), mice were injected i.v. with 5 mg of EdU (in 200 μL, 25 mg/mL). OT1 cells and EdU incorporation were determined using the gating strategy outlined in . (A) Experimental design. (B) Frequency of EdU incorporation in WT SLECs and MPECs following a 12-h EdU pulse. (C) Frequency of EdU incorporation in all WT or OPA1-deficient OT1s during a L. monocytogenes .Ova.ActA or vaccine response following a 12-h EdU pulse. (A–C) n = 4 mice per group, representative of two experiments. Data are from one experiment using B6 donors and recipient mice (shown) and one experiment using mixed-background donors and recipients. (D) Frequency of WT or Opa1 −/− OT1s in the blood on days 5, 7, 9, 11, and 13 following L. monocytogenes .Ova.ActA or vaccine challenge. (E) Ratio of WT to Opa1 −/− cells in the blood over time. (F) Comparison of the WT day 7 peak and the Opa1 −/− day 9 peak following L. monocytogenes .Ova.ActA or vaccine challenge. (D–F) n = 8–9 mice per group, pooled from two experiments using B6 donor and recipient mice. Data shown are means ± SEM. Significance was defined by (B and C) two-tailed paired Student’s t test and (F) two-tailed paired Wilcoxon test, where * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: anti-mouse OPA1 , Cell Signaling , D6U6N.

Techniques: Injection, Comparison, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial protein OPA1 is required for the expansion of effector CD8 T cells

doi: 10.1016/j.celrep.2025.115610

Figure Lengend Snippet: (A) WT and Opa1 −/− OT1s were co-transferred into recipients at varying amounts, followed by L. monocytogenes .Ova.ActA infection. Splenic WT: Opa1 −/− ratio was determined 7 days post infection. n = 4 mice per group, representative of two experiments. One experiment using B6 donors and recipient mice is shown and one experiment used mixed-background donors and recipients. (B) WT: Opa1 −/− and total splenic WT OT1s per mouse on day 7 following various vaccine and infection challenges. n ≥ 4 mice per group, representative of 2 experiments. Data are from two experiments using B6 donors and recipient mice. (C–H) Mice were sublethally irradiated (600 rads), followed by co-transfer of 10 5 CTV-labeled WT and Opa1 −/− OT1s. Ten days later, splenocytes were harvested and analyzed by flow cytometry. (C) Experimental design. (D) Representative flow plot of CTV peaks for WT and Opa1 −/− OT1s. (E) WT and Opa1 −/− cell count per division. (C–H) Data are from two experiments with mixed-background donors and recipients. (E–H) n = 9 mice per group, pooled from two experiments. (F–H) Proliferation statistics calculated with FlowJo proliferation modeling: (F) percent of original population dividing at least once, (G) proliferation index or the average number of divisions by dividing cells, and (H) replication index or the fold expansion of dividing cells. Data shown are means ± SEM. Significance was defined by (A) two-tailed Mann-Whitney test or (F–H) two-tailed Wilcoxon test, where * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: anti-mouse OPA1 , Cell Signaling , D6U6N.

Techniques: Infection, Irradiation, Labeling, Flow Cytometry, Cell Counting, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Mitochondrial protein OPA1 is required for the expansion of effector CD8 T cells

doi: 10.1016/j.celrep.2025.115610

Figure Lengend Snippet: 5 × 10 4 CTV-labeled WT and Opa1 −/− OT1s were transferred into mice, followed by L. monocytogenes .Ova.ActA infection. Two hours prior to euthanasia, mice were injected with EdU (in 200 μL, 25 mg/mL). Splenic OT1s were isolated and analyzed by flow cytometry (B–D) 24 h or (E–I) 36 h later. Cell cycle phases were determined using EdU and DRAQ7 as outlined in . Ghost Dye Red 780 continued to be used to exclude dead cells during analysis. DRAQ7 was only used for DNA quantification. (A) Experimental design. (B) Percentage of cells in S phase 24 h post infection. n = 9 per group, pooled from 2 experiments. (C) EdU geometric mean fluorescence intentity (gMFI) for cells in G1 or S phase 24 h post infection. (D) Size of cells in G1 or S phase 24 h post infection. (C and D) n = 5 per group, representative of the 2 experiments pooled in (B). (E) Representative histogram of CTV 36 h after infection. (F) Percentage of the original OT1 population that has divided at least once 36 h post infection. (G and H) gMFIs of (G) EdU and (H) DRAQ7, shown as a percentage of max in S phase cells 36 h post infection. (I) Cell size of undivided and divided S phase cells 36 h post infection. (E–I) n = 9 per group, pooled from the 2 experiments in (B). (J) Cell counts per division of splenic OT1s at various time points following LIP, as described in . n = 1–3 mice per group, representative of 2 experiments. (K) Viability of OT1s 7 days after various infections or vaccination as outlined in . Mice are pooled from 2–7 different experiments with n = 33 ( L. monocytogenes .Ova.ActA), n = 19 (vaccine), n = 9 ( L. monocytogenes .Ova.Vir), and n = 8 (VV.siin). (A–K) B6 donor and recipient mice used for all experiments. (L) Viability of OT1s, as determined by live/dead flow cytometric staining, at each division 10 days following transfer into irradiated hosts from experiments in and . n = 4 mice per group, representative of 2 experiments. (M–P) 10 4 WT and Opa1 −/− OT1s were co-transferred into mice and vaccinated. Seven days later, OT1s were isolated via negative selection and CTV labeled before being transferred into mice that were 7 days post-vaccination but did not receive an OT1 transfer on day 0. After 17 days following adoptive transfer (24 days after vaccination), splenic OT1s were analyzed by flow cytometry. (M) Experimental design. (N) Representative flow images 17 days after CTV-labeled cells were transferred into vaccine-matched recipients (24 days post vaccination). (O) Percentage of OT1s CTV labeled on day 7 that had undergone at least one division by day 24. (P) As a percentage, dead cells of the undivided and divided OT1 populations 24 days post vaccination. (N–P) Data pooled from two experiments with n = 8 using mixed-background donor and recipient mice. Significance was defined by two-tailed paired Wilcoxon test where * p < 0.05, ** p < 0.01, *** p < 0.001. Data shown are means ± SEM. All experiments used B6 donors and recipients. Significance was defined by either two-tailed paired Wilcoxon test (B and F), two-way ANOVA adjusted for multiple comparisons (C, D, and I), or one-sample t test against 100% EdU gMFI % of max (G and H), where * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: anti-mouse OPA1 , Cell Signaling , D6U6N.

Techniques: Labeling, Infection, Injection, Isolation, Flow Cytometry, Fluorescence, Staining, Irradiation, Selection, Adoptive Transfer Assay, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial protein OPA1 is required for the expansion of effector CD8 T cells

doi: 10.1016/j.celrep.2025.115610

Figure Lengend Snippet: scRNA-seq was performed on either naive or activated WT and Opa1 −/− CD8 OT1 T cells isolated 5 days after L. monocytogenes .Ova.ActA infection. (A) Uniform manifold approximations and projection (UMAP) dimensionality reduction mapping analysis of single-cell gene expression data from naive (gray) and activated (red). (B) UMAP as in (A), with cells color coded for WT (black) or Opa1 −/− (blue). (C) UMAP as in (A), with cells clustered by shared nearest-neighbor analysis, split by Opa1 −/− or WT. (D) Representation of WT and Opa1 −/− T cells per cluster. (E) UMAP as in (A), identifying cells in the phases of the cell cycle, split by WT vs. Opa1 −/− . (F) Total number of cells in each cluster in G1, S, or G2M. (G) cNMF analysis, resulting in identification of 5 gene expression programs (GEPS 1–5), split by WT vs. Opa1 −/− . (H) Representation of WT and Opa1 −/− T cells per GEP. (I) Total number of WT cells in each GEP in G1, S, or G2M. (J) Total number of Opa1 −/− cells in each GEP in G1, S, or G2M. (K) fast GSEA on ranked genes per GEP using gene sets in the MSigDB Hallmark Gene series (mh.all.v2023). (L) fast GSEA on ranked genes per GEP using gene sets in the MSigDB Gene Ontology biological pathway genes sets (m5.go.bp.v2023). B6 donors and recipients used for the experiment are shown. Day 5 WT and Opa1 −/− OT1s were pooled from four recipient mice. Naive WT and Opa1 −/− OT1s were from individual mice.

Article Snippet: anti-mouse OPA1 , Cell Signaling , D6U6N.

Techniques: Isolation, Infection, Gene Expression

Journal: Cell reports

Article Title: Mitochondrial protein OPA1 is required for the expansion of effector CD8 T cells

doi: 10.1016/j.celrep.2025.115610

Figure Lengend Snippet: (A) Boxplots of module scores per cluster or GEP, derived from REACTOME_RESPONSE_OF_EIF2AK4_GCN2_TO_AMINO_ACID_DEFICIENCY.v2023.2.Hs.gmt. (B and C) 5 × 10 4 WT, Opa1 −/− , or Mfn dKO OT1s were transferred into B6 mice, followed by L. monocytogenes .Ova.ActA infection. Five days later, cells were isolated via positive selection, followed by cell sorting and steady-state metabolomics analysis via mass spectrometry. (B) Hierarchical clustering analysis (HCA; Euclidean distance measure, Ward clustering algorithm) of WT, Opa1 −/− , and Mfn dKO OT1s isolated 5 days following L. monocytogenes .Ova.ActA infection. (C) HCA for the top 50 significant metabolites by unpaired t test. (D) Predicted glycolytic dependence (Glyco. Dep) and mitochondrial (FAO and AAO) capacity (Mito. Cap) in day 5 in vivo -activated ( L. monocytogenes .Ova.ActA challenge) WT, Opa1 −/− and Mfn dKO T cells using the SCENITH assay as described in . (E) gMFI of puromycin in control (no inhibitor) WT and Opa1 −/− T cells described in (D). (D and E) n = 5 mice per group, representative of two experiments. (F–L) WT, Opa1 −/− or Mfn dKO cells (5 × 10 4 OT1s) were adoptively transferred into B6 recipients, followed by infection with L. monocytogenes .Ova.ActA. Five days after infection, splenic OT1s were isolated and cultured ex vivo for 3 h in RPMI medium containing a uniformly isotopically labeled amino acid pool. Uptake and catabolism of amino acid tracers were assessed via mass spectrometry. (F) Outline. (G) Abundance of non-oxidative pentose phosphate pathway intermediates. (H) Total isotopolog pool of glutathione in cultured OT1s. (I) Incorporation of labeled carbon atoms from tracer amino acids in TCA cycle intermediates. (J) Extracellular accumulation of isotopically labeled lactate produced by either malic enzyme (ME) or alanine aminotransferase (ALT). (K) Cellular abundances of Nicotinamide adenine dinucleutide (NAD) + , NAD Hydrogen (NADH), NAD Phosphate (NADP + ), and NADP Hydrogen (NADPH) in OT1 cells. (L) Abundance of various amino acids with 15 N 1 labeling derived from the ex vivo -supplied amino acid pool. (M) Abundance of 15 N 1 -labeled alanine found in the cell culture medium. B6 donors and recipients were used for all experiments. 3–8 mice were pooled per n (B, C, F–M). Total n = 3 or 6 for (B) and (C) and n = 3 for (F)–(M). Significance was defined by either (E) two-tailed Mann-Whitney test or (F–M) one-way ANOVA using Tukey’s multiple-comparisons test comparing all groups, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: anti-mouse OPA1 , Cell Signaling , D6U6N.

Techniques: Derivative Assay, Infection, Isolation, Selection, FACS, Mass Spectrometry, In Vivo, Control, Cell Culture, Ex Vivo, Labeling, Produced, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Mitochondrial F0F1-ATP synthase governs the induction of mitochondrial fission

doi: 10.1016/j.isci.2024.109808

Figure Lengend Snippet: Effect of OXPHOS inhibition on OPA1 protein cleavage (A) OPA1 isoforms’ expression profile: Representative images of Western-blots of OPA1 long (L) and short (S) isoforms and SDHA (mitochondrial loading reference) performed on protein extracts from treated cells. (B) Quantification of the OPA1 L/S ratio relatively to the vehicle. n = 5, in five independent biological replicates. Results are presented as means ± SEM. ∗ Indicates significant difference from control conditions (Veh).

Article Snippet: Mouse anti-OPA1 (BD Biosciences, 612607), and Mouse anti-SDHA ([2E3GC12FB2AE2], ab14715, Abcam) primary antibodies were used (dilution at 1:1000).

Techniques: Inhibition, Expressing, Western Blot

Journal: iScience

Article Title: Mitochondrial F0F1-ATP synthase governs the induction of mitochondrial fission

doi: 10.1016/j.isci.2024.109808

Figure Lengend Snippet:

Article Snippet: Mouse anti-OPA1 (BD Biosciences, 612607), and Mouse anti-SDHA ([2E3GC12FB2AE2], ab14715, Abcam) primary antibodies were used (dilution at 1:1000).

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Knock-Out, Double Knockout, Software, Imaging

Journal: Cells

Article Title: Leflunomide Treatment Does Not Protect Neural Cells following Oxygen-Glucose Deprivation (OGD) In Vitro

doi: 10.3390/cells13070631

Figure Lengend Snippet: Leflunomide has limited effects on protein expression in C17.2 cells. HeLa ( a ) or C17.2 ( b – e ) cells were treated with Leflunomide (50 μM) for 16 h. ( a ) HeLa cell lysates were analyzed by Western blot for the expression of MFN2. ( b ) Lysates from C17.2 cells ± Leflunomide ± OGD were analyzed for the expression of OPA1 and MFN2. ( c ) MFN2 protein expression was not altered. ( d ) OPA1 expression increased in control cells (* p < 0.05, one-way ANOVA) after Leflunomide treatment, but this was not maintained following OGD. ( e ) the ratio of long(L)-OPA1: Short(S)-OPA1 was determined, and although there was a decrease by OGD (two-way ANOVA, * p < 0.05, ** p < 0.01), this was not reversed by Leflunomide treatment (mean ± SD, N = 4).

Article Snippet: The following antibodies were used in this study: mouse anti-OPA1 (Clone 18/BD Bioscience 612607), mouse anti-MFN2 ([6A8], Abcam, Cambridge, UK), anti-mouse GAPDH (Merck G8795), IRDye 680RD and 800CW goat anti-mouse secondary antibodies (LI-COR Biosciences).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Extracellular matrix signals promotes actin-dependent mitochondrial elongation and activity

doi: 10.1101/2024.01.22.576703

Figure Lengend Snippet: (A-B) Measure of ATP (A) and lactate (B) levels in MDA-MB-231 cells grown as an adherent monolayer (AM) or as mammospheres in suspension (MS). Each point represents an individual experiment. Bars show the average of 4 independent experiments ± SD. *** p<0.001, ** p<0.01. Two-sided t-test (C-D) Mitochondrial fragmentation in mammospheres in suspension. MDA-MB-231 were grown as AM, MS or MS attached for 6 hours on glass coverslips, and their mitochondria were marked with an antibody against TOM20 (mitochondria, cyan; with nuclei stained with DAPI, yellow). Quantification of 3 independent experiments is shown in (C), with each point represents an individual experiment. Bars show the average ± SD. *** p<0.001. Two-way ANOVA. Representative images are shown in (D). Scale bar 10 µm. (E-F) Western blot showing the expression of mitochondrial dynamics GTPases (E) or OPA1 oligomerization (F). Actin (E) or mtHSP70 (F, lower band – the upper band represents leftover OPA1 signal) were used as loading controls.

Article Snippet: For SDS-PAGE, 30 µg of proteins were mixed with 1X Lammeli buffer containing β-mercaptoethanol, then subjected to SDS-PAGE, transferred to a nitrocellulose membrane and blotted with the indicated antibodies (DRP1 (Anti-Mouse, 1:1000; BD Transduction Laboratories, #611112), Phospho-DRP1 (Ser616) (Anti-Rabbit, 1:1000; Cell signaling technology, (Clone D9A1) #4494S), MFN1 (Anti-Rabbit, 1:1000; Abcam, [EPR21953-74] #ab221661), OPA1(Anti-Mouse, BD Biosciences, #612606), NDUFA9(Anti-Rabbit, 1:1000; Abcam #ab128744), OSCP (Anti-Mouse, 1:1000; Santa Cruz Biotechnology (clone A-8) #sc-365162) UQCRC2 ((Anti-Mouse, 1:1000; Santa Cruz Biotechnology (clone G-10) #sc-390378) Phospho-FAK Y397 (Anti-Rabbit, 1:1000; ; Invitrogen, (clone 31H5L17) # 700255), FAK(Anti-Rabbit, 1:1000; ) TOM20 (Anti-Rabbit, 1:1000; Abcam, ab186735), SOX-2 (Anti-Rabbit, 1:1000 #AB5603 ), ALDH1A1 (Anti-Mouse, 1:1000, # SC-166362), HRP-tagged Actin (1:10,000).

Techniques: Suspension, Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: Extracellular matrix signals promotes actin-dependent mitochondrial elongation and activity

doi: 10.1101/2024.01.22.576703

Figure Lengend Snippet: (A-B) Mitochondrial elongation in response to cellular attachment to ECM substrates. MDA-MB-231 (A) and MCF7 (B) were spun down on coverslips either not coated or coated with poly-D-lysin (PDL) or fibronectin (FN) and incubated for the indicated times. mitochondria were marked with an antibody against TOM20 and mitochondrial length quantified in 3 independent experiments. Each point represents an individual experiment. Bars show the average ± SD. (C-D) Western blot showing the expression of the stem cell marker SOX2 (C) and mitochondrial dynamics GTPases in MDA-MB-231 cells grown as AM, MS or MS attached for 6 hours on fibronectin. Actin (C) or GAPDH (D) were used as loading controls. Western blot showing the lack of OPA1 oligomerization in MS cells in the absence or the presence of fibronectin. mtHSP70 was used as a loading control. (F-H) DRP1 knockdown does not affect mitochondrial structure in MDA-MB-231 mammospheres. (F) Western blot showing the knockdown of DRP1 in MDA-MB-231 cells. (G) Representative images showing mitochondrial structure (marked with an antibody against TOM20) in MDA-MB-231 cells knocked down for DRP1. Scale bar 10 µm. Mitochondrial length was quantified in 3 independent experiments (H) Each point represents an individual experiment. Bars show the average ± SD.

Article Snippet: For SDS-PAGE, 30 µg of proteins were mixed with 1X Lammeli buffer containing β-mercaptoethanol, then subjected to SDS-PAGE, transferred to a nitrocellulose membrane and blotted with the indicated antibodies (DRP1 (Anti-Mouse, 1:1000; BD Transduction Laboratories, #611112), Phospho-DRP1 (Ser616) (Anti-Rabbit, 1:1000; Cell signaling technology, (Clone D9A1) #4494S), MFN1 (Anti-Rabbit, 1:1000; Abcam, [EPR21953-74] #ab221661), OPA1(Anti-Mouse, BD Biosciences, #612606), NDUFA9(Anti-Rabbit, 1:1000; Abcam #ab128744), OSCP (Anti-Mouse, 1:1000; Santa Cruz Biotechnology (clone A-8) #sc-365162) UQCRC2 ((Anti-Mouse, 1:1000; Santa Cruz Biotechnology (clone G-10) #sc-390378) Phospho-FAK Y397 (Anti-Rabbit, 1:1000; ; Invitrogen, (clone 31H5L17) # 700255), FAK(Anti-Rabbit, 1:1000; ) TOM20 (Anti-Rabbit, 1:1000; Abcam, ab186735), SOX-2 (Anti-Rabbit, 1:1000 #AB5603 ), ALDH1A1 (Anti-Mouse, 1:1000, # SC-166362), HRP-tagged Actin (1:10,000).

Techniques: Cell Attachment Assay, Incubation, Western Blot, Expressing, Marker