Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: a Atf3 mRNA levels in white adipose tissue (WAT) of db/db mice, ob/ob mice, high-fat diet (HFD)-fed mice, or Western diet (WD)-fed mice and their control mice ( n = 7–8 per group). b , c Western blot assays were performed ( b ), and protein levels were quantified ( c ). d ATF3 mRNA levels in adipose tissue of lean versus obese individuals ( n = 6–8). e – j Atf3 mRNA and protein levels in white adipocytes ( e – g ) and stromal vascular fraction (SVF) ( h – j ) of male C57BL/6 mice that had been fed a chow (lean) or Western diet (obese) for 4 months ( n = 4). k – m 3T3-L1 cells were treated with DMEM containing IBMX/Dex/Insulin for 2 days, and the media were then changed to DMEM containing Dex/Insulin every 2 days until fully differentiated. After culture in DMEM without FBS for 3 h, the cells were treated with palmitate at indicated concentrations for 24 h. mRNA ( k ) ( n = 6) and protein ( l , m ) levels ( n = 3) were determined. Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t- test ( a , c – e , g , h , j ) or one-way ANOVA ( k , m ). * P < 0.05, ** P < 0.01 versus controls. Additional supporting data can be found in Supplementary Fig. .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Western Blot, Control

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: Aft3 fl/fl mice and Atf3 Adi−/− mice were fed a western diet (WD) for 20 weeks ( n = 7 per group). a Plasma transaminase levels. b Plasma free fatty acid (FFA) levels. c Plasma adiponectin levels. d Hepatic triglyceride (TG) levels. e Hepatic FFA levels. f Hepatic hydroxyproline levels. g NAFLD activity score (NAS). h Hematoxylin (H&E) staining (top panel), oil red O (ORO) staining (middle panel), and picrosirius red staining (bottom panel). i Hepatic mRNA levels. j Western blot assays of hepatic proteins. k Relative protein levels. Scale bars in ( h ): 50 μm. Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t- test ( a – g , i , k ). * P < 0.05, ** P < 0.01 versus controls. Additional supporting data can be found in Supplementary Figs. – .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Western Blot, Clinical Proteomics, Activity Assay, Staining

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: Aft3 fl/fl mice and Atf3 Adi −/− mice were fed a western diet (WD) for 20 weeks ( n = 7 per group). a Body fat content. b H&E staining of epididymal white adipose tissue (eWAT) and brown adipose tissue (BAT). c eWAT adipocyte size. d Energy expenditure over a 48-h period. e Energy expenditure during the full day, day, or night time. f Glucose tolerance test (GTT). g The area under the curve (AUC) of GTT. h Insulin tolerance test (ITT). i Western blot assays of hepatic (top panel) or BAT (bottom panel) proteins. j Quantification of protein levels. k Plasma cytokine levels. l Gene expression in adipose tissues. Scale bar in ( b ): 50 mm. Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t-test ( a , c , g , j – l ) or two-way ANOVA ( d – f , h ). * P < 0.05, ** P < 0.01 versus controls. Additional supporting data can be found in Supplementary Figs. – .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Western Blot, Staining, Clinical Proteomics, Gene Expression

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: Aft3 fl/fl mice and Atf3 Adi−/− mice were fed a chow diet or a high-fat diet (HFD) for 16 weeks ( n = 7–8 per group). a Plasma ALT levels. b Plasma AST levels. c Plasma adiponectin levels. d Plasma free fatty acid (FFA) levels. e Hepatic triglyceride (TG) levels. f Hepatic FFA levels. g Hepatic hydroxyproline levels. h The area under the curve (AUC) of GTT. i Oil red O staining. j Picrosirius red staining. k Fat content. l H&E staining of eWAT. m eWAT adipocyte size. Scale bars in ( i , j , l ): 50 mm. Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t- test ( a – h , k , m ). * P < 0.05, ** P < 0.01. Additional supporting data can be found in Supplementary Figs. – .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Clinical Proteomics, Staining

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: a – k Aft3 fl/fl mice and Atf3 Adi −/− mice were fed a high-fat diet (HFD) for 12 weeks and then injected i.p. with either saline (vehicle) or 5-Aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR, 500 mg/kg/day) for 4 weeks ( n = 7–8 per group). a Western blot assays of WAT proteins (top panel) or hepatic proteins (bottom panel). b Plasma ALT levels. c Plasma AST levels. d Hepatic triglyceride (TG) levels. e Hepatic free fatty acid (FFA) levels. f Hepatic hydroxyproline levels. g H&E staining of liver sections. h Picrosirius red staining of liver sections. i Body fat content (%). j eWAT weight. k H&E staining of eWAT. Statistical analysis was performed using two-way ANOVA ( b – f , i , j ). * P < 0.05, ** P < 0.01. l – u Aft3 fl/fl mice and Atf3 Adi −/− mice were i.v. injected with AAV8-TBG-Null or AAV8-TBG-AMPKa, then fed a western diet (WD) for 16 weeks ( n = 7 per group). l – n Hepatic levels of rAmpka mRNA ( l ) and proteins ( m , n ). o Plasma ALT levels. p Plasma AST levels. q Plasma FFA levels. r Hepatic TG levels. s Hepatic FFA levels. t Hepatic hydroxyproline levels. u H&E staining (top panel) and Picrosirius red staining (bottom panel) of liver sections. v Body fat content (%). w liver-to-body weight ratio (%). Scale bars in ( g , h , k , u ): 50 mm. Statistical analysis was performed using one-way ANOVA ( l , m – t , v , w ). * P < 0.05, ** P < 0.01. Additional supporting data can be found in Supplementary Figs. – .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Injection, Saline, Western Blot, Clinical Proteomics, Staining

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: Aft3 fl/fl mice and Atf3 Adi−/− mice were i.v. injected with AAV8-TBG-shScr or AAV8-TBG-shCd36 and then fed a western diet (WD) for 16 weeks ( n = 7 per group). a Body weight. b Plasma FFA levels. c Liver weight. d Plasma ALT levels. e Plasma AST levels. f Hepatic triglyceride (TG) levels. g Hepatic FFA levels. h Hepatic hydroxyproline levels. i Western blot assays. j Quantification of protein levels. k , l Hepatic mRNA levels. m Hematoxylin-eosin (H&E) staining (top panel) and Picrosirius red staining (bottom panel) of liver sections. Scale bars in ( m ): 50 μm. shScr scramble shRNA. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA ( b – h , j – l ) or two-way ANOVA ( a ). * P < 0.05, ** P < 0.01 versus controls.

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Injection, Western Blot, Clinical Proteomics, Staining, shRNA

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: a – c Aft3 fl/fl mice and Atf3 Adi−/− mice were fed a Western diet (WD) for 20 weeks ( n = 7 per group). a mRNA levels in WAT. b , c Protein levels in WAT. d , e Lipolysis was performed in the presence of isoproterenol (10 μM) and/or Atglistatin (20 μM) for 2 h ( n = 6 per group). d Released free fatty acid (FFA) levels. e Released glycerol levels. f , g Transient transfections were performed by co-transfecting pGL3 (vector) or pGL3-Atgl with CMV-Null or CMV-ATF3 into 3T3-L1 cells ( n = 10). After 36 h, relative luciferase activities (RLU) were determined and normalized to beta-galactosidase activity. WT or Mut refers to wild-type or mutant Atgl promoter. h ChIP assays were performed using 3T3-L1 cell lysates overexpressing ATF3-Flag ( n = 8). After pulldown by a Flag or IgG (control) antibody, the DNA enrichment in the Atgl promoter was determined by qRT-PCR. Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t- test ( a , c , h ) or two-way ANOVA ( d – g ). * P < 0.05, ** P < 0.01. Additional supporting data can be found in Supplementary Fig. .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Control, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: a – d 3T3-L1 cells were infected with Ad-Empty or Ad-ATF3 and then differentiated to adipocytes in the presence of the hormonal cocktail ( n = 6). Oil red O staining ( a ) and de novo lipogenesis in the presence of 14 C-acetic acid ( b ) were performed. The 2-h release of free fatty acid (FFA) ( c ) or glycerol ( d ) in the presence of isoproterenol (10 μM) and/or atglistatin (20 μM) was analyzed. e – h 3T3-L1 cells were infected with Ad-shLacZ or Ad-shAtf3 and then differentiated to adipocytes in the presence of the hormonal cocktail ( n = 6). Oil red O staining ( e ) and de novo lipogenesis in the presence of 14 C-acetic acid ( f ) were performed. The 2-h release of FFA ( g ) or glycerol ( h ) in the presence of isoproterenol (10 μM) and/or atglistatin (20 μM) was analyzed. Scale bars in ( a , e ): 50 mm. Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed, unpaired t- test ( b , f ) or two-way ANOVA ( c , d , g , h ). * P < 0.05, ** P < 0.01. Additional supporting data can be found in Supplementary Figs. , .

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Infection, Staining

Journal: Communications Biology

Article Title: Loss of adipose ATF3 promotes adipose tissue lipolysis and the development of MASH

doi: 10.1038/s42003-024-06915-x

Figure Lengend Snippet: a , b Fat pads from Aft3 fl/fl mice or Atf3 Adi−/− mice were treated with vehicle or isoproterenol (10 μM) for 2 h. The conditioned media were then used to treat primary hepatocytes for 24 h ( a ). b mRNA levels of Srebp1c , Acc , Fasn , Mcp1 , and Tnfa were determined ( n = 6 per group). c Primary hepatocytes were treated with bovine serum albumin (BSA; control) or 100 mM palmitic acid, oleic acid, or linoleic acid for 24 h, and mRNA levels were quantified ( n = 6). d A model for ATF3 in white adipose tissue (WAT) or brown adipose tissue (BAT) to inhibit the development of MASLD. Adipocyte ATF3 is induced in obesity, which in turn prevents MASLD via blocking FFA flux to hepatocytes by inhibiting ATGL-mediated lipolysis. ATF3 also inhibits lipogenesis and inflammation in WAT and induces thermogenesis in BAT, thus preventing obesity and insulin resistance (IR). Data are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA ( b ) or one-way ANOVA ( c ). * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies against activating transcription factor 3 (ATF3) (cat # NBP1-85816) or sterol regulatory element-binding protein 1 (SREBP-1) (cat # NB600-582) were purchased from Novus.

Techniques: Control, Blocking Assay

Journal: BMC Complementary and Alternative Medicine

Article Title: Cytotoxic activity of the twigs of Cinnamomum cassia through the suppression of cell proliferation and the induction of apoptosis in human colorectal cancer cells

doi: 10.1186/s12906-018-2096-x

Figure Lengend Snippet: Contribution of ROS-dependent ATF3 activation to TC-HW-mediated apoptosis. a and b HCT116 and SW480 cells were treated with TC-HW at the indicated concentrations for 24 h. For Western blot analysis, cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against ATF3. Actin was used as internal control for Western blot analysis. For RT-PCR analysis of the gene expression of ATF3, total RNA was prepared. GAPDH was used as internal control for RP-PCR. b HCT116 cells were co-transfected with ATF3 promoter and pRL-null for 24 h, and then treated with TC-HW at the indicated concentrations for 24 h. Luciferase activity for ATF3 promoter activity was measured as a ratio of firefly luciferase signal/renilla luciferase signal using a dual luciferase assay kit. * P < 0.05 compared to cell without TC-HW. c HCT116 cells were treated with TC-HW (100 μg/ml) in presence/absence of NAC. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against ATF3. Actin was used as internal control for Western blot analysis. d HCT116 cells were co-transfected with ATF3 promoter and pRL-null for 24 h, and then treated with TC-HW (100 μg/ml) in presence/absence of NAC. Luciferase activity for ATF3 promoter activity was measured as a ratio of firefly luciferase signal/renilla luciferase signal using a dual luciferase assay kit. * P < 0.05 compared to cell without TC-HW. e HCT116 cells were transfected with control and ATF3 siRNA and then treated with TC-HW (100 μg/ml). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against ATF3 or cleaved PARP. Actin was used as internal control for Western blot analysis

Article Snippet: Antibody for activating transcription factor (ATF3) was purchased from Santa Cruz Inc. (Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot, SDS Page, Control, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Transfection, Luciferase, Activity Assay