Journal: Cell Death & Disease

Article Title: IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype

doi: 10.1038/s41419-023-05957-z

Figure Lengend Snippet: a , b Representative electron microscopy images of colonic epithelium. A white arrow in ( b ) shows an inclusion body. c Representative blot of MIC60 expression in two independent preparations of the colon of CRL and IF1-KO mice is shown. α-Tubulin is shown as a loading control. Box-and-whisker plot shows MIC60 expression relative to α-Tubulin ( n = 4). d Box-and-whisker plots show the quantification of inclusion bodies per mitochondria ( n = 37–43 mitochondria in 4 mice per genotype). e Histograms show mean ± SEM of the intramitochondrial Ca 2+ content of CRL and IF1-KO mice ( n = 3). f Box-and-whisker plots show adenine nucleotide content in colon mitochondria of CRL and IF1-KO mice ( n = 4). g Histograms show mean ± SEM of mitochondrial membrane potential (∆Ψm) in colonocytes from CRL ( n = 4) and IF1-KO ( n = 3) mice. The effect of oligomycin (OL) is shown. h Ca 2+ efflux rates in colon mitochondria. Left, representative traces of the fluorescence intensity of Ca 2+ -green after 3 pulses of 2 nmol Ca 2+ . The rate of Ca 2+ efflux was recorded after the addition of 0.2 µM ruthenium red (RR) in mitochondria of CRL ( n = 5 assayed in duplicate) and IF1-KO ( n = 4 assayed in duplicate) mice. Right, histograms show the Ca 2+ efflux rate mean ± SEM. i Ca 2+ retention capacity (CRC) in colon mitochondria of CRL ( n = 3–5) and IF1-KO ( n = 5) mice. Left, representative traces of the fluorescence intensity of Ca 2+ -green in the presence or absence of cyclosporine A (CsA). Mitochondria were challenged with the addition of 2 nmol Ca 2+ . Right, box-and-whisker plots show the amount of Ca 2+ required to induce PTP opening. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 when compared according to the Student’s t -test. See also Supplementary Fig. S .

Article Snippet: Ca 2+ content of isolated mitochondria was determined by fluorescence using the Ca 2+ sensitive fluorescent probe Calcium-Green 5N (Thermo Fisher, C3737) (250 nM, excitation 506 nm, emission 532 nm) in a hypotonic solution (0.5 mM Tris-HCl pH 7.5) [ ].

Techniques: Electron Microscopy, Expressing, Control, Whisker Assay, Membrane, Fluorescence

Journal: Cell Death & Disease

Article Title: IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype

doi: 10.1038/s41419-023-05957-z

Figure Lengend Snippet: IF1 knockout murine colon carcinoma cell lines CT26 and MC38 were developed. a Representative blots of IF1 expression in wild type (wt) and cells co-transfected with Cas9 and non-targeted sgRNA (N. T.) or guided to the Atp5if1 gene (IF1-KO). β-F1-ATPase is used as loading control. b BN-PAGE of Complex V in mitochondria from CRL and IF1-KO CT26 and MC38 cells. β-F1 (Complex V) blots are shown. VDAC is shown as loading control. c Representative images of Proximity Ligation Assay (PLA) using γ-F1-ATPase as target (green dots). DAPI (blue) stained nuclei. Upper histograms show the number of PLA signals per cell in CRL ( n = 4) and IF1-KO ( n = 3) CT26 cells. d Calcium retention capacity (CRC) in mitochondria of CT26 and MC38 CRL ( n = 2) and IF1-KO ( n = 2–3) cells. Left, representative traces of the CRC. Right, histograms show the mean ± SEM of the amount of Ca 2+ necessary to induce PTP opening. e Box-and-whisker plots show cellular levels of AMP in CT26 and MC38 CRL and IF1-KO cells ( n = 3). f Box-and-whisker plots show the levels of adenosine released into the culture medium in CT26 and MC38 CRL and IF1-KO cells ( n = 4). g , h Box-and-whisker plots show the activity of phosphoribosyl pyrophosphate synthase (PRPP synthase) ( g ) and hypoxanthine-guanine phosphoribosyl transferase (HPRT) ( h ) in CT26 and MC38 CRL and IF1-KO cells ( n = 3-4). i Representative blot showing the expression of adenine phosphoribosyl transferase (APRT) in two independent preparations of CT26 CRL and IF1-KO cells. The box-and-whisker plot shows the corresponding quantitation ( n = 4). β-Actin is shown as loading control. j , k Representative images and traces of mitochondrial ATP levels measured by the mitGO-ATeam2 probe at different times before and after the addition of oligomycin (OL) at 50 s ( n = 11–14 cells per genotype assayed in three independent days) ( j ) or carboxyatractyloside (CAR) at 300 s ( n = 27–36 cells per genotype assayed in three independent days) ( k ) in CT26 CRL and IF1-KO cells. Box-and-whisker plots show the rates of mitochondrial ATP turnover due to OL ( i ) or CAR ( j ) addition. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 when compared by Student’s t -test. See also Supplementary Fig. S .

Article Snippet: Ca 2+ content of isolated mitochondria was determined by fluorescence using the Ca 2+ sensitive fluorescent probe Calcium-Green 5N (Thermo Fisher, C3737) (250 nM, excitation 506 nm, emission 532 nm) in a hypotonic solution (0.5 mM Tris-HCl pH 7.5) [ ].

Techniques: Knock-Out, Expressing, Transfection, Control, Proximity Ligation Assay, Staining, Whisker Assay, Activity Assay, Quantitation Assay