gfp mcherry cox8  (Addgene inc)

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A) NTC or APOL3 -/- HeLa cells primed overnight with IFN-γ, were pulsed with LLOME (600 μM, 2 hr unless otherwise indicated), recovered for 8 h and analyzed by RNAseq. Volcano plots show differential expression between untreated (DMSO) and LLOME treated for both genotypes. Significant genes (P < 0.01, log 2 -fold change (LFC) > 2) are depicted in rose (upregulated) or purple (downregulated). Selected genes of interest were annotated (ISGs in teal). Statistical significance was calculated using the Wald test, with P values adjusted by the Benjamini-Hochberg procedure. (B) NTC or APOL3 -/- HeLa cells expressing pMSCV-empty or pMSCV- APOL3 were pulsed with LLOME and IFN-β protein levels in the supernatants determined by ELISA after 12 hr. (C) HeLa genotypes expressing IRF3-luciferase +/- IFN-γ priming pulsed with LLOME or treated with Poly(I:C) and luminescence measured after 6 hr. Data is fold change relative to mock treated. (D) IFN-β in the supernatants of primed or unprimed NTC or APOL3 -/- HeLa cells after the indicated insult. (E and F) IFN-β (E) or IL-1β (F) in the supernatants of IFN-γ/IL-1β-primed THP-1 macrophages 24 hr after being fed SiO 2 nanoparticles (50 μg/ml for 4 hr) to trigger phagosomal rupture. APOL3 was expressed in trans via lentivirus transduction. (G) IFN-β levels in the supernatants of NTC or APOL3 -/- cells infected (8 hr) with invasive (SPI-1), hyper-invasive (Δ sifA ), or non-invasive (SPI-2) Salmonella Typhimurium ( Stm ), invasive adenovirus (AdV), or non-invasive AdV- ts1 . (H) HeLa genotypes +/- IFN-γ priming infected with AdV-GFP (24 hr) and imaged (representative images shown). Poly(I:C) or anti-IFNAR antibodies were co-incubated for the duration. Percentage values denote mean ± SD, n = 3) (I) IFN-β in the supernatants of IFN-γ-primed HeLa cells of the indicated genotypes 24 hr after LLOME pulse. Representative immunoblots for each genotype are shown. Data represent mean ± SEM from 3 or 4 independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Quantitative Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Luciferase, Transduction, Infection, Incubation, Western Blot, Comparison

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A) IFN-γ-primed HeLa genotypes expressing FLAG-cGAS or FLAG-GFP were treated with LLOME followed by extraction of DNA from FLAG immunoprecipitants and mitochondrial ( ND1 , ND2 ), nuclear ( L1 , PG ) or ribosomal ( 18S ) transcripts. Fold change (FC) is relative to mock treated. (B) IFN-β produced by IFN-γ-primed APOL3 -/- cells rescued with retroviral- APOL3 and cultured with ddC to deplete mtDNA prior to treatment. (C) qPCR quantification of mtDNA in the cytosol relative to whole cell extract (WCE) after treatment with LLOME or ABT-737/Q. Shown are floating bars (mean, min/max, n = 5 independent experiments) (D) APOL3 mnGFP knock-in cells expressing OMP25 JF646 and Lamp1-mCherry were pulsed with LLOME and imaged at the indicated time. Fractional overlap for APOL3 is shown (n = 25 cells, error bars ± SD). Results are representative of 3 independent experiments. (E) IFN-γ/IL-1β-primed THP-1 macrophages expressing APOL3-GFP were fed SiO 2 particles (50 μg/ml, 3 hr) and immunostained for Tom20 or Lamp1. (F) APOL3 mnGFP cells infected with SPI-1 Stm (DAPI) for 3 hr and mitochondria labeled with OMP25 JF646 . Images are representative single z planes of Thunder deconvolved widefield images. Scale bar, 5 μm. Data are mean ± SEM from 3 (A) or 4 (B) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 determined by two-tailed unpaired Student’s t tests (A, D) or one-way ANOVA with Tukey’s multiple comparison test (B, C).

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Expressing, Extraction, Produced, Retroviral, Cell Culture, Knock-In, Infection, Labeling, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A, B) IFN-γ-primed HeLa cells expressing cytosolic GFP (CytoGFP) and mitochondrial mCherry (MitoCherry) were treated with LLOME or ABT-737/Q with heterodimerizer and imaged. Total mitochondrial volume (MitoCherry) overlapping with cytoGFP is shown (mean ± SD from 15 (A) or 50 (B) cells. (C) IFN-γ-primed NTC or BAK/BAX -/- cells expressing APOL3-GFP, OMP25 646 , or Lamp1-mCherry treated with LLOME. Fractional overlap for APOL3 is shown (n = 25 cells, error bars ± SD). (D) qPCR of cytosolic mtDNA relative to WCE in IFN-γ-primed NTC or BAK/BAX -/- cells treated with LLOME. (E) IFN-γ-primed BAK/BAX -/- cells as in (C) were treated with LLOME with or without CCCP. Fractional overlap for APOL3 is shown (n = 11 cells, error bars ± SD). (F) qPCR quantification of mtDNA in the cytosol relative to WCE of IFN-γ-primed BAK/BAX -/- cells with the indicated agonist. (G) Representative immunoblots of cytosolic (cyto) or mitochondrial (mito) fractions prepared from IFN-γ-primed HeLa genotypes treated with LLOME or ABT-737/Q. (A), (B), (C), (E) are representative or >3 independent experiments. (D) and (F) are floating bars (mean, min/max, n = 3 or 4 independent experiments). ** P < 0.01; *** P < 0.001 determined by one-way ANOVA. Images are maximum intensity projections (A, B, E) or single z planes (C) of Thunder deconvolved widefield images. Scale bar, 5 μm.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A) IFN-γ-primed HeLa cells co-expressing the indicated variant of APOL3-GFP and OMP25 JF646 treated with LLOME. Volume of OMP25 overlapping with APOL3 is shown (n = 20 cells, mean ± SD). Images are single z planets of Thunder deconvolved widefield images and are representative of 3 independent experiments. Scale bar, 5 nm. (B and C) IFN-γ-primed APOL3 -/- cells rescued with the indicated APOL3 variant were treated with LLOME (2 hr) and mtDNA in the cytosol relative to WCE determined by qPCR (B) or luminescence detected in cells expressing IRF3-luciferase after an additional 4 hr (C). (D) APOL3 -/- or NTC cells +/- IFN-γ priming rescued with the indicated APOL3 variant and infected with Stm Δ sifA for 6 hr or AdV for 12 hr. FC relative to mock treated for each gene was determined by qPCR (n = 2). (E and F) IFN-γ/IL-1β-primed APOL3 -/- THP-1 macrophages rescued with the indicated APOL3 variant were fed SiO 2 particles to induce phagosomal rupture. Cytosolic mtDNA relative to WCE was determined by qPCR (E), or IFN-β measured by ELISA after 24 hr (F). (B) and (E) are floating bars (mean, min/max, n = 5 independent experiments). (C) and (F) are mean ± SEM from 3 or 4 independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Expressing, Variant Assay, Luciferase, Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A, B) IFN-γ-primed HeLa cells pulsed with ABT-737 or LLOME with or without pan-caspase inhibition (Q-VD-Oph) and analyzed by Flow cytometry for Annexin-V staining or IFN-β production by ELISA, both after 24 hr. (C) Time course immunoblots of cytosolic or mitochondrial fractions from IFN-γ-primed HeLa cells incubated with LLOME or ABT-737. Blots are representative of 3 independent experiments. (D) IFN-γ-primed NTC or APOL3 -/- cells expressing cytochrome c – GFP and TFAM-mScarlet treated with ABT-737/Q or LLOME. Images are maximum intensity projections, representative of 3 independent experiments. Quantification is mean ± SEM from 3 independent experiments (n = 20 cells per replicate). Scale bar, 5 μm. (E) IFN-γ-primed NTC or APOL3 -/- HeLa cells expressing FLAG-cGAS were pulsed with LLOME (2 hr) at the indicated concentration. 2 hr later cell death was measured by PI uptake (right y axis, rose) followed by qPCR for mtDNA in FLAG immunoprecipitants (left y axis, black). Where indicated treatments included Q-VD-Oph. (B) and (E) are mean ± SEM from 3 independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Inhibition, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A and B) Cardiolipin immunofluorescence (single z planes) in IFN-γ-primed wildtype HeLa cells (+/- LLOME) permeabilized with saponin or Trition-X100 (A) or in NTC and PLSCR3 -/- cells + LLOME using saponin permeabilization (B). Quantification represents mean ± SEM from 3 independent experiments (n = 20 cells per replicate). Immunoblots (IB) are representative of 2 experiments. (C) Representative Airyscan image of IFN-γ-primed LLOME-treated APOL3 mnGFP cells stained for OMP25 with JF646 and CL. Single z slices and line profile is shown. (D) APOL3-GFP expressed in IFN-γ-primed NTC or PLSCR3 -/- cells treated with LLOME and immunostained for Tom20 and Lamp1. Fractional overlap for APOL3 is shown (n = 12 cells, representative of 3 independent experiments, error bars ± SD). Maximum intensity projections of Thunder deconvolved z stacks are shown. (E) qPCR quantification of mtNDA in the cytosol versus WCE in IFN-γ-primed HeLa genotypes treated with LLOME. Shown are floating bars (mean, min/max, n = 5 independent experiments). (F) Mitochondria isolated from NTC or PLSCR3 -/- cells treated with rAPOL3 +/- rBAX and mtDNA release detected by PicoGreen fluorescence. Data are mean ± SEM (n = 3). (G, H, I) Primary human airway epithelial cells (AECs) primed with IFN-γ (or not) were treated with TiO2 nanoparticles (24 hr) and IFN-β measured in the supernatants by ELISA (G). AECs were transduced with lentivirus encoding Cas9 and sgRNAs targeting APOL3 , PLSCR3 , or cGAS or cultured with ddC prior to treatment (H). Alternatively, AECs were stained with Tetramethylrhodamine Methyl Ester (TMRM) after 12 hr to probe mitochondrial membrane potential (ΔΨm) and quantified by flow cytometry (MFI: median fluorescence intensity). (G, H, I) represent the mean ± SEM with AECs prepared from three different organ donors. * P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA (B, E, G, H, I) or two-tailed unpaired t test (D).

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Immunofluorescence, Western Blot, Staining, Isolation, Fluorescence, Enzyme-linked Immunosorbent Assay, Transduction, Cell Culture, Membrane, Flow Cytometry, Two Tailed Test

Journal: Cell reports. Medicine

Article Title: Bioengineering the metabolic network of CAR T cells with GLP-1 and Urolithin A increases persistence and long-term anti-tumor activity.

doi: 10.1016/j.xcrm.2025.102021

Figure Lengend Snippet: Figure 1. Functional and metabolic characterization of CAR T cells after repeated tumor re-challenge (A) Schematic of the lymphoma model. Mice were injected with Raji cells (0.5 3 106) on day 0, followed by CAR T cell (5 3 106) administration on day 5. TR was done on days 12, 19, and 26 with 0.2 3 106 Raji cells. Blood was collected on days 14, 21, and 28. (B) Sorting and flow cytometry gating of human CD3+ CAR T cells from blood samples across time points (3 mice were pooled for a total of 24 in each group to obtain n = 8 per condition). (C–F) Flow cytometric analysis showing expression of Granzyme B (C), Perforin (D), PD-1 (E), and LAG-3 (F) in CON and TR groups over time (n = 8; mean ± SD). The data are represented as mean fluorescence intensity (MFI) calculated from the flow cytometry histograms. (G–I) Percentage of CAR T cells in blood tracked over time (G). IL-2 (H) and IFN-g (I) secretion quantified from co-culture of CAR T cells with Raji cells for 24 h (n = 8). (J and K) Human CD8+ T cell subsets (Tn, Tscm, Tcm, Tem, and Teff) in CD3+ T cells measured by flow cytometry in TR vs. CON groups at each time point. The pie chart shows the percentage of Tex in the Teff population. (L and M) Sorting of human CD8+ cells from pooled blood samples (3 mice were pooled for a total of 24 in each group to obtain n = 8 per condition). Representative panels showing the purity of CD3+ and CD8+ cells. (N and O) Live-cell Ca2+ imaging showing representative images (after 300 s) of cells transduced with the mitochondrial Ca2+ indicator (GCaMP6f) and cytosolic (RCaMP), upon histamine stimulation, which was confirmed by quantitative analysis over time. (P and Q) OCR and ECAR measured using Seahorse XF in CON and TR CAR T cells with respective bar graphs showing the basal respiration (n = 6). (R1) Representative image of mtDNA, indicated by staining the cells anti-TFAM (Mitochondrial transcription factor A). (R2) Quantitative analysis of images (n = 11 images) was done and represented as integrated density of TFAM signal. (S) Fluorescent assessment of mitophagy by tracking LTDR/MTG ratios in CON and TR CAR T cells following FCCP treatment for 60 min. (T1) Representative images of CAR T cells, which were transduced with LC3-mCherry and mito-GFP showing co-localization of LC3 (red) with mitochondria (green). Blue arrowheads show co-localization, and white arrowheads indicate lack of co-localization. Cells were treated with FCCP for 30 min. (T2) Quantitative analysis of the LC3 images (n = 10 images). (U) p62, Beclin-1, and Atg14 expression measured by flow cytometry in CON and TR CAR T cells (n = 6). Data represents mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. A non-parametric t test and one-way ANOVA were used for statistical analysis. Scale bars: 10 mm.

Article Snippet: The mitophagy reporter was generated by PCR amplification of the Cox8 EGFP mCherry from pCLBW Cox8 EGFP mCherry (Addgene #78520) followed by cloning in our in-house LV vector backbone.

Techniques: Functional Assay, Injection, Cytometry, Expressing, Co-Culture Assay, Imaging, Transduction, Staining

Journal: Cell Reports Medicine

Article Title: Bioengineering the metabolic network of CAR T cells with GLP-1 and Urolithin A increases persistence and long-term anti-tumor activity

doi: 10.1016/j.xcrm.2025.102021

Figure Lengend Snippet:

Article Snippet: pCLBW cox8 EGFP mCherry , Rojansky et al. , Addgene #78520.

Techniques: Virus, Recombinant, Lysis, Modification, Activity Assay, Cobalt Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, shRNA, Plasmid Preparation, Sequencing, Software, In Vivo Imaging