Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: a – c HepG2 cells were treated with 500 μM H 2 O 2 for 48 h to induce senescent hepatocytes: 9-HODE and 13-HODE levels in control and senescent hepatocytes ( a ) and in the conditioned medium from these cells ( b ); ( c ) Western blot analysis of the protein level of ALOX15 in control and senescent hepatocytes; n = 5 independent experiments. d Immunofluorescence staining of ALOX15 and γH2AX in liver sections from mice aged 2.5 or 12 months; scale bar = 5 μm. e – g HepG2 cells were treated with 500 μM H 2 O 2 for 48 h to induce senescence and were transfected with si- AlOX15 or si-NC. e Western blot analysis of the protein level of ALOX15; n = 4 independent experiments. f , g The conditioned medium of these cells was collected: Oil Red O staining of HepG2 cells treated with the indicated conditioned medium; n = 5 independent experiments; scale bar = 50 μm. h , i HepG2 cells were treated with 2 μM Doxorubicin (DOX) for 2 h and then cultured in DOX-free medium for 6 days to induce senescence: western blot analysis of the protein levels of ALOX15, P21, and P53 ( h ); 9-HODE and 13-HODE levels in the conditioned medium from these cells ( i ). j , k RAW264.7 cells were treated with 2 μM DOX for 2 h and then cultured in DOX-free medium for another 4 days to induce senescence: western blot analysis of the protein levels of ALOX15, P21, and P53 ( j ); 9-HODE and 13-HODE levels in the conditioned medium from these cells ( k ). h – k n = 3 independent experiments. Data represent the mean ± SEM. Two-tailed student’s t test was performed for ( a – c , h – k ); One-way ANOVA with Fisher’s LSD was performed for e . mo month, Sen senescent. Figure 2f was created with Biorender.com.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

Techniques: Control, Western Blot, Immunofluorescence, Staining, Transfection, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: Eight-week-old mice were treated with a mixture of 9-HODE and 13-HODE (equal amounts of 9-HODE and 13-HODE and a combined dose of 0.5 μg/g body weight) once a day for 9 days: ( a ) body weight, liver weight, and liver/body weight; ( b ) fasting blood glucose level; ( c ) H&E, Oil Red O, and Nile Red staining of liver sections; scale bar = 100 μm for H&E and Oil Red O; scale bar = 20 μm for Nile Red; ( d ) liver TG and CHO content; ( a – d ) n = 9 mice in Ctrl group; n = 7 mice in HODE group; for liver weight and liver/body weight, n = 7 mice per group. e – h RNA sequencing was performed: ( e ) PCA analysis; ( f ) heatmap of differentially expressed genes; ( g ) GO biofunction enrichment analysis of differentially expressed genes; ( h ) expression level of differentially expressed genes that promote liver steatosis. n = 3 mice per group. i qPCR analysis of the mRNA levels of Fasn , Thrsp , Cd36 , Cyp4a14 , and Elovl6 ; n = 9 mice in Ctrl group; n = 7 mice in HODE group. Data represent the mean ± SEM. Two-tailed student’s t test was performed for ( a , d ) and ( Fasn , Thrsp , Cd36 and Cyp4a14 in i ); Two-tailed Mann-Whitney test for ( b ) and ( Elovl6 in i ). HODEs 9/13-HODEs, TG triglyceride, CHO total cholesterol, FBG fasting blood glucose.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

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

Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: a Western blot analysis of the protein levels of cleaved SREBP1, full-length SREBP1, and FASN in 9/13-HODEs-treated mice or control mice; n = 9 mice in Ctrl group; n = 7 mice in HODE group. b Western blot analysis of the protein levels of cleaved SREBP1, full-length SREBP1, and FASN in middle-aged or control mice; n = 6 mice per group. c Western blot analysis of the protein levels of cleaved SREBP1, full-length SREBP1, and FASN in aged or control mice; n = 5 mice per group. d Oil Red O staining of primary mouse hepatocytes treated with a mixture of 9-HODE and 13-HODE (equal amounts of 9-HODE and 13-HODE with a total concentration of 1 μM) for 48 h; n = 5 independent experiments; scale bar = 50 μm. e – g Primary mouse hepatocytes treated with 9-HODE or 13-HODE (1 μM) for 48 h: ( e ) Oil Red O staining; scale bar = 50 μm; ( f ) western blot analysis of the protein levels of cleaved SREBP1 and full-length SREBP1; ( g ) qPCR analysis of the mRNA levels of Fasn , Thrsp , Cd36 , Cyp4a14 , and Elovl6 ; n = 5 independent experiments. h Primary mouse hepatocytes were pretreated with or without 13-HODE (1 μM) for 24 h and then co-treated with 25-HC (1 μg/mL) for 24 h: western blot analysis of the protein level of cleaved SREBP1; n = 5 independent experiments. i Primary mouse hepatocytes were pre-treated with or without 13-HODE (1 μM) for 36 h and then co-treated with MG132 (10 μM) for 12 h: western blot analysis of the protein level of cleaved SREBP1; n = 4 independent experiments. Data represent the mean ± SEM. Two-tailed Student’s t test was performed for ( a , cleaved SREBP1 and FASN in b , c , g ); Two-tailed Mann–Whitney test for (Full-length SREBP1 in b , h ); One-way ANOVA with Fisher’s LSD was performed for f , i . HODEs: 9/13-HODEs; mo: months; 25-HC: 25-Hydroxycholesterol.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

Techniques: Western Blot, Control, Staining, Concentration Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: a Primary mouse hepatocyte proteins were pulled down by biotin-labeled 13(S)-HODE followed by proteomics; ( b ) top 10 proteins based on the Vina scores from in silico molecular docking; ( c ) structural overview of CAT monomer-13(S)-HODE and CAT tetramer-13(S)-HODE model. d Surface plasmon resonance assay for interaction of 13(S)-HODE was passed over the Biacore chip surfaces immobilized with recombinant CAT protein: Biacore diagram and estimated dissociation constant value (KD) for 13(S)-HODE binding CAT. e Western blot analysis of the CAT protein levels in 13-HODE treated hepatocytes; n = 5 independent experiments. f CAT activity of hepatocytes treated with 13-HODE (1 μM) for 2, 6, or 12 h; n = 5 independent experiments. g Recombinant CAT protein was incubated with 13-HODE for 20 min, and CAT activity was measured; data are from 3 repeats. h CAT activity in 12-month-old or 2.5-month-old mouse livers; n = 6 mice per group. i ROS level demonstrated by DCFH-DA and fluorescence analysis of primary mouse hepatocytes treated with 13-HODE; scale bar = 250 μm; n = 5 independent experiments. j primary mouse hepatocytes were treated with 1 μM 13-HODE for 6 h; cells were pretreated with 100 μM DSS crosslinker for 1 h before protein extraction: Western blot analysis of protein level of tetramer and monomer of CAT; n = 4 independent experiments. Data are presented as the mean ± SEM. Two-tailed student’s t test was performed for e , f , h , i . Figure 5a was created with Biorender.com.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

Techniques: Labeling, In Silico, SPR Assay, Recombinant, Binding Assay, Western Blot, Activity Assay, Incubation, Fluorescence, Protein Extraction, Two Tailed Test

Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: a – d Primary mouse hepatocytes were transfected with Cat -expressing plasmid with or without 13-HODE treatment. a Western blot analysis of protein level of CAT. b Oil Red O staining; scale bar = 50 μm. Western blot analysis of protein levels of cleaved SREBP1 ( c ) and FASN ( d ). e–h Primary mouse hepatocytes were transfected with si- Cat or si-NC. e Western blot analysis of protein level of CAT. f Oil Red O staining; scale bar = 50 μm. Western blot analysis of protein levels of cleaved SREBP1 ( g ) and FASN ( h ). i – k Primary mouse hepatocytes were treated 13-HODE (1 μM) with or without NAC (10 mM). i Oil Red O staining; scale bar = 50 μm. Western blot analysis of protein levels of cleaved SREBP1 ( j ) and FASN ( k ). a – d , f – k n = 5 independent experiments; ( e ) n = 3 independent experiments. Data are presented as the mean ± SEM. Two-tailed Student’s t test was performed for ( a , e , g , h ); One-way ANOVA with Fisher’s LSD was performed for c , d , j , k . CAT OE catalase overexpression.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Two Tailed Test, Over Expression

Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: a – e Eight-week-old mice were injected with AAV- Cat -flag bearing TBG promoter or control AAV; 10 days after AAV injection, mice were treated with or without 13-HODE (0.5 μg/g body weight) once a day for 9 days. a Western blot analysis of protein level of CAT in liver, pancreas, eWAT, iWAT, and skeletal muscle. b H&E, Oil Red O and Nile Red staining of liver sections; scale bar = 100 μm for H&E and Oil Red O; scale bar = 20 μm for Nile red. c Liver TG and CHO content. d , e Western blot analysis of protein level of cleaved SREBP1 and FASN; ( b – e ) n = 6 mice in Ctrl group and 13-HODE group; n = 7 mice in 13-HODE + CAT group. f – i Eight-month-old mice were injected with AAV- Cat -flag or control AAV, and mice were sacrificed 2 months after AAV injection. f H&E, Oil Red O, and Nile Red staining of liver sections; scale bar = 100 μm for H&E and Oil Red O; scale bar = 20 μm for Nile red. g Liver TG and CHO content. h , i Western blot analysis of protein levels of cleaved SREBP1 and FASN; n = 8 mice per group. Data represent the mean ± SEM. One-way ANOVA with Fisher’s LSD was performed for c , e ; Two-tailed Student’s t test was performed for g , i . eWAT epididymal white adipose tissue, iWAT inguinal WAT, TG triglyceride, CHO total cholesterol, CAT OE catalase overexpression.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

Techniques: Injection, Control, Western Blot, Staining, Two Tailed Test, Over Expression

Journal: Nature Communications

Article Title: Senescence-associated 13-HODE production promotes age-related liver steatosis by directly inhibiting catalase activity

doi: 10.1038/s41467-023-44026-z

Figure Lengend Snippet: Increased ALOX15 in senescent hepatocytes and macrophages produces more 9-HODE and 13-HODE. Increased 13-HODE acts on the hepatocytes surrounding the senescent hepatocytes, binding to CAT and decreasing CAT activity. Decreased CAT activity activates SREBP1 further by increasing H 2 O 2 levels and promoting liver steatosis. The illustration was created with Biorender.com.

Article Snippet: To study the role of tetramerization of CAT in 13-HODE-induced hepatocyte steatosis, plasmid expressing CAT lacking N-terminal arm (residues 5-70; ΔCAT) was constructed to disrupt the tetramerization of CAT. Primary mouse hepatocytes were transfected with a his-tagged Cat - plasmid, a Δ Cat plasmid, or control pcDNA 3.1 plasmid, after 6 h, the cells were treated with 1 μM 13-HODE for another 48 h. For SREBP1 cleavage inhibition, hepatocytes were pretreated with 1 μM 13-HODE for 24 h before being co-treated with 1 μg/mL 25-HC (HY-113134, MedChemExpress, NJ) for another 24 h. For SREBP1 degradation assays, hepatocytes were pretreated with 1 μM 13-HODE for 36 h before being co-treated with 10 μM MG132 (HY-13259, MedChemExpress, NJ) for another 12 h. For Nr2c2 , Pparg and Rictor knockdown, hepatocytes were transfected for 48 h with siRNA targeting Nr2c2 , Pparg , Rictor , or negative control siRNA (Sangon Biotech, Shanghai).

Techniques: Binding Assay, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes express 15-lipoxygenase-1 and -2: potential role in osteoarthritis

doi: 10.1186/ar2652

Figure Lengend Snippet: 13-HODE and 15-HETE suppressed IL-1β-induced MMP-1/MMP-13 production in a PPARγ dependent manner. (a) 13-HODE and 15-HETE activate endogenous PPARγ in human chondrocytes. Chondrocytes were transiently transfected with a reporter construct containing three copies of a consensus PPRE placed upstream from the Tk-luciferase reporter (PPRE 3 -Tk-Luc) along with the internal control pSV40-β-gal using FuGene 6 transfection reagent. Six hours later, the cells were washed and changed to medium containing 0.5% fetal calf serum for an additional 18 hours. Transfected cells were then treated with the control vehicle dimethyl sulfoxide or increasing concentrations of 13-HODE or 15-HETE for 18 hours. Luciferase activity values were determined and normalized to β-galactosidase activity. Results are expressed as fold changes, considering 1 as the value of unstimulated cells, and are the mean ± standard deviation of three independent experiments. * P < 0.05 versus unstimulated cells. (b) PPARγ antagonist (GW9662) prevented the suppressive effect of 13-HODE and 15-HETE on IL-1β-induced MMP-1 and MMP-13 release. Chondrocytes were pretreated with increasing concentrations (1, 5, and 10 μmol/l) of GW9662 for 30 minutes. Then, the cells were treated with or without IL-1β (100 pg/ml) for 24 hours in the absence or the presence of 50 μmol/l 13-HODE (panel a) or 50 μmol/l 15-HETE (panel b). The levels of MMP-1 and MMP-13 proteins in conditioned media were measured using ELISA. Results are expressed as the percentage of control, considering 100% as the value of cells treated with IL-1β alone, and are the mean ± standard deviation of four independent experiments. * P < 0.05 versus cells treated with IL-1β and 13-HODE or 15-HETE. HETE, hydroxyeicosatetraenoic acid; HODE, hydroxy octadecadienoic acid; MMP, matrix metalloproteinase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor-responsive element.

Article Snippet: GW9662, 13(S)-HODE, 15(S)-HETE, anti-15-LOX-1 and 15-LOX-2 antibodies were from Cayman Chemical Co. (Ann Arbor, MI, USA).

Techniques: Transfection, Construct, Luciferase, Control, Activity Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay