Journal: Scientific Reports

Article Title: Nardilysin in adipocyte regulates insulin sensitivity via HIF1α and PPARγ

doi: 10.1038/s41598-025-21276-z

Figure Lengend Snippet: NRDC associates with PPARγ and may regulate its target genes. ( A ) Volcano plot showing transcription factor enrichment analysis of differentially expressed genes (DEGs) based on public ChIP-seq datasets from murine WAT, analyzed using the ChIP-Atlas platform. The DEGs were identified by RNA-seq in NRDC-knockdown 3T3-L1 adipocytes. Each point represents a transcription factor: blue indicates enriched factors, and red indicates those with P < 0.05. P values were calculated using a two-tailed Fisher’s exact probability test. The x-axis shows log 2 -transformed fold enrichment of transcription factors in the DEGs relative to all coding genes. The y-axis shows the -log 10 -transformed P values for enrichment. The dashed line indicates a significance threshold of P = 0.05; the dotted line indicates the Bonferroni-adjusted threshold ( P = 0.05/53). ( B ) Immunoprecipitation and Western blot analysis showing the formation of a complex of NRDC, PPARγ, and PGC-1α in COS7 cells transfected with expression vectors for NRDC-V5, HA-PGC1α and FLAG-PPARγ. ( C ) Relative mRNA expression levels of Nrd1 , Pparγ , Hif1a , and key Pparγ and Hif1a target genes in eWAT from HFD-fed Floxed and Adipo-KO mice, which were normalized to β-actin mRNA levels. N = 6/group. Data presented as mean ± SE; * P < 0.05, *** P < 0.0001.

Article Snippet: Antibody against PGC1α (#16) was developed in our laboratory as previously described , and anti-PPARγ antibody was obtained from the Santa Cruz Biotechnology.

Techniques: ChIP-sequencing, RNA Sequencing, Knockdown, Two Tailed Test, Transformation Assay, Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Animal Nutrition

Article Title: Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes

doi: 10.1016/j.aninu.2023.07.001

Figure Lengend Snippet: Primer sequences for RT-qPCR of cattle.

Article Snippet: Cells were maintained in RPMI 1640 basic medium containing 2% BSA and treated with different concentrations of PA (0, 100, 200, or 400 μM) and NaP (0, 1, 2.5, or 5 mM), alone or in combination, for 12 h. A 2 × 2 factorial arrangement was applied for the experiments: primary hepatocytes were treated with NaP (2.5 mM), the mTORC1 inhibitor rapamycin (100 nM) (V900930; Sigma Aldrich, MO, USA), the mTORC1 activator MHY1485 (2 μM) (S7811; Selleck, Shanghai, China), and the PGC1α inhibitor SR-18292 (20 μM) (S8528; Selleck) for 12 h to observe the effect of mTORC1 and PGC1α on the mRNA expression of gluconeogenic genes.

Techniques: Sequencing

Journal: Animal Nutrition

Article Title: Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes

doi: 10.1016/j.aninu.2023.07.001

Figure Lengend Snippet: Increased expression of key transcription factors of gluconeogenesis in calf hepatocytes treated with propionate. Calf hepatocytes were treated with indicated concentration of NaP for 12 h, and expression levels of hepatocyte nuclear factor 4 ( HNF4A ) (A), forkhead box O1 ( FOXO1 ) (B), c-AMP response binding protein ( CREB ) (C), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha ( PGC1α ) (D) were detected by RT-qPCR. Calf hepatocytes (E, F), HepG2 (G, H), and LO2 (I, J) cells were treated with indicated concentration of NaP for 12 h, the indicated proteins were detected by Western blotting (E, G, I), and quantified by ImageJ (F, H, J). Data were analyzed by one-way ANOVA. a, b, c, d Bars with a different letter mean a significant difference ( P < 0.05).

Article Snippet: Cells were maintained in RPMI 1640 basic medium containing 2% BSA and treated with different concentrations of PA (0, 100, 200, or 400 μM) and NaP (0, 1, 2.5, or 5 mM), alone or in combination, for 12 h. A 2 × 2 factorial arrangement was applied for the experiments: primary hepatocytes were treated with NaP (2.5 mM), the mTORC1 inhibitor rapamycin (100 nM) (V900930; Sigma Aldrich, MO, USA), the mTORC1 activator MHY1485 (2 μM) (S7811; Selleck, Shanghai, China), and the PGC1α inhibitor SR-18292 (20 μM) (S8528; Selleck) for 12 h to observe the effect of mTORC1 and PGC1α on the mRNA expression of gluconeogenic genes.

Techniques: Expressing, Concentration Assay, Binding Assay, Quantitative RT-PCR, Western Blot

Journal: Animal Nutrition

Article Title: Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes

doi: 10.1016/j.aninu.2023.07.001

Figure Lengend Snippet: Role of mammalian target of rapamycin complex 1 (mTORC1) and PGC1α in propionate-mediated regulation of the expression of gluconeogenesis-related genes in calf hepatocytes. (A–C). Calf hepatocytes were treated with NaP and rapamycin (100 nM). The expression levels of FBP1 (A), PCK1 (B), and G6PC (C) were detected by RT-qPCR. (D–F). Calf hepatocytes were treated with NaP and MHY1485 (2 μM). The expression levels of FBP1 (D), PCK1 (E), and G6PC (F) were detected by RT-qPCR. (G–I). Calf hepatocytes were treated with NaP and SR18292 (20 μM). The expression levels of FBP1 (G), PCK1 (H), and G6PC (I) were detected by RT-qPCR. Data were analyzed by two-way ANOVA. a, b, c Bars with a different letter mean a significant difference ( P < 0.05).

Article Snippet: Cells were maintained in RPMI 1640 basic medium containing 2% BSA and treated with different concentrations of PA (0, 100, 200, or 400 μM) and NaP (0, 1, 2.5, or 5 mM), alone or in combination, for 12 h. A 2 × 2 factorial arrangement was applied for the experiments: primary hepatocytes were treated with NaP (2.5 mM), the mTORC1 inhibitor rapamycin (100 nM) (V900930; Sigma Aldrich, MO, USA), the mTORC1 activator MHY1485 (2 μM) (S7811; Selleck, Shanghai, China), and the PGC1α inhibitor SR-18292 (20 μM) (S8528; Selleck) for 12 h to observe the effect of mTORC1 and PGC1α on the mRNA expression of gluconeogenic genes.

Techniques: Expressing, Quantitative RT-PCR

Journal: Animal Nutrition

Article Title: Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes

doi: 10.1016/j.aninu.2023.07.001

Figure Lengend Snippet: Regulation of mTOR activity and expression of gluconeogenesis-related genes in calf hepatocytes treated with palmitic acid (PA). Calf hepatocytes were treated with indicated concentration of PA for 12 h, the indicated proteins were detected by Western blotting (A), and quantified by ImageJ (B–E). Calf hepatocytes were treated with PA for 12 h, and expression levels of FOXO1 (F), CREB (G), PGC1α (H), FBP1 (I), FBP2 (J), PCK1 (K), PCK2 (L), G6PC (M), ACCS1 (N), SUCLG2 (O), MCEE (P), MMUT (Q), and PCCA (R) were detected by RT-qPCR. Data were analyzed by one-way ANOVA (B–E) and t -test (F–R). a, b, c, d Bars with a different letter mean a significant difference ( P < 0.05).

Article Snippet: Cells were maintained in RPMI 1640 basic medium containing 2% BSA and treated with different concentrations of PA (0, 100, 200, or 400 μM) and NaP (0, 1, 2.5, or 5 mM), alone or in combination, for 12 h. A 2 × 2 factorial arrangement was applied for the experiments: primary hepatocytes were treated with NaP (2.5 mM), the mTORC1 inhibitor rapamycin (100 nM) (V900930; Sigma Aldrich, MO, USA), the mTORC1 activator MHY1485 (2 μM) (S7811; Selleck, Shanghai, China), and the PGC1α inhibitor SR-18292 (20 μM) (S8528; Selleck) for 12 h to observe the effect of mTORC1 and PGC1α on the mRNA expression of gluconeogenic genes.

Techniques: Activity Assay, Expressing, Concentration Assay, Western Blot, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Senegenin Attenuates Pulmonary Fibrosis by Inhibiting Oxidative-Stress-Induced Epithelial Cell Senescence through Activation of the Sirt1/Pgc-1α Signaling Pathway

doi: 10.3390/antiox13060675

Figure Lengend Snippet: The primer sequence.

Article Snippet: The membranes were incubated with 5% skimmed milk, which was followed by incubation with β-actin, α-SMA, Pgc-1α, and Sirt1 antibodies (Proteintech) and with collagen I, p21, and p16 antibodies (Abcam, Cambridge, UK).

Techniques: Sequencing

Journal: Antioxidants

Article Title: Senegenin Attenuates Pulmonary Fibrosis by Inhibiting Oxidative-Stress-Induced Epithelial Cell Senescence through Activation of the Sirt1/Pgc-1α Signaling Pathway

doi: 10.3390/antiox13060675

Figure Lengend Snippet: Senegenin improved the pathological changes in lung tissue morphology and structure and reduced the levels of lung fibrosis markers in mice with pulmonary fibrosis. High (20 mg/kg), medium (5 mg/kg), and low (1 mg/kg) doses of senegenin were intraperitoneally injected into mice on days 15–28 after tracheal injection of bleomycin to examine the therapeutic effects of senegenin on pulmonary fibrosis. ( A , B ) HE and Masson staining were used to assess the changes in lung tissue structure and extracellular matrix in mice . ( C ) The Ashcroft scoring method was used to comprehensively analyze the degree of lung fibrosis in each group of mice . ( D ) Biochemical assay was used to determine the hydroxyproline content in mouse lung tissues. ( E , F ) qPCR was used to analyze the mRNA expression levels of ACTA2 and Col1a1 in lung tissues. ( G ) Western blotting was used to determine the levels of α-SMA and type I collagen in mouse lung tissues. Control—control group; BLM—lung fibrosis model group; BLM + L—low-dose treatment group; BLM + M—medium-dose treatment group; BLM + H—high-dose treatment group. (Data are expressed as the mean ± standard deviation; n = 8, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The membranes were incubated with 5% skimmed milk, which was followed by incubation with β-actin, α-SMA, Pgc-1α, and Sirt1 antibodies (Proteintech) and with collagen I, p21, and p16 antibodies (Abcam, Cambridge, UK).

Techniques: Injection, Staining, Expressing, Western Blot, Control, Standard Deviation

Journal: Antioxidants

Article Title: Senegenin Attenuates Pulmonary Fibrosis by Inhibiting Oxidative-Stress-Induced Epithelial Cell Senescence through Activation of the Sirt1/Pgc-1α Signaling Pathway

doi: 10.3390/antiox13060675

Figure Lengend Snippet: Sirt1 inhibitor EX527 inhibited the effect of senegenin on lung-fibrosis-related indices. EX527 (5 mg/kg) was intraperitoneally injected 30 min prior to high-dose senegenin treatment in pulmonary fibrosis mice modeled using bleomycin, and the results were compared with those in high–dose senegenin–treated pulmonary fibrosis mice . ( A , B ) HE and Masson staining were used to assess the changes in lung tissue structure and extracellular matrix in mice . ( C ) The Ashcroft score was used to comprehensively analyze the degree of lung fibrosis in each group of mice . ( D ) Biochemical assay was used to detect the hydroxyproline content in mouse lung tissues. ( E , F ) qPCR was used to analyze the mRNA expression levels of ACTA2 and Col1a1 in lung tissues. ( G ) Western blotting was used to detect the levels of α-SMA and type I collagen in mouse lung tissues. Control—control group; BLM—lung fibrosis model group; BLM + H—high-dose treatment group; EX527—group treated with EX527 before high-dose treatment. (Data are expressed as the mean ± standard deviation; n = 8; **** p < 0.0001).

Article Snippet: The membranes were incubated with 5% skimmed milk, which was followed by incubation with β-actin, α-SMA, Pgc-1α, and Sirt1 antibodies (Proteintech) and with collagen I, p21, and p16 antibodies (Abcam, Cambridge, UK).

Techniques: Injection, Staining, Expressing, Western Blot, Control, Standard Deviation

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet: ( A ) Heatmap of genes driving the de-enrichment of ‘GO:0022900 electron transport chain’. Heatmap is annotated for electron transport chain complexes and target genes induced by the transcription factor Ppargc1a . ( B ) Normalized RNA-seq expression of Ppargc1a in healthy (n = 5) and acute EAE (n = 5) motor neurons. Bars show mean values ± s.e.m. ( C ) Relative qPCR mRNA expression of Ppargc1a to Tbp in healthy (n = 3) and acute EAE (n = 3) motor neurons. Bars show mean values ± s.e.m. ( D ) Quantification of PGC-1α protein in cervical spinal cords of healthy (n = 5) and acute (n = 5) EAE mice. Each sample was normalized to its β-actin. Bars show mean values ± s.e.m. ( E ) Representative immunoblots of PGC-1α, phosphorylated PGC-1αS570 (pPGC-1α), and corresponding β-actin of cervical spinal cords of healthy and acute EAE mice. ( F ) Quantification of phosphorylated PGC-1αS570 (pPGC-1α) in relation to PGC-1α total protein in cervical spinal cords of healthy (n = 5) and acute (n = 5) EAE mice. Each sample was normalized to its β-actin. Bars show mean values ± s.e.m. Statistical analysis in B and C was performed by unpaired, two-tailed Student’s t-test, and in D and F by unpaired, two-tailed Mann–Whitney test; *p<0.05.

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: RNA Sequencing, Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet: ( A ) Relative qPCR mRNA expression of Thy-1 to Tbp in hippocampal primary neuronal culture (hPNC) and cortical primary neuronal cultures (cPNC) (DIV14) of wild-type (n = 6 per group) mice. Bars show mean values ± s.e.m. ( B ) Relative diploid nuclear chromosomal DNA copy numbers of Ppargc1a in tail biopsy of wild-type (n = 9) and Thy1-Ppargc1a (n = 16) mice. Bars show mean values ± s.e.m. ( C ) Relative qPCR mRNA expression of Ppargc1a in hippocampus (Hip), cortex, and cervical spinal cord (SC) of wild-type (n = 5) and Thy1-Ppargc1a (n = 4) mice. Bars show mean values ± s.e.m. ( D ) Relative qPCR mRNA expression of Ppargc1a in hPNC and cPNC (DIV14) of wild-type (n = 3) and Thy1-Ppargc1a (n = 3) mice. Bars show mean values ± s.e.m. ( E ) Relative qPCR mRNA expression of Ppargc1a target genes in hPNC (DIV14) of wild-type (n = 3) and Thy1-Ppargc1a (n = 3) mice. Bars show mean values ± s.e.m. ( F ) Relative qPCR mRNA expression of Ppargc1a target genes in cPNC (DIV14) of wild-type (n = 3) and Thy1-Ppargc1a (n = 3) mice. Bars show mean values ± s.e.m. ( G ) Relative qPCR mRNA expression of Ppargc1a target genes in hippocampus of wild-type (n = 5) and Thy1-Ppargc1a (n = 4) mice. Bars show mean values ± s.e.m. ( H ) Relative qPCR mRNA expression of Ppargc1a target genes in cortex of wild-type (n = 5) and Thy1-Ppargc1a (n = 4) mice. Bars show mean values ± s.e.m. ( I ) Relative qPCR mRNA expression of Ppargc1a target genes in cervical spinal cord of wild-type (n = 5) and Thy1-Ppargc1a (n = 5) mice. Bars show mean values ± s.e.m. ( J ) Representative immunocytochemical staining of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in cPNC (DIV14) of wild-type and Thy1-Ppargc1a mice. Co-stainings for microtubule-associated protein 2 (Map2) and 4′,6-diamidino-2-phenylindole (DAPI). Scale bar 10 µm. ( K ) Representative immunohistochemical staining of PGC-1α in spinal cord of wild-type and Thy1-Ppargc1a mice. Co-stainings for neuronal nuclei (NeuN), FLAG, and DAPI. Scale bar 20 µm. ( L ) Mean fluorescence intensity (MFI) quantification of calcein of hPNC (DIV14) of wild-type (n = 1) and Thy1-Ppargc1a (n = 1) mice (three wells per culture) to determine cell viability. Bars show mean values ± s.e.m. Statistical analysis in C and D was performed by unpaired, one-tailed Student’s t-test, and in B , E–I , and L by unpaired, two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: Expressing, Staining, Immunohistochemical staining, Fluorescence, One-tailed Test, Two Tailed Test

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet: ( A ) Relative qPCR mRNA expression in hippocampal primary neuronal cultures (hPNC) (DIV14) of wild-type (n = 4) and Thy1-Ppargc1 a (n = 4) mice of Ppargc1a -regulated electron transport chain genes that were detected to be downregulated during experimental autoimmune encephalomyelitis. Bars show mean values ± s.e.m. ( B ) Mitochondrial DNA copy numbers (mtDNA) relative to diploid nuclear chromosomal DNA (nDNA) in hPNC (DIV14) of wild-type (n = 3) and Thy1-Ppargc1 a (n = 3) mice. Bars show mean values ± s.e.m. ( C ) mtDNA relative to nDNA in muscle, liver, hippocampus (Hip), cortex, and cervical spinal cord (SC) of wild-type (n = 3) and Thy1-Ppargc1 a (n = 3) mice. Bars show mean values ± s.e.m. ( D ) Representative images and quantification of cytochrome c oxidase (COX) histochemistry of cervical spinal cord gray matter (GM) or ventral horn (VH) of wild-type (n = 5) and Thy1-Ppargc1 a (n = 6) mice (2–3 stainings per mice) normalized to neuronal nuclei (NeuN)-positive neurons. Bars show mean values ± s.e.m. Scale bar 250 µm. ( E ) Profile and quantification of oxygen consumption rate in hPNC (DIV14) of wild-type (n = 4) and Thy1-Ppargc1 a (n = 4) mice. Yellow: basal respiration (Basal); blue: ATP-dependent respiration (ATP dependent); orange: maximal respiratory capacity (Max); green: non-mitochondrial respiration (NMR). Bars show mean values ± s.e.m. ( F ) Representative images of hPNC and mean fluorescence intensity quantification of tetramethylrhodamin-ethylester mitochondrial membrane potential assay of hPNC (DIV14) and cortical primary neuronal culture of wild-type (n = 3) and Thy1-Ppargc1 a (n = 3) mice (five cells per culture). FCCP(carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) was used as ionophore uncoupler of oxidative phosphorylation. Bars show mean values ± s.e.m. Scale bar: 10 µm. Statistical analysis in A was performed by unpaired, one-tailed Student’s t-test, and in B–E by unpaired, two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 3—source data 1. Analysis of oxygen consumption rate in primary neurons of Thy1-Ppargc1a mice.

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: Expressing, Fluorescence, Membrane, Phospho-proteomics, One-tailed Test, Two Tailed Test

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet: ( A ) Experimental approach for analysis of Ca 2+ signaling. GCamp6f fluorescence was recorded for 10 minutes in spontaneously active cortical primary neuronal culture (cPNC) (DIV15) prior to ionomycin application used for signal normalization. ( B ) Representative cytosolic calcium transients of GCaMP6f-transduced spontaneously active cPNC (DIV15) of wild-type and Thy1-Ppargc1 a mice normalized to fluorescence of cytosolic calcium-saturated conditions (Fmax). ( C ) Quantification of calcium transient decay constant Tau (T) as an indicator of cytosolic calcium clearance time, calcium transient amplitude (A), mean cytosolic calcium signal intensity and of the number of cytosolic calcium transients presented as firing rate of GCaMP6f-transduced cPNC (DIV15) of wild-type (n = 74 cells from three different mice) and Thy1-Ppargc1a (n = 51 cells from three different mice). 478s of the recorded trace were analysed. Bars show mean values ± s.e.m. ( D ) Representative cytosolic calcium trace of Fluo-4-stained cPNC (DIV14–16) of wild-type and Thy1-Ppargc1a mice. Statistical analysis in C was performed by unpaired, two-tailed Student’s t-test. **p<0.01.

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: Fluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet: ( A ) Relative qPCR mRNA expression of Eno2 to Tbp in hippocampal primary neuronal cultures (hPNC) (DIV14) of wild-type (n = 3) mice. Bars show mean values ± s.e.m. ( B ) Relative qPCR mRNA expression of Ppargc1a in sorted immune cells in relation to hPNC (DIV14) of wild-type (n = 3) mice. Bars show mean values ± s.e.m. ( C ) Relative qPCR mRNA expression of Ppargc1a in hippocampus (Hip), cortex, and cervical spinal cord (SC) of Ppargc1a flx/flx (n = 3) and Ppargc1a flx/flx × Eno2 Cre+ (n = 3) mice. Bars show mean values ± s.e.m. ( D ) Relative qPCR mRNA expression of Ppargc1a target genes in hippocampus of Ppargc1a flx/flx (n = 3) and Ppargc1a flx/flx × Eno2 Cre+ (n = 3) mice. Bars show mean values ± s.e.m. ( E ) Relative qPCR mRNA expression of Ppargc1a target genes in cortex of Ppargc1a flx/flx (n = 3) and Ppargc1a flx/flx × Eno2 Cre+ (n = 3) mice. Bars show mean values ± s.e.m. ( F ) Relative qPCR mRNA expression of Ppargc1a target genes in cervical spinal cord of Ppargc1a flx/flx (n = 3) and Ppargc1a flx/flx × Eno2 Cre+ (n = 3) mice. Bars show mean values ± s.e.m. ( G ) Analysis of immunohistochemical stainings and quantification of HuC/HuD-positive neurons in cervical spinal cord ventral horn (VH) or gray matter (GM) of Ppargc1a flx/flx (n = 4) and Ppargc1a flx/flx × Eno2 Cre+ (n = 4) mice (two areas per mouse) at day 40 post-immunization. Bars show mean values ± s.e.m. ( H ) Analysis of immunohistochemical stainings and quantification of neuronal nuclei (NeuN)-positive neurons in cervical spinal cord VH or GM of healthy Ppargc1a flx/flx (n = 5) and Ppargc1a flx/flx × Eno2 Cre+ (n = 5) mice (two areas per mouse). Bars show mean values ± s.e.m. ( I ) Analysis of immunohistochemical stainings and quantification of surviving HuC/HuD-positive neurons in cervical spinal cord VH or GM of wild-type (n = 5) and Thy1-Ppargc1a (n = 6) (two areas per mice for NeuN, three areas per mice for neurofilament) at day 40 post immunization. Bars show mean values ± s.e.m. ( J ) Analysis of immunohistochemical stainings and quantification of NeuN-positive neurons in cervical spinal cord VH or GM of healthy wild-type (n = 5) and Thy1-Ppargc1a (n = 5) (two areas per mice) immunization. Bars show mean values ± s.e.m. Statistical analysis in B was performed by multiple comparisons test following one-way ANOVA, and in C–J by unpaired, two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: Expressing, Immunohistochemical staining, Two Tailed Test

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet: ( A ) Mean clinical scores of Ppargc1a flx/flx (n = 6) and Ppargc1a flx/fl × Eno2 Cre+ (n = 6) mice undergoing EAE. Curves show mean ± s.e.m. ( B ) Representative images and analysis of immunohistochemical stainings of surviving NeuN-positive neurons in cervical spinal cord ventral horn (VH) or gray matter (GM) of Ppargc1a flx/flx (n = 4) and Ppargc1a flx/flx × Eno2 Cre+ (n = 4) mice (two areas per mice) at day 40 post-immunization with quantification. Bars show mean values ± s.e.m. Scale bar 100 µm. ( C ) Mean clinical scores of wild-types (n = 31) and Thy1-Ppargc1a (n = 22) mice undergoing EAE. Curves show mean ± s.e.m., all pooled from three independent experiments. ( D ) Histopathological stainings of T cells (CD3), microglia (IBA1), and macrophages (MAC-3) in cervical spinal cord sections of wild-type (n = 6) and Thy1-Ppargc1a (n = 5) mice (2–3 stainings per mice) at day 15 post immunization with quantifications. Bars show mean values ± s.e.m. Scale bar 250 and 100 µm. ( E ) Quantification of cytochrome c oxidase (COX) histochemistry of cervical spinal cord GM or VH of wild-type (n = 6) and Thy1-Ppargc1a (n = 4) mice (2–3 stainings per mice) at acute stage of EAE normalized to neuronal nuclei (NeuN)-positive neurons. Bars show mean values ± s.e.m. Scale bar 250 µm. ( F ) Representative images and analysis of immunohistochemical stainings of surviving NeuN-positive neurons in cervical spinal cord VH or GM and surviving neurofilament-positive axons in the dorsal columns of wild-type (n = 5) and Thy1-Ppargc1a (n = 5) (two areas per mice for NeuN, three areas per mice for neurofilament) at day 40 post immunization. Bars show mean values ± s.e.m. Scale bar: 100 µm and 50 µm. Statistical analysis in A and C was performed by one-tailed Mann–Whitney U test of area under the curve (AUC) starting at peak (day 15) of disease; in B , D , E , and F by unpaired, two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 5—source data 1. Numerical data of clinical scores of Ppargc1a flx/flx (flx/flx) ( n = 6) and Ppargc1a flx/flx × Eno2 Cre+ (flx/flx × Eno2Cre) (n = 6) mice undergoing experimental autoimmune encephalomyelitis (EAE). Figure 5—source data 2. Numerical data of clinical scores of wild-types (WT) ( n = 31) and Thy1-Ppargc1a (TT) (n = 22) mice undergoing experimental autoimmune encephalomyelitis (EAE).

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: Immunohistochemical staining, One-tailed Test, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis

doi: 10.7554/eLife.61798

Figure Lengend Snippet:

Article Snippet: Copy numbers were analyzed by quantitative real-time PCR performed in an ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan Copy Number Assays (Thermo Fisher) for Ppargc1a ( Pgc-1α, Mm00164544_cn, FAM) and Tfrc (TaqMan Copy Number reference assay for mouse, VIC) in a duplex PCR.

Techniques: Staining, Adjuvant, Membrane, Recombinant, Sequencing, Transfection, Construct

Journal: PLoS ONE

Article Title: Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0005624

Figure Lengend Snippet: MCF-7 breast cancer cells were treated with Compound A for either 24 or 48 hours. No change in ERRα or ERα mRNA levels were measured after 24 and 48 hours of treatment with the ERRα antagonist verses vehicle (DMSO), while other ERRα target genes medium-chain acyl coenzyme ( ACADM ), aromatase ( CYP19A1 ), pyruvate dehydrogenase kinase 4 ( PDK4 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) were all significantly (P<0.001) down modulated upon treatment with ERRα antagonist at 24 and/or 48 hours. Additionally, peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) ( PPARGC1A ), is also significantly (P<0.001) down modulated upon treatment with Compound A. These results are representative of three independent experiments performed in triplicate. Differences in relative mRNA expression between vehicle (DMSO) and Cmpd A were measured by ANOVA followed by a student t-test with a 0.05 significance level.

Article Snippet: Primer/probe sets for target genes: human ERRα ( ESRRA ) (Hs00607062_gH), human ERα ( ESR1 ) (Hs00174860_m1), human PGC-1α ( PPARGC1A ) (Hs00173304_m1), human PDK4 ( PDK4 ) (Hs00176875_m1), human osteopontin ( SPP1 ) (Hs00959010_m1), human pS2 ( TFF1 ) (Hs00170216_m1), human ACADM ( ACADM ) (Hs00163494_m1) and 18S rRNA endogenous control (4308329) were purchased from Applied Biosystems.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0005624

Figure Lengend Snippet: Short hairpin plasmids expressing short hairpin RNA (shRNA) specific for knocking-down ERRα were transfected separately into MCF-7 cells. In addition, a shRNA expressing a scrambled artificial non-specific sequence was transfected. (-) control. (A) MCF-7/shRNA ERRα3 cells underwent four rounds (I–IV) of fluorescent activated cell sorting (FACS) to enrich for GFP expressing cells. FACS is described in Materials and Methods . (B) ERRα ( ESRRA ), ACADM , and PGC-1α ( PPARGC1A ) mRNA expression was measured by real-time RT-PCR. After 4 rounds of FACS, MCF-7/shRNA ERRα3 cells ERRα ( ESRRA ) mRNA levels were significantly reduced by 79%, ACADM levels by 75%, and PGC-1α ( PPARGC1A ) by 71% (*, P<0.05). Differences in relative mRNA expression between MCF-7/shRNA (-) and MCF-7/shRNA ERRα3 were measured by ANOVA followed by a student t-test with a 0.05 significance level. (C) MCF-7, MCF-7/shRNA (-), MCF-7/shRNA ERRα2, and MCF-7/shRNA ERRα3 cells ERRα protein expression was measured by Western blot, equal loading of protein was assessed by Coomassie blue staining of gels, and densitometric quantification are described in Materials and Methods . Results shown are representative of three independent experiments. MCF-7/shRNA ERRα2 exhibited 59% less protein verses the negative control, while MCF-7/shRNA ERRα3 cells ERRα protein levels were reduced by 69%. (D) ERα ( ESR1 ), aromatase ( CYP19A1 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) mRNA expression was measured in MCF-7/shRNA (-), MCF-7/shRNA ERRα2, and MCF-7/shRNA ERRα3 cells by real-time RT-PCR after 4 rounds of FACS. Knocking-down ERRα by shRNA (RNAi) led to significant decrease (*, P<0.05) in expression of ERRα target genes aromatase ( CYP19A1 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) while ERα ( ESR1 ) levels were not affected. All real-time RT-PCR results are representative of three independent experiments performed in triplicate.

Article Snippet: Primer/probe sets for target genes: human ERRα ( ESRRA ) (Hs00607062_gH), human ERα ( ESR1 ) (Hs00174860_m1), human PGC-1α ( PPARGC1A ) (Hs00173304_m1), human PDK4 ( PDK4 ) (Hs00176875_m1), human osteopontin ( SPP1 ) (Hs00959010_m1), human pS2 ( TFF1 ) (Hs00170216_m1), human ACADM ( ACADM ) (Hs00163494_m1) and 18S rRNA endogenous control (4308329) were purchased from Applied Biosystems.

Techniques: Expressing, shRNA, Transfection, Sequencing, Control, FACS, Quantitative RT-PCR, Western Blot, Staining, Negative Control