Journal: Bioactive Materials

Article Title: Near infrared enhanced palladium loaded siraitia grosvenorii carbon dots amplify mitophagy for acute lung injury immunotherapy

doi: 10.1016/j.bioactmat.2026.02.040

Figure Lengend Snippet: ALI therapeutic mechanism. A) Co-immunofluorescent staining images of cells incubated with Cy5-CPs or Cy5-CPs@SS31 by confocal staining microscope: DAPI (blue), Mito-tracker green (green), and Cy5-CPs or Cy5-CPs@SS31 (red). (Scale bar = 10 μm) B) MMP of treated cells by flow cytometry. C) Heatmap of DEGs between control group and CPs@SS31+NIR: genes with relatively high (red) and low (blue) expression levels. D) Volcano plot of DEGs: 542 upregulated and 499 downregulated genes between control group and CPs@SS31+NIR. E) Anti-inflammation pathways related DEGs by KEGG enrichment analysis (red). F) Anti-inflammation pathways related differential biological functions by GO enrichment analysis (red): molecular function (MF), biological process (BP) and cell component (CC). G) Protein-protein interaction network of mitophagy related proteins. H) P62, Parkin and PINK1 expression levels of treated cells by WB. I) P62, Parkin and PINK1 expression levels in the lung tissue of treated rats. (Scale bar = 100 μm).

Article Snippet: Furthermore, the membranes were separately incubated with the primary antibody (anti-P62, Parkin, PINK1 and GAPDH, Proteintech, China) overnight at 4 °C.

Techniques: Staining, Incubation, Microscopy, Flow Cytometry, Control, Expressing

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Proteomic workflow in HEK293 cells with doxycycline-inducible expression of PKA Cα wild-type (WT) or W197R mutant (MUT). (B) The top 8 significantly enriched KEGG pathways within the mitochondrial quality control category identified from the proteomic dataset. (C) Schematic of the mt-mKeima mitophagy reporter. (D) Representative Airyscan live-cell imaging of THP-1 macrophages expressing mt-mKeima sensor treated with 1 µM PGE 2 in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi) for 16 hours. Scale bar, 10 µm. (E) Quantification of mitolysosome numbers shown in (D) (n = 10). Data were quantified from one representative experiment of three. (F) Representative images of live-cell 4D lattice light sheet imaging on THP-1 macrophages treated as in (D) . Scale bar, 20 µm. (G-H) Quantification of net mitolysosome displacement (n = 10) (G) and average mitolysosome speed (n = 12) (H) from imaging in (F) . (I) THP-1 macrophages treated with PGE 2 at indicated concentrations in the presence or absence of 1 µM PKA inhibitor (PKAi) for 16 hours. Mitochondrial fractions were isolated and subjected to immunoblotting with indicated antibodies. Band intensities were quantified and normalized to COX4 expression for PINK1 (J) and pUB Ser65 (K) (n = 3). (L) Working model illustrating that PGE 2 induces mitophagy and mitochondrial biogenesis to enhance mitochondrial homeostasis in a EP4- and PKA-dependent manner. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibody for PINK1 (BC100-494) was purchased from Novus Biologicals, and Flag-M2 (F3165) from Millipore-Sigma.

Techniques: Expressing, Mutagenesis, Control, Live Cell Imaging, Imaging, Isolation, Western Blot

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) THP-1 macrophages were treated with 1 µM PGE 2 alone, 1 µM forskolin and 50 µM IBMX alone, or infected with HSV-1 (MOI = 1) in the presence or absence of PGE 2 or forskolin/IBMX. At 16 h.p.i, cell lysates were collected and subjected to immunoblotting with indicated antibodies. (B) Representative Airyscan live-cell imaging of THP-1 macrophages transfected with either control siRNA (siCTRL) or TFAM siRNA (siRNA) in the absence or presence of 1 µM PGE 2 for 16 hours. Scale bars, 2 µm. (C) Quantification of cytosolic PicoGreen from images in (B) (n = 10). Data were quantified from one representative experiment of three. (D) Schematic showing the workflow to quantify cytosolic mitochondrial DNA using qPCR. (E) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siRNA) in the presence or absence of 1 µM PGE 2 for 16 hours. Cytosol fractions were isolated and subjected to qPCR to assess the presence of mt-Dloop and mt-ND1 regions (n = 3). (F) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, cytosol fractions were isolated and subjected to qPCR to assess the presence of mt-Dloop and mt-ND1 regions (n = 3). (G) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or PINK1 siRNA (siPINK1), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, cytosol fractions were isolated and subjected to qPCR to assess the presence of mt-Dloop and mt-ND1 regions (n=3). (H) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or PINK1 siRNA (siPINK1), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, whole cell lysates were collected and subjected to RT-qPCR to assess mRNA levels of IFNβ (n = 3). (I) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or PINK1 siRNA (siPINK1), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, whole cell lysates were collected and subjected to qPCR to assess HSV-1 UL30 genomic abundance (n=3). All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibody for PINK1 (BC100-494) was purchased from Novus Biologicals, and Flag-M2 (F3165) from Millipore-Sigma.

Techniques: Infection, Western Blot, Live Cell Imaging, Transfection, Control, Isolation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Network of mitochondrial quality control proteins interacting with wild-type (WT) and mutant (MUT) PKA Cα with BFDR σ; 0.2. Interactors are colored by log 2 fold change (WT/MUT spectral counts) with WT-specific interactors in dark purple and MUT-specific interactors in dark green. Edges to the central bait (yellow) represent interactions detected in this study. (B) Whole cell lysates of THP-1 macrophages were subjected to pulldown using 8-AHA-cAMP (RIα), Rp-8-AHA-cAMPS (holoenzyme), or HaloLink resin (control), followed by immunoblotting with indicated antibodies. (C-D) THP-1 macrophages were stimulated with 1 µM PGE 2 for 6 hours, followed by incubation with 1 µM cycloheximide for the indicated times. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. Representative blots from three independent experiments are shown (C) . Band intensities for STOML2 were quantified and normalized to HSP90 intensity (n = 3) (D) . (E-G) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by treatment with PGE 2 at indicated concentrations for 16 hours. Mitochondrial fractions were isolated and subjected to immunoblotting with the indicated antibodies (E) . Band intensities for PINK1 (F) and pUB Ser65 (G) were quantified and normalized to COX4 intensity (n = 3). (H) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, cytosol fractions were isolated and subjected to qPCR to assess the presence of mt-Dloop and mt-ND1 regions (n=3). (I) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, whole cell lysates were collected and subjected to RT-qPCR to assess mRNA levels of IFNβ (n=3). (J) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or STOML2 siRNA (siSTOML2), followed by mock or HSV-1 infection in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, whole cell lysates were collected and subjected to qPCR to assess HSV-1 UL30 genomic abundance (n=3). All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibody for PINK1 (BC100-494) was purchased from Novus Biologicals, and Flag-M2 (F3165) from Millipore-Sigma.

Techniques: Control, Mutagenesis, Western Blot, Incubation, Transfection, Isolation, Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) HEK293 cells expressing STOML2-3xFlag were treated with 1 µM forskolin and 50 µM IBMX for the indicated times. Whole cell lysates were subjected to immunoprecipitation using DYKDDDDK Fab-Trap agarose and immunoblotting with the indicated antibodies. (B) Top: domain architecture of STOML2 and predicted PKA phosphorylation sites (Scansite 4.0; PhosphoSitePlus). Bottom: ClustalW alignment showing evolutionary conservation surrounding STOML2 Ser29. (C) Representative structural model of the interaction between the active pocket of PKA Cα and STOML2 N-terminal (aa 18 to 37) (ipTM = 0.9). Gray: PKA Cα, green: PKA Cα residue D167, yellow: STOML2, magenta: STOML2 residue S29. (D) HEK293 cells expressing either STOML2 wild-type (WT) or S29A mutant (MUT) were treated with 1 µM forskolin and 50 µM IBMX for 6 hours. Cell lysates were subjected to immunoprecipitation using DYKDDDDK Fab-Trap agarose and immunoblotting with the indicated antibodies. (E) Schematic illustrating the generation of THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) via lentivirus transduction. (F) THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) were stimulated with 1 µM PGE 2 for 16 hours. Cell lysates were subjected to immunoprecipitation using DYKDDDDK Fab-Trap agarose and immunoblotting with the indicated antibodies. (G) Representative Airyscan live-cell imaging of THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) together with the mt-mKeima sensor were stimulated with 1 µM PGE 2 for 16 hours. Scale bar, 10 µm. (H) Quantification of mitolysosome numbers from (G) (n = 10). (I) THP-1 macrophages expressing either STOML2 wild-type (WT) or S29A mutant (MUT) were stimulated with 1 µM PGE 2 for 16 hours. Mitochondrial fractions were collected and subjected to immunoblotting with indicated antibodies. (J) A schematic illustrating that PKA activation promotes STOML2 phosphorylation at Ser29 which subsequently induces PINK1-mediated mitophagy in response to PGE 2 stimulation. All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: Antibody for PINK1 (BC100-494) was purchased from Novus Biologicals, and Flag-M2 (F3165) from Millipore-Sigma.

Techniques: Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Residue, Mutagenesis, Transduction, Live Cell Imaging, Activation Assay