Fig 1: PACT suppresses mitobiogenesis through mature miR-181c.A, list of mitochondrial targets of miR-181. B and C, Prkra +/+ or Prkra −/− MEFs were transfected with empty vector (Empty Vec.) or FLAG-PACT and total RNA extracts were analyzed by qRT-PCR to determine (B) miR-181c and U6 small nuclear RNA (U6) (n = 6) and (C) pre-miR-181c and U6 RNA expression (n = 6). D and E, HEK293T cells were transfected with scrambled or PRKRA siRNA and total RNA extracts were analyzed by qRT-PCR for (D) miR-181c and U6 RNA and (E) pre-miR-181c and U6 RNA expression (n = 3). F, left panel, DICER cleavage assay performed using synthetic pre-miR-181c (10 μM) as substrate with recombinant DICER or PACT (0.2 μg) at 37 °C for 4 h. Samples are separated in 15% Urea-PAGE and detected with SYBR gold staining. M indicates microRNA marker. The average band intensities for the mature miR product are indicated at the top of the gel. Right panel, 5 ml of same samples (in the left panel) were analyzed by Western blotting using specific antibodies for DICER and PACT (n = 3). G–I, HEK293T cells were transfected with scrambled or miR-181c mimic (100 nM) (n = 3); (G) Mitochondria enriched fraction (MF) and total cell lysates (CL) proteins were analyzed by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, SIRT1, NRF1, and β-actin. H, total genomic DNA was analyzed by qRT-PCR to determine mtDNA (mitochondrial major and minor arc): nucDNA (B2M) ratio (n = 3). I, mitochondrial respiration was analyzed by OCR. Arrows indicate time for drug injections (n = 5; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). J–L, HEK293T cells were transfected with control or miR-181c AntagomiR (100 nM). J, MF and CL protein lysates were analyzed by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, SIRT1, NRF1, and β-actin (n = 3). K, total genomic DNA was analyzed by qRT-PCR to determine mtDNA (mitochondrial major and minor arc): nucDNA (B2M) ratio (n = 3). L, mitochondrial respiration was analyzed by OCR. Arrows indicate time for drug injections (n = 5; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). M, MEF cells were transfected with scrambled or miR-181c mimic (100 nM) and complex I (mOD/min), III (Units/μg), and IV (mOD/min) activity was measured by ELISA (n = 3). N, MEF cells were transfected with control or miR-181c AntagomiR (100 nM) and complex I (mOD/min), III (Units/μg), and IV (mOD/min) activity was measured by ELISA (n = 3). O and P, Prkra +/+ or Prkra−/− MEF cells transfected with scrambled or miR-181c mimic (100 nM). O, total genomic DNA was analyzed by qRT-PCR for mtDNA (mitochondrial Cox1 or Nd4): nucDNA (nuclear ApoB) ratio (n = 3). P, mitochondrial respiration was analyzed by OCR. Arrows indicate time for drug injections (n = 5; data were normalized to mitochondrial mass that was quantified from total OXPHOS protein levels from the same samples and represented as arbitrary units (A.U)). Protein expression was calculated relative to β-actin for whole cell lysate and Ponceau S. for mitochondrial fraction and depicted at the top of each blot. Data are mean ± SD. Unpaired t test with Welch’s correction or one-way ANOVA. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ns: not significant. CL, cell lysate; Cyto, cytoplasmic fraction; ETC, electron transfer chain; MEF, mouse embryonic fibroblast; MF, mitochondrial fraction; mtDNA, mitochondrial DNA; nucDNA, nuclear DNA; OCR, oxygen consumption rate; siRNA, silencer RNA.
Fig 2: Loss of PACT augments CL316,243-induced brown adipose mitobiogenesis.A, schematic representation of primary brown adipocyte differentiation protocol (upper panel). Mitochondrial protein levels were analyzed from protein lysates of undifferentiated (Day 0) or differentiated Prkra+/+ or Prkra+/− brown adipocytes by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, TOM70, NRF1, and β-actin (n = 3). B, the genomic DNA obtained from differentiated brown adipocytes in (A) was analyzed by qRT-PCR for mtDNA (Cox1 or Nd4): nucDNA (ApoB) ratio (n = 3). C, mitochondrial respiration was analyzed by OCR in differentiated Prkra+/+ or Prkra+/− brown adipocytes (n = 6; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). D–F, Prkra+/+ or Prkra+/− mice were injected with CL316,243 (1 mg/kg/day) for 6 days and sacrificed 24 h after the final injection (n = 8). D, BAT protein lysates were analyzed by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, TOM70, NRF1, UCP1, and β-actin. E, mtDNA (Cox1 or Nd4): nucDNA (ApoB) ratio was analyzed by qRT-PCR from total genomic DNA. F, total BAT RNA extracts were analyzed by qRT-PCR for miR-181c and U6 levels. G–I, metabolic parameters of Prkra+/+ or Prkra+/− animals (n = 4): (G) energy expenditure (EE), (H) oxygen consumption (VO2), and (I) respiratory exchange ratio (RER). Protein expression was calculated relative to β-actin (for whole cell lysate) or Ponceau S. (for mitochondrial fraction) and depicted at the top of each blot. Data are mean ± SD. Unpaired t test with Welch’s correction or one-way ANOVA. ∗p ≤ 0.05, ∗∗p ≤ 0.01. BAT, brown adipose tissue; ETC, electron transfer chain; mtDNA, mitochondrial DNA; nucDNA, nuclear DNA; OCR, oxygen consumption rate.
Fig 3: Phagocytosis receptor deletion causes sudden cardiac death.a, scRNA-seq data from n = 3 mice each on days 1, 2 and 4 after MI. Uniform manifold approximation and projection (UMAP) indicates cell subset. b, UMAP of Mertk expression by cell populations shown in a. c, UMAP of Cd36 expression by cell populations shown in a. d, Mertk and Cd36 expression in flow-sorted macrophages (Macs) (n = 8 mice), monocytes (n = 8 mice) and neutrophils (n = 7 mice) 5 hours after MI, normalized to Gapdh. One-way ANOVA followed by Tukey’s multiple comparisons test were used for statistical analysis. e, Experimental outline. C57BL/6, Cd36−/− and Mertk−/− mice underwent STORM protocol. f, Kaplan–Meier survival curve of wild-type (n = 14 mice, left, and n = 11 mice, right), Cd36−/− (n = 14) and Mertk−/− (n = 10) mice. P values were calculated using the log-rank (Mantel–Cox) test. g, ECG recordings from C57BL/6, Cd36−/− and Mertk−/− mice, all after STORM procedure. h, VT burden and Vfib burden in C57BL/6 mice (n = 9) and Cd36−/− mice (n = 9) after STORM procedure, within 6 hours after MI. A two-sided Mann–Whitney test was used for statistical analysis. i, VT burden and Vfib burden in C57BL/6 mice (n = 8) and Mertk−/− mice (n = 9) after STORM procedure, within 6 hours after MI. Two-sided Mann–Whitney tests were used for statistical analysis. j, Experimental outline. Bone marrow donors were either wild-type or Cd36−/− mice. Recipient wild-type mice underwent STORM protocol. k, VT burden and Vfib burden in wild-type controls (Cd36+/+, n = 8 mice) or Cd36−/− (n = 9) bone marrow chimeras 24 hours after MI. Two-sided Mann–Whitney tests were used for statistical analysis. l, Experimental outline. Cx3cr1CreERt2;Mertkfl/fl mice and Cx3cr1CreERt2 control mice were fed a tamoxifen (tamox) diet for 10 days. All mice underwent STORM protocol after tamoxifen exposure. m, VT burden and Vfib burden in Cx3cr1CreERt2 controls (n = 8 mice) or Cx3cr1CreERt2;Mertkfl/fl mice (n = 8) 24 hours after MI. A two-sided unpaired t-test (VT burden) and a Mann–Whitney test (Vfib burden) were used for statistical analysis. Data are mean ± s.e.m.
Fig 4: Exercise and anti-PD-1 treatment do not alter growth rate of B16-F10 or EO771 tumours.Survival curves for mice bearing B16-F10 (a) or EO771 (b) tumours (endpoint due to tumour size only). Data analysed by Log rank (Mantel-Cox) test. Time from treatment begin to euthanasia for mice bearing B16-F10 (c) or EO771 (d) tumours. B16-F10: n = 8–10 per group; EO771: n = 8–10 per group. Data are shown as individual data points and mean with 95% CI. Data analysed by two-way ANOVA.
Fig 5: SqorΔN/ΔN mice had decreased complex IV activity, increased systemic H2S levels, and an impaired metabolic status.(A) Histochemical staining was used to measure cytochrome c oxidase (COX) activity in SqorΔN/ΔN and control mice. Control mouse brain, liver, and muscle sections are stained dark brown; the same tissues from SqorΔN/ΔN mice are light brown, indicating decreased complex IV activity. Scale bars: 100 μm. (B) Complex IV activity in isolated mitochondria of the brain, liver, and muscle. The mean values of complex IV activity in control mice were set to 100%. Comparisons were made using unpaired 2-tailed t test. n = 5–6 mice for each group. Data are presented as means with SD. (C) Sulfide levels in plasma, brain, liver, and muscle were measured using HSip-1. The sulfide levels in SqorΔN/ΔN mice were compared with those in control mice (the sulfide level for control mice was set to 1). Data were analyzed using Mann-Whitney test for plasma, and unpaired 2-tailed t test for brain, liver, and muscle. n = 5–8 mice for each group. Data are presented as medians with interquartile range for plasma, and as means with SD for brain, liver, and muscle. (D) Compared with control mice, the blood lactate levels in SqorΔN/ΔN mice were higher at postnatal ages 30, 40, and 50 days. Comparisons between SqorΔN/ΔN and control mice at postnatal ages 30, 40, and 50 days were made using unpaired 2-tailed t test. Mixed-effects analysis with Dunnett’s multiple-comparison test was performed to compare the blood lactate levels at postnatal ages 30, 40, and 50 days in SqorΔN/ΔN mice. n = 4–5 mice for each group. Data are presented as means with SD.
Supplier Page from Abcam for Complex IV Rodent Enzyme Activity Microplate Assay Kit