Journal: iScience

Article Title: Exogenous sphingomyelinase mediates MSC-derived EV biogenesis and enhances potency via repackaging of molecular cargo

doi: 10.1016/j.isci.2026.114992

Figure Lengend Snippet: SMase-treated MSC-derived EVs enhance angiogenic potential and immunomodulatory function (A) Representative phase-contrast images showing microvascular network formation in response to control media, untreated (UT) and SMase-treated MSC conditioned media, and their respective EVs (scale bars, 500 μm). (B–E) Quantitative analysis of network formation parameters showing enhanced (B) number of nodes, (C) number of meshes, (D) number of branches, and (E) total length in all conditions. (F) Representative images of macrophage response to LPS stimulation in the presence of UT or SMase MSC-derived EVs (scale bars, 200 μm). (G) Quantification of TNF-α production following treatment and LPS-stimulation in macrophages. Single-cell flow cytometry performed on wounded tissues collected from mice treated with hydrogels, UT-EV hydrogels, or SMase-EV hydrogels at day 7 post-injury. (H) UMAP projection of single-cell flow cytometry data illustrating lymphoid cell marker expression intensity from low (black) to high (copper) and corresponding cell density from low (blue) to high (red). (I–L) (I) Stacked bar graph depicting quantified CD11b − immune cell populations. T cell populations were identified and classified as (J) CD3 + T cells, (K) CD4 + helper T cells, and (L) CD8 + cytotoxic T cells. Data presented as mean ± SEM. Statistical significance indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n = 3–4.

Article Snippet: RAW 264.7 (TIB-71, ATCC) murine macrophages were seeded at 40,000 cells/cm 2 and cultured in RPMI 1640 medium (11875093, GibcoTM) supplemented with 10% heat inactivated FBS and 1% penicillin-streptomycin.

Techniques: Derivative Assay, Control, Single Cell, Flow Cytometry, Marker, Expressing

Journal: bioRxiv

Article Title: Reversible Dissociation of Mitochondrial Complex V Balances Anabolic and Energy-Generating Needs in Cancer

doi: 10.1101/2025.08.05.668642

Figure Lengend Snippet: (A). mtDNA content of livers and HBs generated by Y S127A + 14 patient-derived β-catenin mutants (including B [Δ90]) . Each point shows the mean of duplicate TaqMan assays for mtDNA content, performed in triplicate with 2 different sets of mtDNA probes , . (B). Complex I activities of the tissues depicted in A measured as OCRs in response to pyruvate, malate, glutamate and ADP. The results were adjusted to account for the mtDNA differences shown in A. OCRs were quantified with an Oroboros respirometer as previously described , . (C). Pyruvate dehydrogenase (PDH) activity as measured by the oxidation of 1- 14 C-pyruvate to acetyl-CoA and 14 CO 2 , , , . Results were adjusted to account for mtDNA content differences as in (B). (D). Composite heat map showing relative expression levels of transcripts encoding subunits of CI-CIV of the ETC in livers and 4 sets of HBs from panels A-C. (E). Composite heat map showing relative expression of the transcripts encoding subunits of CI-CIV of the ETC during the course of MYC -induced HCC induction, regression and recurrence . (F). Transcripts encoding mitochondrial proteins (other than those for CI-CV) in a subset of the murine livers and HBs shown in (A-C). (G). Transcripts encoding mitochondrial proteins (other than those for CI-CV) in livers and murine HCCs.

Article Snippet: To evaluate the efficiency of TALED-mediated mutagenesis, a 504 bp fragment of murine mitochondrial DNA (mtDNA) spanning the Mt-atp6/8 gene was amplified with Platinum SuperFi DNA polymerase (Thermo Fisher) from 10 ng of total cellular DNA in a 50 μL PCR reaction using the following primers : FWD: 5’-CACATACATTTACACCTACTACCC-3’ and REV: 5’- GTTAGAAGGAGGGCTGAAAAGG-3’.

Techniques: Generated, Derivative Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Reversible Dissociation of Mitochondrial Complex V Balances Anabolic and Energy-Generating Needs in Cancer

doi: 10.1101/2025.08.05.668642

Figure Lengend Snippet: (A). NDGE and in situ ATPase assays performed on ETC complexes isolated from BY1 and BY3 HB cell lines . (B). Immunoblot analysis for Mt-atp6 and Atp5f1a (α subunit of CV) in BY1 and BY3 cells. Control liver and BY HB lysates were used as controls to show that the cell lines retained the low-level expression of Mt-atp6 associated with tumors . (C). Over-exposure of the ATPase assay performed in (A). The adjacent cartoons depict the CV-related proteins that were identified in the bands excised from the indicated regions of the gel using qualitative protein MS. (D). In vitro growth curves of BY1 and BY 3 cells. Each point represents the mean of 6 replicas +/- 1 S.E. (E). BY1 and BY3 tumor growth rates. 10 6 cells of each type were grown as subQ tumors in FVB mice. Tumor volumes were measured at the times indicated. n=4 mice/group. (F). Structural relationships between Mt-atp6 and the c-subunit ring (from ref. 27). The cradling of the c-ring by the α5 and α6 helices of Mt-atp6 forms part of the proton channel , . (G). Mt-atp8 and Mt-atp6 immunoblots in BY1 cells. Lane 2: total lysate from BY1 cells stably expressing murine Mt-atp8-Flag tag protein. The precursor protein (not seen) contained a mitochondrial targeting sequence (MTS) at its N-terminus. Only the completely processed C-terminal Flag-tagged protein is seen. Lane 3: total lysate of BY1 cells stably transfected with a control, empty SB vector. Lane 4: Total lysate of BY1 cells transiently transfected with a SB vector encoding a murine Mt-atp6-c subunit-V5 fusion protein. As with the Mt-atp8 protein, no precursor protein containing the localization signal was detected. Different regions of the blot were probed with anti-FLAG and anti-V5 antibodies to allow for detection of both MT-ATP8-FLAG and MT-ATP6-V5 proteins. (H). Stable expression of Mt-atp6-c-V5 fusion protein from A in purified BY1 and BY3 mitochondria. Lane 1: a mitochondrial lysate from WT BY1 cells transfected with an empty vector served as a negative transfection control. The Mt-atp6-c blot was probed with an anti-V5 antibody. (I). TALED-generated mutations of the Mt-atp6 gene. BY3 cells were transiently transfected with TALED vectors and a control vector expressing GFP. The EGFP+ population was then purified by FACS on day 2 and evaluated for Mt-atp6 heteroplasmy at the times indicated. For this, a 504 bp fragment of mtDNA spanning the Mt-atp6 gene coding sequence was amplified from BY3-TALED cells or WT-BY3 cells. The fragments were melted, re-annealed and either digested with T7 endonuclease (+) or not (-) followed by 2% agarose gel electrophoresis. Sequencing of the PCR products obtained on ∼day 23 documented the expected mutations at a total frequency of 37% (Supplementary File 1). (J). Immunoblots of endogenous Mt-atp6 protein in WT BY3 and BY3-TALED cells on day 14.

Article Snippet: To evaluate the efficiency of TALED-mediated mutagenesis, a 504 bp fragment of murine mitochondrial DNA (mtDNA) spanning the Mt-atp6/8 gene was amplified with Platinum SuperFi DNA polymerase (Thermo Fisher) from 10 ng of total cellular DNA in a 50 μL PCR reaction using the following primers : FWD: 5’-CACATACATTTACACCTACTACCC-3’ and REV: 5’- GTTAGAAGGAGGGCTGAAAAGG-3’.

Techniques: In Situ, Isolation, Western Blot, Control, Expressing, ATPase Assay, In Vitro, Stable Transfection, FLAG-tag, Sequencing, Transfection, Plasmid Preparation, Purification, Generated, Amplification, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Reversible Dissociation of Mitochondrial Complex V Balances Anabolic and Energy-Generating Needs in Cancer

doi: 10.1101/2025.08.05.668642

Figure Lengend Snippet: (A). Appearance of free F 1 in NIH3T3 fibroblasts during Rho0 cell generation. Cells were maintained in 30 or 60 ng/ml of EtBr for 2 days (lanes 2 & 3) and then switched to EtBr-free medium for 2 wks to allow for recovery (lanes 4&5). At each point, in situ ATPase assays were performed. (B). mtDNA content of EtBr-treated cells described in (A) and normalized to that of the nuclear-encoded Mlx gene . (C). Immuno-blot showing that mitochondrial-encoded Mt-atp6 but not nuclear-encoded Atp5f1a α subunit is depleted in a dose-dependent manner by EtBr (+EtBr) and then restored following subsequent recovery in EtBr-free medium (-EtBr). (D). Immunoblot showing time-dependent Mt-atp6 depletion in NIH3T3 cells in response to doxycycline or chloramphenicol treatment. (E). In situ ATPase assays performed on NIH3T3 cells exposed to doxycycline (10 μg/ml) or chloramphenicol (25 μg/ml) for 48 hr. (F). Stable expression and mitochondrial localization of Mt-atp6-c in NIH3T3 cells. Mitochondria from control (WT) cells or those stably expressing the Mt-atp6-c fusion protein were purified as described in . Loading control immuno-blots were performed for the mitochondrial-localized proteins Pdha1a and ATP5fa1. (G). Identification of TALED mutations in NIH3T3 cells using the T7 endonuclease assay described in . (H). Enforcing Mt-atp6-c or mutating endogenous Mt-atp6 with TALEDs, respectively, alters NIH3T3 cell tsusceptibility to EtBr-mediated F o -F 1 dissociation. In situ ATPase assays were performed on control, untreated NIH3T3 cells or on cells cultured in EtBr (30 ng/ml) as described in (A). (I) Hypoxia and chloramphenicol reduce ATP t 1/2 in BY3 cells. Cells stably expression Mito-targeted iATPSnFR2HaloTag were exposed to 1% oxygen or 25 mg/ml chloramphenicol for 48 hr. They were then stained with Janelia Fluor JFX650 HaloTag® Ligand and subjected to moving average continuous flow cytometry as described in .

Article Snippet: To evaluate the efficiency of TALED-mediated mutagenesis, a 504 bp fragment of murine mitochondrial DNA (mtDNA) spanning the Mt-atp6/8 gene was amplified with Platinum SuperFi DNA polymerase (Thermo Fisher) from 10 ng of total cellular DNA in a 50 μL PCR reaction using the following primers : FWD: 5’-CACATACATTTACACCTACTACCC-3’ and REV: 5’- GTTAGAAGGAGGGCTGAAAAGG-3’.

Techniques: In Situ, Western Blot, Expressing, Control, Stable Transfection, Purification, Cell Culture, Staining, Flow Cytometry