Fig 1: PACT suppresses mitobiogenesis.A and B, mitochondrial mass was determined from MitoTracker Green–stained cells (quantified by measuring mean fluorescence intensity (MFI) with ImageJ from at least 200 cells; a representative image shown. The scale bar represents 50 μm): (A) Prkra+/+, Prkra−/−, or Prkra−/− MEFs reconstituted with empty vector (Empty Vec.) or PACT (n = 8) and (B) HEK293T cells transfected with 100 nM scrambled or PRKRA siRNA (n = 5). C and D, HEK293T cells transfected with 100 nM scrambled or PRKRA siRNA: (C) Protein lysates were analyzed by Western blotting using specific antibodies for PGC1α, TOM70, antibody cocktail against ETC proteins, NRF1, PACT, and β-actin (n = 3) and (D) Total genomic DNA was isolated and mtDNA (mitochondrial minor and major arc): nucDNA (B2M) ratio was analyzed by qRT-PCR (n = 3). E and F, HEK293T cells transfected with empty vector (Empty Vec.) or PACT plasmid: (E) Protein lysates were analyzed by Western blotting using specific antibodies for PGC1α, TOM70, antibody cocktail against ETC proteins, NRF1, PACT, and β-actin (n = 3) and (F) Total genomic DNA was isolated and mtDNA (mitochondrial minor and major arc): nucDNA (B2M) ratio was analyzed by qRT-PCR (n = 3). Protein expression was calculated relative to β-actin (for CL) or Ponceau S. (for MF) 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. CL, cell lysate; Cyto, cytoplasmic fraction; ETC, electron transfer chain; MEF, mouse embryonic fibroblast; MF, mitochondrial fraction; mtDNA, mitochondrial DNA; nucDNA, nuclear DNA; 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: PACT regulates mitochondrial respiratory metabolism.A–E, mitochondrial respiration was analyzed in Prkra+/+ or Prkra-/- MEFs reconstituted with either empty vector (Empty Vec.) or FLAG-PACT by measuring (A) oxygen consumption rate (OCR), (B) extracellular acidification rate ECAR, and (C) ATP production after oligomycin (Oligo; 1 µM) injection. Oligomycin: ATP synthase inhibitor; FCCP: mitochondrial uncoupler; R/A: rotenone and antimycin A mix (inhibitors for ETC complex I and III, respectively). D, maximal respiration (as the highest OCR after FCCP injection; 1 µM). E, basal respiration (as OCR before oligomycin injection). Arrows indicate time for drug injections (n = 4; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). F and G, mitochondrial fuel oxidation measurements of Prkra+/+, Prkra-/-, and Prkra-/- MEFs reconstituted with FLAG-PACT to determine (F) dependency on and (G) capacity to oxidize glucose, glutamine, or fatty acids during mitochondrial respiration (n = 5). H, Prkra+/+ MEFs or Prkra-/- MEFs reconstituted with either empty vector (Empty Vec.) or FLAG-PACT were analyzed by Western blotting using antibody cocktail against ETC proteins (n = 3). Protein expression was calculated relative to ß-actin and depicted at the top of each blot. I, Prkra+/+ MEFs or Prkra-/- MEFs reconstituted with either empty vector (Empty Vec.) or FLAG-PACT and complex I (mOD/min), III (Units/µg), and IV (mOD/min) activity was measured by ELISA (n = 3). 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. ETC, electron transfer chain; MEF, mouse embryonic fibroblast.
Fig 4: 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.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Recombinant Human PACT His Protein