Journal: Advanced Science

Article Title: Selenoprotein H Functions as a PPARα Coactivator to Link Selenium Homeostasis to Hepatic Lipid Metabolism and Protect against Steatohepatitis

doi: 10.1002/advs.202519563

Figure Lengend Snippet: SELENOH binds to and activates fatty acid oxidation gene transcription in a PPARα‐dependent manner. (A) Western blot analysis of liver cytoplasmic and nuclear proteins. (B) Distribution of SELENOH signals in different genomic locations relative to known genes. (C) Barplot showing GO enrichment analysis of binding sites occupied by SELENOH. (D) Motif analysis of SELENOH‐bound sequences in mouse liver. (E) Venn diagram representing the overlapped SELENOH and PPARα peaks detected by CUT&Tag in mouse livers. (F) CUT&Tag tracks of SELENOH and PPARα peaks at the Cpt1a and Acox1 loci. (G) Luciferase reporter experiments using the PPAR response element (PPRE) in HEK293 cells. n = 3 independent culture experiments. (H) Heatmap showing ATAC‐seq signals at PPARα‐activated FAO genes in MASH control and MASH SELENOH‐OE livers. Genes shown in rows were sorted in decreasing order by signal intensity in each condition. (I and J) ATAC‐seq and RNA‐seq tracks of Cpt1a and Acox1 loci. (K) Relative mRNA levels of indicated FAO genes in liver tissues from mice injected with AAV‐TBG‐control or AAV‐TBG‐ Selenoh ‐sgRNA. n = 6 per group. (L) GO enrichment analysis of downregulated genes in SELENOH‐depleted livers. (M) Hepatic TG level of animals as indicated in (K). n = 5 per group. (N) Schematic of the experimental design: GW6471 treatment on SELENOH overexpression mice. (O) Blood AST and ALT levels, and hepatic TG level of animals as indicated. n = 5 per group. (P) Representative H&E staining of liver sections from animals as indicated. (Q) FAO activities of liver tissues from animals as indicated. n = 6 per group. (R) Relative mRNA levels of indicated FAO genes in liver tissues. n = 6 per group. (S) CUT&Tag signals of liver SELENOH at PPARα‐activated FAO genes. CUT&Tag tracks of SELENOH at Cpt1a and Acox1 loci.Values are mean ± SEM. The one‐way ANOVA with post hoc Bonferroni multiple‐comparison test (G, K, M, O, Q, and R) was used for statistical analysis.

Article Snippet: The biotinylated Acox1 ‐p probes were immobilized on Streptavidin Magnetic Beads (HY‐K0208, MCE) in DNA binding buffer (5 mmol/L Tris pH 7.5, 0.5 mmol/L EDTA, 1 mol/L NaCl).

Techniques: Western Blot, Binding Assay, Luciferase, Control, RNA Sequencing, Injection, Over Expression, Staining, Comparison

Journal: Advanced Science

Article Title: Selenoprotein H Functions as a PPARα Coactivator to Link Selenium Homeostasis to Hepatic Lipid Metabolism and Protect against Steatohepatitis

doi: 10.1002/advs.202519563

Figure Lengend Snippet: SELENOH interacts with activated PPARα and facilitates the genomic recruitment of PPARα‐P300 complex. (A) Representative immunofluorescence of SELENOH and PPARα in U2OS, Scale bars, 5 µm. (B) Endogenous co‐immunoprecipitation (co‐IP) analysis between PPARα and SELENOH in nuclear proteins from mouse livers under fasting or fed conditions. (C) Co‐IP of HA‐PPARα and FLAG‐SELENOH in HEK293T cells treated with vehicle or wy‐14643. (D) Co‐IP of different PPARα truncations with FLAG‐SELENOH in HEK293T cells treated with wy‐14643. (E) Endogenous co‐IP of PPARα and P300 in liver nuclear proteins from MASH control and MASH SELENOH‐OE mice. (F) Heatmap showing CUT&Tag signals of P300 at PPARα‐activated FAO genes in MASH control and MASH SELENOH‐OE livers. (G) Endogenous co‐IP of PPARα and P300 in liver nuclear proteins from mice injected with AAV‐TBG‐control or AAV‐TBG‐ Selenoh ‐sgRNA under the fasting condition. (H) Heatmap showing CUT&Tag signals of P300 at PPARα‐activated FAO genes in livers from mice injected with AAV‐TBG‐control or AAV‐TBG‐ Selenoh ‐sgRNA under the fasting condition. (I) Schematic of DNA pulldown assay. (J) Western blot analysis of biotin‐ Acox1 promoter ( Acox1 ‐p) pull‐down of nuclear protein extracts from indicated liver tissues. (K) Heatmap showing CUT&Tag signals of PPARα in MASH control and MASH SELENOH‐OE livers. (L) CUT&Tag tracks of P300 and PPARα peaks at the Cpt1a locus. (M) Heatmap showing CUT&Tag signals of PPARα in livers from mice injected with AAV‐TBG‐control or AAV‐TBG‐ Selenoh ‐sgRNA under the fasting condition. (N) CUT&Tag tracks of P300 and PPARα peaks at the Cpt1a locus. (O) Co‐IP of HA‐PPARα and FLAG‐SELENOH SXXS in HEK293T cells treated with vehicle or wy‐14643. (P) Relative mRNA levels of indicated FAO genes in primary hepatocytes from mice injected with AAV‐TBG‐control, AAV‐TBG‐SELENOH, or AAV‐TBG‐SELENOH SXXS . n = 6. (Q) Luciferase reporter experiments using the PPAR response element (PPRE) in HEK293T cells. Cells were transfected with indicated plasmid with or without 1 µM selenium (Se) treatment. n = 3 independent culture experiments.Values are mean ± SEM. The one‐way ANOVA with post hoc Bonferroni multiple‐comparison test (P and Q) was used for statistical analysis.

Article Snippet: The biotinylated Acox1 ‐p probes were immobilized on Streptavidin Magnetic Beads (HY‐K0208, MCE) in DNA binding buffer (5 mmol/L Tris pH 7.5, 0.5 mmol/L EDTA, 1 mol/L NaCl).

Techniques: Immunofluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Injection, Western Blot, Luciferase, Transfection, Plasmid Preparation, Comparison

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: The metabolic remodeling process in natural kidney aging. A Representative images of Oil Red O staining in the 24-month-old and 6-month-old group ( n = 6), scale bar = 50 μm; B , C the downregulated protein levels of FAO markers (i.e., PPARα, ACOX1, and CPT1A) and upregulated protein levels of glycolysis markers (i.e., HK2 and PDK1) by western blot, and their semi-quantitative analyses ( n = 6); D immunostaining for GLIS1 (red) and PPARα (green), with DAPI (blue) counterstaining by IF staining in the 24-month-old and 6-month-old group ( n = 6), scale bar = 50 μm; E lactate levels in the 24-month-old and 6-month-old group ( n = 6); F the downregulated protein levels of PPARα, ACOX1, and CPT1A, and up-regulated protein levels of HK2 and PDK1 in the 24-month-old group by IHC assay and their semi-quantitative analyses ( n = 6), scale bar = 50 μm. The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the 6-month-old group by Student’s t -test

Article Snippet: Specific primary antibodies targeting GLIS1 (Proteintech 23,138–1-AP), P16INK4A (Thermo Fisher Scientific Cat# MA5-17,142), γ-H2AX (Proteintech 10,856–1-AP), FN (Abcam ab2413), α-SMA (Abcam ab5694), PPARα (Novus NB300-537), ACOX1 (Novus NBP1-80950), CPT1A (Abcam ab234111), HK2 (Sigma SAB2108077), PDK1 (Sigma SAB4502160), METTL3 (Abcam ab195352), and YTHDF1 (Abcam ab252346) were incubated overnight at 4 °C.

Techniques: Staining, Western Blot, Immunostaining

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: GLIS1 regulated metabolic remodeling from FAO to glycolysis pathway by binding to the PPARα promoter. A Representative images of Oil Red O staining in the control vector and siGLIS1 group ( n = 3), scale bar = 50 μm; B , C the ECAR levels after culturing with glucose followed by oligomycin and 2-DG in the control vector and siGLIS1 group, and its semi-quantitative analysis ( n = 3); D metabolic component analysis in control and siGLIS1 group ( n = 3); E , F downregulated protein levels of GLIS1, PPARα, ACOX1, and CPTA1, and upregulated protein levels of HK2 and PDK1 in siGLIS1 group by western blot, and their semi-quantitative analyses ( n = 3); G – I cellular free fatty acid (FFA), triglyceride (TG) and total cholesterol (TC) levels in HK-2 cells in control and siGLIS1 group ( n = 3); J immunostaining for PPARα, ACOX1, CPT1A, HK2, and PDK1 (green), with DAPI (blue) counterstaining by IF staining ( n = 3), scale bar = 50 μm; K the interaction of PPARα and GLIS1 confirmed by ChIP and PCR assay ( n = 3); L the protein levels of FN and HK2 by western blot in HK-2 cells treated with D-gal in the presence of OE-GLIS1, OE-GLIS1 + CPTA1 inhibitor (etomoxir), OE-GLIS1 + PPARα (MK886), and OE-GLIS1 + siACOX1( n = 3). The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the vector group ( C , D , F , H , and I ), or IgG group ( K ) by Student’s t -test

Article Snippet: Specific primary antibodies targeting GLIS1 (Proteintech 23,138–1-AP), P16INK4A (Thermo Fisher Scientific Cat# MA5-17,142), γ-H2AX (Proteintech 10,856–1-AP), FN (Abcam ab2413), α-SMA (Abcam ab5694), PPARα (Novus NB300-537), ACOX1 (Novus NBP1-80950), CPT1A (Abcam ab234111), HK2 (Sigma SAB2108077), PDK1 (Sigma SAB4502160), METTL3 (Abcam ab195352), and YTHDF1 (Abcam ab252346) were incubated overnight at 4 °C.

Techniques: Binding Assay, Staining, Control, Plasmid Preparation, Western Blot, Immunostaining

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: The over-expressed GLIS1 suppressed metabolic remodeling from FAO to glycolysis in the accelerated aging mouse model. A The representative images of mouse kidney tissue in the control, AAV-Vector and AAV-GLIS1 group stained with Oil Red O ( n = 6), scale bar = 50 μm; B , C downregulated protein levels of PPARα, ACOX1, and CPT1A in the accelerated aging mouse model were reversed in the presence of AAV-GLIS1, while upregulated protein levels of HK2 and PKD1 in the accelerated aging mouse model were reduced by introducing AAV-GLIS1 ( n = 6); D lactate levels in the control, AAV-Vector and AAV-GLIS1 group ( n = 6); E the reversed effect of PPARα, ACOX1 and CPT1A levels, as well as HK2 and PDK1 levels in the presence of AAV-GLIS1 by IHC assay, and their semi-quantitative analyses ( n = 6), scale bar = 50 μm; F immunostaining for PPARα, ACOX1, CPTA1, HK2, and PDK1 (green), with DAPI (blue) counterstaining by IF staining in accelerated aging mouse model ( n = 6), scale bar = 50 μm. The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the AAV-vector group by one-way ANOVA

Article Snippet: Specific primary antibodies targeting GLIS1 (Proteintech 23,138–1-AP), P16INK4A (Thermo Fisher Scientific Cat# MA5-17,142), γ-H2AX (Proteintech 10,856–1-AP), FN (Abcam ab2413), α-SMA (Abcam ab5694), PPARα (Novus NB300-537), ACOX1 (Novus NBP1-80950), CPT1A (Abcam ab234111), HK2 (Sigma SAB2108077), PDK1 (Sigma SAB4502160), METTL3 (Abcam ab195352), and YTHDF1 (Abcam ab252346) were incubated overnight at 4 °C.

Techniques: Control, Plasmid Preparation, Staining, Immunostaining

Journal: BMC Biology

Article Title: N6-methyladenosine regulates metabolic remodeling in kidney aging through transcriptional regulator GLIS1

doi: 10.1186/s12915-024-02100-y

Figure Lengend Snippet: The ablation of METTL3 triggered metabolic remodeling and aggravated cell senescence renal fibrosis. A Protein levels of GLIS1, PPARα, ACOX1, CPT1A, HK2, PDK1, FN, α-SMA, and P16 INK4A by western blot in the vector and siMETTL3 group, and their semi-quantitative analyses ( n = 3); B representative images of Oil Red O staining in the vector and siMETTL3 group ( n = 3), scale bar = 50 μm; C double immunostaining for GLS1 and PPARα, ACOX1, CPT1A, HK2, PDK1, and α-SMA by IF staining ( n = 3) scale bar = 50 μm. The data are expressed as the mean ± SD of three independent experiments. ** P < .01 or *** P < .001 versus the vector group by Student’s t -test

Article Snippet: Specific primary antibodies targeting GLIS1 (Proteintech 23,138–1-AP), P16INK4A (Thermo Fisher Scientific Cat# MA5-17,142), γ-H2AX (Proteintech 10,856–1-AP), FN (Abcam ab2413), α-SMA (Abcam ab5694), PPARα (Novus NB300-537), ACOX1 (Novus NBP1-80950), CPT1A (Abcam ab234111), HK2 (Sigma SAB2108077), PDK1 (Sigma SAB4502160), METTL3 (Abcam ab195352), and YTHDF1 (Abcam ab252346) were incubated overnight at 4 °C.

Techniques: Western Blot, Plasmid Preparation, Staining, Double Immunostaining

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Changes in lipid metabolism in ft/ft mouse epidermis. ( a ) Microarray analysis showing a subset of genes involved in FA metabolism in mouse epidermis. The full gene list and respective fold changes are provided in Table 3 . ( b ) Relative mRNA level of Acox1 and Hsd17b4 in the epidermis of mice (n = 9–10). ( c, d ) Protein abundance of ACOX1 in mouse epidermis. The dashed line indicates the dermal‒epidermal boundary. Bar = 50 μm ( e ) ACOX activity measured in mouse epidermal cells (expressed as mU per μg of proteins, n = 5–7). Relative mRNA expression of ( f ) Acot5 , Acot8 , ( g ) Crot , and ( h ) PPAR mRNA, Ppar , isoforms in epidermal samples from CTRL and ft/ft mice (n = 9–10). Data were analyzed with a Student’s t -test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. CTRL, control; FA, fatty acid; H 2 O 2 , hydrogen peroxide; PPAR, peroxisome proliferator–activated receptor.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Microarray, Quantitative Proteomics, Activity Assay, Expressing, Control

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: PCR Array Gene List and Respective Fold Changes

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Sequencing, Binding Assay, Transferring

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Detection of ACOX1 in human AD skin and overexpression of ACOX1 in HEEs. ( a, b ) Representative immunostaining showing the protein abundance of ACOX1 in ADL) and in ADNL epidermis compared with the epidermis of healthy donors (CTRL). The dashed line indicates the dermal‒epidermal boundary. Bar = 30 μm. ( c ) Representative H&E staining of HEEs generated with KCs infected with lentivirus containing either pHR-SFFV-ACOX1 (ACOX1 OE) or pHR-SFFV-Puro CTRL(Puro) vector. Bar = 50 μm. ( d ) mRNA and ( e, f ) protein levels of ACOX1 in HEEs overexpressing ACOX1 compared with those in their Puro CTRLs (n = 8). The dashed line indicates the basal epidermal layer. Bar = 50 μm. Data were analyzed with a paired Student’s t -test. ∗∗∗ P < 0.001. AD, atopic dermatitis; ADL, lesional atopic dermatitis; ADNL, nonlesional atopic dermatitis; CTRL, control; HEE, human epidermal equivalent; KC, keratinocyte.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Over Expression, Immunostaining, Quantitative Proteomics, Staining, Generated, Infection, Plasmid Preparation, Control

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Analysis of HEEs overexpressing ACOX1. ( a ) Ultrastructural analysis showing LB secretion (upper panel, arrows), LB numbers (lower panel, arrows), and morphology (insets) in HEEs overexpressing ACOX1 (right panel) and in their Puro controls (left panel). Osmium tetroxide after fixation. Bar = 250 nm or 125 nm (inset). ( b ) LB numbers and ( c ) quantified secretion areas in HEEs in eight randomly selected fields per group (n = 2). ( d ) TEER and ( e ) LY penetration assay (green) in HEEs. Nuclei were counterstained with DAPI (blue). Bar = 50 μm. (n = 3). ( f ) Representative Ki-67 staining and the number of Ki-67‒positive nuclei in HEEs overexpressing ACOX1 compared with those in their Puro controls (n = 5). ( g ) Heat map showing the fold changes in the mRNA level of inflammation-related genes in HEEs (n = 5–7). Data were analyzed with a paired Student’s t -test. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. HEE, human epidermal equivalent; LB, lamellar body; LY, Lucifer yellow; SC, stratum corneum; SG, stratum granulosum; TEER, transepithelial electrical resistance.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Staining

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Lipidomic analysis of HEEs overexpressing ACOX1. ( a ) d18:1 Cers(NS) (n = 3) and ( b ) FFA (n = 3) species in HEEs generated with KCs infected with lentivirus containing either pHR-SFFV-ACOX1 (ACOX1 OE) or pHR-SFFV-Puro control (Puro) vector. Data are shown as analyte/IS ratio (AU) per mg protein or as the relative percentage of total lipid species. Data were analyzed with a Student’s t -test. ∗ P < 0.05. AU: arbitrary unit; Cer, ceramide; FFA, free fatty acid; HEE, human epidermal equivalent; IS, internal standard; KC, keratinocyte; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Generated, Infection, Control, Plasmid Preparation

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Fatty acid synthesis and elongation in ft/ft mouse epidermis. ( a ) Relative mRNA levels of Fasn , Elov11 , Elov4 , and Elov6 in the epidermis of ft/ft mice compared with those in the epidermis of the CTRL mice (n = 9–10). ( b ) Representative western blot and immunostaining showing protein abundance of ELOVL1 in the epidermis of CTRL and ft/ft mice. ( c ) qPCR showing the relative mRNA level of Acox1 (left panel) and its protein abundance (right panel) in the epidermis of ft/ft mice uncovered (CTRL) or covered with an occlusive dressing to reduce TEWL (n = 5). The dashed line indicates the dermal‒epidermal boundary. Bar = 50 μm. Data were analyzed with a Student’s t -test. ∗ P < 0.05 and ∗∗∗ P < 0.001. CTRL, control; TEWL, transepidermal water loss.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Western Blot, Immunostaining, Quantitative Proteomics, Control

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Metabolic and ultrastructural analysis of Flg -KO mouse epidermis. ( a ) Microarray analysis showing fold changes of a subset of genes involved in FA metabolism in the epidermis of Flg -KO mice compared with those in the epidermis of CTRLs. ( b ) Relative Acox1 mRNA level (n = 10) and protein abundance as well as ( c ) ACOX activity in CTRL and Flg -KO mouse epidermis (n = 5). ( d ) Relative levels of PPAR mRNA, Ppar , isoforms in mouse epidermis (n = 10). ( e ) Ultrastructural analysis of Flg -KO mouse epidermis showing LB morphology and secretion (arrows). Osmium tetroxide after fixation. Bar = 250 nm. ( f ) Relative mRNA level of Glut1 in mouse epidermis (n = 5). ( g ) Glucose consumption and ( d ) lactate production by epidermal sheets of CTRL and Flg -KO mice (n = 5). ( h ) Intracellular levels of metabolites and Krebs cycle intermediates quantified by LC‒MS in the epidermis of Flg -KO mice compared with those in the epidermis of CTRLs. Fold changes between the mean values of five mice per group are shown (n = 5). Data were analyzed with a Student’s t -test. The list of genes featured on the array and of all quantified metabolites as well as respective fold changes are provided in Tables 3 and . CTRL, control; Flg -KO, Flg -knockout; KO, knockout; LB, lamellar body; LC‒MS, liquid chromatography‒mass spectrometry; PPAR, peroxisome proliferator–activated receptor; SC, stratum corneum; SG, stratum granulosum.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Microarray, Quantitative Proteomics, Activity Assay, Control, Knock-Out

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: MC903 mouse model of ADL ( a ) Representative H&E staining of ear sections (bar = 200 μm) of MC903-treated mice compared with that of the vehicle-treated CTRLS (n = 7) and ( b ) TEWL values measured on the ears of mice (n = 7). ( c ) qPCR and immunostaining showing mRNA and protein level of ACOX1 in the epidermis of mice treated with MC903 or vehicle. The dashed line indicates the dermal‒epidermal boundary. Bar = 50 μm. mRNA level of ( d ) Acot5 ; ( e ) Crot ; ( f ) PPAR mRNA, Ppar , isoforms; and Fabp5 in the epidermis of mice treated with MC903 or vehicle (n = 7). ( g ) mRNA level and protein abundance of GLUT1 and ( h ) mRNA level of key enzymes of glycolysis as well as of ( i ) Ldha and Pdk1 in the epidermis of mice treated with MC903 or vehicle (n = 7). The dashed line indicates the dermal‒epidermal boundary. Bar = 50 μm. Data were analyzed with a Student’s t -test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. ADL, lesional atopic dermatitis; CTRL, control; PPAR, peroxisome proliferator–activated receptor; TEWL, transepidermal water loss.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Staining, Immunostaining, Quantitative Proteomics, Control

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: IMQ mouse model and biopsies from patients with PSO. ( a ) Representative H&E staining of ear sections (bar = 200 μm) of mice treated with 5% IMQ cream or with petroleum jelly (CTRL) (n = 7) and ( b ) TEWL values measured on the ears of mice (n = 7). ( c ) qPCR and immunostaining showing mRNA and protein level of ACOX1 in the epidermis of mice treated with 5% IMQ or with petroleum jelly. The dashed line indicates the dermal‒epidermal boundary. Bar = 50 μm. ( d ) Immunostaining of ACOX1 in the epidermis of patients with PSO and healthy donors (CTRL). The dashed line indicates the dermal‒epidermal boundary. Bar = 30 μm. Data were analyzed with a Student’s t -test. ∗∗∗ P < 0.001. CTRL, control; IMQ, imiquimod; PSO, psoriasis; TEWL, transepidermal water loss.

Article Snippet: The lentiviral vector containing human ACOX1 (NM_004035) was generated by cloning human ACOX1 cDNA from the pCMV-ACOX1 (OriGene Technologies, Rockville, MD) plasmid into the BamHI/NotI site of the lentiviral pHR-SIN-CSGW vector (kindly provided by Mary Collins, University College London, London, United Kingdom), thereby generating pHR-SFFV-hACOX1.

Techniques: Staining, Cream, Immunostaining, Control

Journal: PLoS Pathogens

Article Title: Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism

doi: 10.1371/journal.ppat.1006256

Figure Lengend Snippet: (A.) Overview of Essential Fatty Acids (EFA) and peroxisome pathway. Numbers indicate genes altered by KSHV (time post infection in black and fold change in red) as identified by RNA-seq in orange ( PLA2G4A ), a previously published metabolomics screen in blue and proteomic screen in red. siRNA treatments of ABCD3 and ACOX1 for panel B are indicated in blocked red sign. (B.) TIME cells were transfected with a control siRNA (siSCRB) or siRNA to ABCD3 or ACOX1. siABCD3 and siACOX1 treatments lead to greater than 70% reduction in ABCD3 and ACOX1 expression as determined by qRT-PCR normalized to the housekeeping genes GAPDH and HPRT. (C.) TIME cells were transfected with siRNAs as in panel B and 24 later were Mock- or KSHV-infected. 96 hpi (120 hours post transfection) cells were harvested and % cell death was measured using Trypan blue stain. In parallel, cells were treated with 20 μM QVD, a pan-caspase inhibitor. Data shown is from three independent experiments. Student’s t-test ( D .) Data shows the average fold change in % dead cells over control siRNA transfected cells from three independent experiments from panel C. (E.) IncuCyte microscopy images identifying dead cell nuclei (YOYO-1) for Mock- and KSHV-infected cells transfected with siSCRB, siABCD3 or siACOX1 at 96 hpi. Essen software was used to identify cell nuclei by size and fluorescent intensity, with background subtracted. YOYO-1 positive nuclei are in fluorescent green. All the data are represented as mean +/- SEM.

Article Snippet: siRNAs specifically targeting ACOX1 (pre-validated) were purchased from Santa Cruz Biotechnology (Cat. Sc-94104).

Techniques: Infection, RNA Sequencing, Transfection, Control, Expressing, Quantitative RT-PCR, Staining, Microscopy, Software