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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 <t>of</t> <t>S-palmitoylated</t> (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.
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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 <t>of</t> <t>S-palmitoylated</t> (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.
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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 <t>of</t> <t>S-palmitoylated</t> (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.
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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.

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