Journal: bioRxiv

Article Title: CPT1A loss promotes lung metastasis in immune-competent mice via a mechanism of mtDNA release and chronic activation of STING pathway

doi: 10.64898/2026.05.01.722261

Figure Lengend Snippet: a , Immunofluorescence staining of dsDNA (red) and Tom20 (green) in control (sgNTC) and Cpt1a -KO (sgCpt1a) 4T1 cells. Scale bar 20µM. Cytosolic dsDNA coverage was determined as a percent (%) of total area. Unpaired Student’s t-test was performed. b-c , Fractionation of 4T1 control (Ctrl sg or sgNTC) or Cpt1a -KO (Cpt1a sg or sgCpt1a) cells was performed to collect the cytosolic fraction. b , Immunoblot to demonstration fractionation. Markers to denote mitochondrial (Mito., CPT1A and VDAC) and cytosolic (Cyto., cGAS and VINCULIN) fractions are shown. Whole cell lysates (WCL) serve as an input control. c , Relative levels of mitochondrial DNA (mtDNA) regions (Dloop1, Dloop2, mt-16S, mt-Nd4) (left) or nuclear DNA (nDNA, Tert) (right) in the cytosolic fraction was determined using RT-PCR. Unpaired Student’s t-tests were performed. d-e , Control (sgNTC) or Cpt1a -KO (sgCpt1a) 4T1 cells were treated with vehicle (VEH) or dideoxycytidine (ddC) to deplete mtDNA. n=3 per group. d , Relative levels of mtDNA regions (left) or nDNA (right) in the cytosolic fraction was determined using RT-PCR. Two-way ANOVA with Tukey’s post hoc; p=0.0181 (Dloop1), p=0.0109 (mt-16S), p=0.0312 (mt-Nd4), p=0.886 (Tert). e , Relative expression of Ifi44, Irf7, Lcn2 , and Slpi was determined by RT-PCR. Two-way ANOVA with Tukey’s post hoc; p=0.0104 (Ifi44), p=0.00637 (Irf7), p=5.45×10 -4 (Lcn2), p=0.00357 (Slpi). f , Schematic model of mtDNA release from the mitochondrial permeability transition pore (mPTP). CypD acts as regulatory subunit of the mPTP, located within the inner mitochondrial space. VDAC acts as a channel on the outer mitochondrial membrane (OMM). Palmitoylation of CypD at C202 (C203 in humans) prevents CypD from interacting with the mPTP to keep the pore in a closed state. g , Immunoblot of S-palmitoylated (S-pal) CypD in control (sgNTC) and Cpt1a -KO (sgCpt1a) 4T1 cells. Input shows total CPT1A and CypD levels. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ns, not significant.

Article Snippet: S-palmitoylation of CypD was assessed using the CAPTUREome S-Palmitoylated Protein Kit (Badrilla #K010-311) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Control, Fractionation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Permeability, Membrane

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: The second-generation hC3 rats express human, but not rat C3 in their plasma and tissues, at levels higher than those in the first-generation hC3 rats. (A) Plasma from both human and rat samples were collected, and the presence of human (hC3) and rat C3 (rC3) were evaluated by western blots. Purified human C3 (hC3), rat C3 (rC3), human C3 depleted serum (hC3-dpl), and C3 knockout rat serum rat plasma (rC3-KO) were used as controls. Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls. 1st gen, first generation; 2nd gen, second generation.

Article Snippet: In these assays, human and rat C3 proteins were detected using a complement C3 polyclonal antibody (MP Biomedicals, catalog no. 0855117) and a secondary anti-goat immunoglobulin G (IgG) (heavy chain+light chain [H+L]) polyclonal antibody (SouthernBiotech, catalog no. 6160-05).

Techniques: Clinical Proteomics, Western Blot, Purification, Knock-Out, Enzyme-linked Immunosorbent Assay, Reverse Transcription

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: C3 inhibitor AMY-101 inhibits the alternative but not classical pathway complement-mediated hemolysis in the second-generation hC3 rats in vitro. (A) The C3 inhibitor AMY-101 (0.3-20μM) was incubated with rabbit RBCs in the presence of 80% WT rat serum, or second-generation hC3 rat serum in Mg 2+ -EGTA buffer at 37°C for 30 minutes, then hemolysis was quantitated, showing that AMY-101 almost completely inhibited the second-generation hC3 rat alternative pathway-mediated hemolysis at a concentration as low as 2.5μM while did not affect the WT rat alternative pathway-mediated hemolysis at a concentration as high as 20μM. (B) The C3 inhibitor AMY-101 (0.3-10μM) were incubated with antibody-sensitized sheep RBCs in the presence of 10% second-generation hC3 rat serum in GVB ++ buffer at 37°C for 30 minutes, then hemolysis was quantitated, showing that AMY-101 did not have any effect on the second-generation hC3 rat classical pathway-mediated hemolysis at a concentration as high as 10μM. 2nd gen, second generation.

Article Snippet: In these assays, human and rat C3 proteins were detected using a complement C3 polyclonal antibody (MP Biomedicals, catalog no. 0855117) and a secondary anti-goat immunoglobulin G (IgG) (heavy chain+light chain [H+L]) polyclonal antibody (SouthernBiotech, catalog no. 6160-05).

Techniques: In Vitro, Incubation, Concentration Assay