Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: Random insertion mutagenesis screens identify new cholesterol regulatory genes a-d , Plasma membrane (PM) cholesterol was measured by mNeonGreen-ALOD4 (mNG-ALOD4) staining followed by flow cytometry. Wild type (WT) HAP1 cells are compared to GRAMD1A, GRAMD1B, and GRAMD1C triple knockout HAP1 cells ( GRAMD1 TKO) either left untreated ( a ) or treated with 4 µM 25-hydroxycholesterol (25HC) for the times indicated ( b ). WT cells were treated with or without LXR inhibitor (LXRi; 1 µM GSK2033) for 16 hrs in 5% FBS and either left untreated ( c ) or treated with 25HC for the times indicated ( d ). e , Schematic showing cholesterol trafficking from the PM to the ER membrane where it binds to proteins including SCAP and ACAT1 or ACAT2 (ACAT). ACAT esterifies cholesterol with a fatty acid (FA), enabling it to be stored in lipid droplets (LD). Cholesterol is transported to the PM from the Golgi . f , Schematic of genetic screening pipelines. The mutant cell library was treated with either of NPC1i (1 μM U18666A) for 20 hrs or 4 μM 25HC for 6 hrs and then cells were stained with mNG-ALOD4. The top 10% of mNG-ALOD4 fluorescent cells were isolated for sequencing. g,h, Screen results showing enriched genes with an FDR-corrected p -value of less than 2E-4 for cells treated with NPC1i ( g ) or 25HC ( h ). IGTIOB is a measure of the inactivating potential of the mapped insertions, and circle size shows the number of insertions for each gene. i,j , mNG- ALOD4 flow cytometry analysis of PM cholesterol in WT or ACC1 KO HAP1 cells treated with or without 1 μM of NPC1i for 20 hrs ( i ) or WT and NPC1 KO HAP1 cells treated with or without ACC1i (30 μM Firsocostat) for 16 hrs ( j ). k-m , Cells were left untreated ( k ) or treated for 16 hrs with 30 µM ACC1i and then treated with 4 µM 25HC for the times indicated ( l and m ). The amount of mNG-ALOD4 bound to the PM was measured by flow cytometry. Data is normalized to cells not treated with 25HC for each condition, and each circle is the mean of three biological replicates; error bars are the standard deviation. Statistical tests compare two conditions at the same time point of 25HC treatment ( d and k-m ). Statistical significance was determined by Student’s t-test with a Welch’s correction ( a-d and j- m ) or an Ordinary one-way ANOVA ( i ). Exact p -values are reported in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Mutagenesis, Clinical Proteomics, Membrane, Staining, Flow Cytometry, Triple Knockout, Isolation, Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: ACC1 functions independently of ACAT1/2 to maintain plasma membrane cholesterol levels. a , Fluorescence microscopy image showing wild-type (WT) 3T3 cells left untreated or treated with 0.3 mM MβCD:cholesterol or ACC1i (30 μM Firsocostat) and stained with mNG-ALOD4 and DAPI. Scale bar is 10 microns. b-d , Cell lines were left untreated ( b ) or treated with 30 µM Firsocostat ( c ) or 20 μM CP-640186 for 16 hrs ( d ) and then stained with mNG-ALOD4 and analyzed by flow cytometry. e , Cells were treated with 4 µM 25HC for 4 hrs and then media was replaced with PFO* for 30 minutes at 37°C before cell viability was measured with an XTT assay. f , Schematic of cholesterol ester synthesis. ACAT1 and ACAT2 (ACAT) enzymes transfer a fatty acid from acyl-CoA to cholesterol, forming cholesterol esters. g , Western blot (ACAT1) and PCR ( ACAT2 ) analysis of ACAT1/2 DKO clones. h , WT and ACAT1/2 DKO cell lines were treated with 4 μM 25HC for 6 hrs and the esterified cholesterol was quantified using the Amplex Red assay. i , j , Cells were left untreated ( i ) or treated for 16 hrs with ACC1i (30 μM Firsocostat) ( j ); cells were then treated with 4 µM 25HC for the times indicated and then the amount of mNG-ALOD4 bound to the PM was measured by flow cytometry. k , Western blot (ACAT1 and ACC1) or PCR ( ACAT2 ) analysis of ACC1/ACAT1/2 TKO clones . l , Cells were treated with 4 µM 25HC for the indicated times and then mNG-ALOD4 staining was measured by flow cytometry. m , mNG-ALOD4 flow cytometry analysis of PM cholesterol in WT and ACAT1/2 DKO HAP1 cells left untreated or treated with ACC1i (30 μM Firsocostat) for 16 hrs. n , Quantification of total cellular free (unesterified) cholesterol in WT HAP1 cells or ACC1 KO HAP1 cells treated with or without 4 μM 25HC for 8 hrs. Cells ( b , d , and m ) were grown in lipoprotein depleted serum media for 16 hours prior to treatments. For each condition ( i , j and l ), data is normalized to cells not treated with 25HC, and each data point represents the average of three biological replicates, with error bars denoting the standard deviation. Statistical significance was determined by a Student’s t-test with a Welch’s correction ( b , c , h - j , l and n ) or an Ordinary one-way ANOVA ( d , and m ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Microscopy, Staining, Flow Cytometry, XTT Assay, Western Blot, Clone Assay, Amplex Red Assay, Standard Deviation

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: Lipid droplet catabolism increases plasma membrane cholesterol. a , Plasma membrane (PM) cholesterol was measured by mNG-ALOD4 staining followed by flow cytometry in WT HAP1 cells treated with ACC1i (30 µM Firsocostat) for the indicated times. b , c , Microscopy images of HAP1 cells left untreated or treated with ACC1i (30 μM Firsocostat), and then stained with ACC1 monoclonal antibody ( b ) or 647-conjugated ACC1 polyclonal antibody ( c ), Bodipy 493/503 to measure lipid droplets (LDs) and DAPI (blue). Scale bar is 5 microns. d , Schematic showing that enzymes involved in the synthesis of fatty acyl-CoAs, which are then converted into triacylglycerol (TAG), cholesterol esters (CE), sphingolipids (SL) and phospholipids (PL). TAG and CE are the main components of the LD core (green oval). Inhibitors targeting enzymes in this process are shown in gray. e - g mNG-ALOD4 flow cytometry analysis of PM cholesterol (blue curves) and the corresponding fluorescence microscopy quantification of the number of LDs per cell (purple curves) in HAP1 cells grown in 5% LDS ( e ) or 5% FBS ( f , g ) and then treated with ACC1i (30 μM Firsocostat) ( e , f ) or ACSLi (5 μM Triacsin-C) ( g ). LDs were quantified by taking the mean number of LDs per cell in four fields of view with ∼75 cells per field. h , i , mNG-ALOD4 flow cytometry analysis of PM cholesterol in WT HAP1 cells treated with or without ACSLi (5 μM Triacsin-C) for 16 hrs in 5% LDS, followed by treatment with 4 μM 25HC ( h ) or 30 μM ACC1i ( i ) for the indicated times. j , k , LD quantification ( j ) and mNG-ALOD4 flow cytometry analysis of PM cholesterol ( k ) of WT HAP1 cells treated with or without 30 μM ACC1i or 5 μM ACSLi for 16 hrs, followed by 200 μM oleic acid treatment for 6 hrs in 5% FBS. Each flow cytometry data point ( e - i ) represents the mean of three biological replicates, with error bars indicating the standard deviation. Values were normalized to the untreated control at the corresponding time point. Statistical significance was determined by Ordinary one-way ANOVA ( a ) or Student’s t-test with a Welch’s correction ( h - k ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Staining, Flow Cytometry, Microscopy, Fluorescence, Standard Deviation, Control

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: ACC1 inhibition triggers lipid droplet catabolism through ATGL. a , Schematic illustrating the formation of fatty acyl-CoAs from de novo synthesis of fatty acids (top) or from the breakdown of triacylglycerol (TAG) into diacylglycerol and a fatty acid by ATGL (bottom). ATGL localizes at the lipid droplet (LD) surface. b , Western blot analysis of ATGL KO HAP1 cells compared to a wild-type (WT) control. c , d , Quantification of LD numbers per cell ( c ) or LD area ( d ) in WT and ATGL KO cells. e - g Microscopy images ( e , f ) and quantification ( g ) of LDs in WT ( e ) and ATGL KO ( f ) HAP1 cells treated with or without ACC1i (30 μM Firsocostat) for 6 hrs. Cells were stained with Bodipy 493/503 and DAPI (blue). Scale bar is 5 microns. Each data point is the total number of LDs per cell in a single field of view, with ∼75 cells per field ( c and g ). h , i , mNG-ALOD4 flow cytometry analysis of PM cholesterol in WT and ATGL KO cells ( h ) or WT cells treated with or without ATGLi (50 μM NG-497) ( i ), followed by treatment with ACC1i (30 μM Firsocostat) for the indicated times. All experiments were carried out in cells pre-cultured for 16 hours in lipoprotein depleted serum. Data points represent the average of three biological replicates, with error bars indicating the standard deviation. All values were normalized to the untreated control ( h , i ). Statistical significance was determined by a Student’s t-test with a Welch’s correction ( c , d and g - i ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Control, Microscopy, Staining, Flow Cytometry, Cell Culture, Standard Deviation

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: Increasing lipid droplet abundance reduces plasma membrane cholesterol. a - c , Wild-type (WT) HAP1 cells were left untreated or treated with ACSLi (5 μM Triacsin-C), ATGLi (50 μM NG-497) or both for 16 hrs and then analyzed by microscopy to quantify lipid droplet (LD) numbers per cell ( a , b ) or by mNG-ALOD4 flow cytometry analysis to determine plasma membrane (PM) cholesterol levels ( c ). d , Schematic demonstrating that AMPK inactivates ACC1 and activates ATGL. Inhibitors for each enzyme are shown in gray. e , Western blot analysis of ACC1 phosphorylation in WT HAP1 cells left untreated or treated with AMPKi (2.5 μM Dorsomorphin), ACC1i (30 μM Firsocostat) or both. f - h WT HAP1 cells were left untreated or treated with 30 μM ACC1i, 2.5 μM AMPKi or both and then analyzed by microscopy to quantify LD numbers per cell ( f , g ) or by mNG-ALOD4 flow cytometry analysis to determine PM cholesterol levels ( h ). i , j , LD quantification ( i ) and flow cytometry analysis of PM cholesterol using mNG-ALOD4 staining ( j ) in ACAT1/2 DKO cells left untreated or treated with 2.5 μM AMPKi, 30 μM ACC1i or both. All cells were grown in lipoprotein depleted serum media for 16 hours prior to drug treatments. Lipid droplets are visualized with Bodipy 493:503 and nuclei are visualized with DAPI. Scale bars are 5 microns. Statistical significance was determined by an Ordinary one-way ANOVA ( b - c and g - j ). Each data point is the total number of LD per cell in a single field of view ( b , g and i ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Microscopy, Flow Cytometry, Western Blot, Phospho-proteomics, Staining

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: ACC1 loss traps cholesterol at the plasma membrane and trigger de nova cholesterol synthesis in a mouse model. a , Western blot analysis of ACC1 and ACC2 proteins in primary hepatocytes isolated from Acc1/Acc2 flox/flox and Albumin-Cre- Acc1/Acc2 dLKO mice. b - d , Hepatic fatty acid synthesis rates ( b ), hepatic triacylglycerol (TAG) levels ( c ), and hepatic cholesterol ester (CE) levels ( d ) in Acc1/Acc2 flox/flox and Albumin-Cre- Acc1/Acc2 dLKO mice. e , Fluorescence microscopy images of primary hepatocytes stained with mNG-ALOD4 and DAPI. Scale bar is 10 microns. Quantification is shown in ( f ). g , h , Bubble plot showing differential gene expression from RNA sequencing in WT HAP1 cells. Cells were pre-cultured in lipoprotein depleted serum (LDS) media for 16 hours and then left untreated or treated for 6 hrs with HMGCRi (10 µM Lovastatin), ACC1i (30 μM Firsocostat), ACSLi (5 μM Triacsin-C) or FASNi (10 μM C75). Differential gene expression was computed by comparing each treatment to the untreated (LDS only) control. Bubble size shows the FDR-corrected p -value and bubble color shows the fold change (see legends) for each SREBF2 ( g ) and SREBP1 ( h ) target gene. SREBP1 target genes are ranked based on their enrichment in a ChIP-Seq dataset. i , Gene Set Enrichment Analysis from RNA sequencing data using the top 200 genes sorted by the FDR-corrected p -value. “nf” refers to not found. j - l , Hepatic sterol synthesis rate ( j ), hepatic cholesterol content ( k ), or fecal cholesterol measurement ( l ) in chow-fed Acc1/Acc2 flox/flox and Albumin-Cre- Acc1/Acc2 dLKO mice. m , Schematic showing a proposed model: ACC1 inhibits ATGL, blocking the breakdown of TAG to diacylglycerol (DAG) and fatty acids (FA). When ACC1 is inhibited (right), ATGL is activated, leading to TAG hydrolysis. Cell diagrams show that lipid droplets (LD) control cholesterol trafficking from the PM to the ER. When LDs are depleted (right), cholesterol becomes trapped at the PM, and SREBP2 drives the expression of cholesterol biosynthesis genes. Statistical significance was determined by a Student’s t-test with a Welch’s correction ( b - d, f and j - l ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Western Blot, Isolation, Fluorescence, Microscopy, Staining, Gene Expression, RNA Sequencing, Cell Culture, Control, ChIP-sequencing, Blocking Assay, Expressing

Journal: The FASEB Journal

Article Title: Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis

doi: 10.1096/fj.201600845r

Figure Lengend Snippet: Figure 3. Full p53 stabilization under starvation requires AMPK signaling. A) HepG2 cells were starved for indicated times, and Western blot analysis was performed to determine protein levels of AMPK subunits a and b and their respective phosphorylated forms. b-Actin (ACTB) served as loading controls. B) Primary human hepatocytes from 2 donors were treated for 24 h with 0.5 mM AICAR or with DMSO as vehicle control and analyzed for p53 protein expression. Phosphorylated form of ACC1 is shown to validate AMPK activation; ACTB served as loading control. C) HepG2 cells were treated for 24 h with 10 mM nutlin-3a, 0.5 mM AICAR, or respective vehicle controls and analyzed for p53 protein expression. D) qPCR analysis of p53 target gene expression after starvation or treatment with 0.5 mM AICAR or 10 mM nutlin-3a. Expression levels are set to 1 in control. *P , 0.05 compared to control. E) Western blot analysis validating knock-down of AMPKa and decreased AMPK activity by reduced phosphorylation of downstream target ACC1. F) Two days after transfection with control or AMPKa siRNA, HepG2 cells were starved for 6 h or received fresh medium. Western blot analysis was used to determine p53 protein levels. G) Quantification of p53 protein expression from 3 independent experiments (F). Protein abundance was set to 1 for starved cells transfected with control siRNA. *P , 0.05 compared to siControl.

Article Snippet: The following antibodies were used: human p53 (sc-126; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse p53 (2524; Cell Signaling Technology, Danvers, MA, USA), b-actin (ab6276; Abcam, Cambridge, MA, USA), ras-related nuclear protein, (RAN) (610340; BD Biosciences, San Jose, CA, USA), p21 (2947; Cell SignalingTechnology), andAMPK/acetyl-CoA carboxylase 1 (ACC1) total and phosphorylated from Cell Signaling Technology (9957; AMPK and ACC1 antibody sampler kit).

Techniques: Western Blot, Control, Expressing, Activation Assay, Targeted Gene Expression, Knockdown, Activity Assay, Phospho-proteomics, Transfection, Quantitative Proteomics

Journal: RSC Advances

Article Title: Lipid-lowering and antioxidative effects ofApium graveolensL. root flavonoid extracts

doi: 10.1039/c9ra04481g

Figure Lengend Snippet: Fig. 8 FDFT1, SREBP1, HMGCR and ACC1 protein status and gene expression. P < 0.05 indicates significant difference between two groups. *, compare with Control group, #, compare with Model group.

Article Snippet: View Article Online Shanghai Trading Co. Ltd. (Shanghai, China), the anti-acetyl-CoA carboxylase 1 (ACC1) antibody from Proteintech (Rosemont, IL, USA), and the anti-GAPDH and secondary antibodies from Elabscience Biotechnology Co. Ltd. (Wuhan, China).

Techniques: Gene Expression, Control

Journal: Frontiers in Pharmacology

Article Title: Mechanisms of Puerariae Lobatae Radix in regulating sebaceous gland secretion: insights from network pharmacology and experimental validation

doi: 10.3389/fphar.2024.1414856

Figure Lengend Snippet: Docking information of Puerariae Lobatae Radix with key pathway targets.

Article Snippet: Antibodies such as ACC1 (Proteintech, catalog number 67373-1-Ig) and PPAR-γ (Proteintech, catalog number 16643-1-AP) were used for specific protein detection.

Techniques: Activation Assay, Gene Expression

Journal: Frontiers in Pharmacology

Article Title: Mechanisms of Puerariae Lobatae Radix in regulating sebaceous gland secretion: insights from network pharmacology and experimental validation

doi: 10.3389/fphar.2024.1414856

Figure Lengend Snippet: Analyze the docking situation. (A) 5281708-PPARG, (B) 5281708-ACC1, (C) 5281807-PPARG, (D) 5281807-ACC1.

Article Snippet: Antibodies such as ACC1 (Proteintech, catalog number 67373-1-Ig) and PPAR-γ (Proteintech, catalog number 16643-1-AP) were used for specific protein detection.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Mechanisms of Puerariae Lobatae Radix in regulating sebaceous gland secretion: insights from network pharmacology and experimental validation

doi: 10.3389/fphar.2024.1414856

Figure Lengend Snippet: (A–D) Effects of Puerariae Lobatae Radix on the expression of PPARG and ACC1 in the sebaceous glands of golden hamsters (* p < 0.05, ** p < 0.01, both compared with the blank control group).

Article Snippet: Antibodies such as ACC1 (Proteintech, catalog number 67373-1-Ig) and PPAR-γ (Proteintech, catalog number 16643-1-AP) were used for specific protein detection.

Techniques: Expressing, Control

Journal: GeroScience

Article Title: A chemical biology approach to identifying molecular pathways associated with aging

doi: 10.1007/s11357-020-00238-5

Figure Lengend Snippet: CAD031 maintains several parameters of mitochondrial metabolism that are altered with aging in SAMP8 mice. a Heatmap of the fold gene expression for 9 months/13 months, 13 months/13 months+CAD031, regarding the genes that are common to the KEGG pathways Parkinson’s disease, oxidative phosphorylation, Huntington’s disease, and Alzheimer’s disease. Most are associated with the mitochondrial ETC. The log2 fold-change is plotted in red–blue color scale with red indicating upregulation and blue indicating downregulation. ETC, electron transport chain complex; CI, complex I; CIII, complex III; CIV, complex IV; CV, complex V. b Heatmap of the fold gene expression for 9 months/13 months, 13 months/13 months+CAD031, regarding all genes from the mitochondrial DNA with detectable expression levels. Color key = log2FC. c Transcriptomic drift of genes that encode for all proteins known to be associated with the mitochondria (Mitocarta) and proteins associated with the ETC. Values are expressed as box-and-whisker plots. Brown-Forsythe test. n = 5–6/group. d RIPA-soluble fractions from hippocampal tissue of 9 months, 13 months, and 13 months+CAD031 SAMP8 mice were analyzed by Western blotting for the ETC markers NDUFB8 (CI), SDHB (CII), UQCRC2 (CIII), COX4 (CIV), MTCO1 (CIV), ATP5a (CV), and the mitochondrial protein VDAC. Quantifications were normalized to actin. One-way ANOVA followed by Tukey-Kramer post hoc test (n = 5/group). All data are mean ± SD

Article Snippet: The primary antibodies HRP-conjugated rabbit anti-actin (#5125, 1/20,000), phospho-ACC1 (#3661, 1/2000), total ACC1 (#4190, 1/1000), phospho-AMPK (#2535, 1/ 1000), total AMPK (#2793, 1/1000), COX4 (#11967, 1/2000), and VDAC-1 (#4866, 1/2000) were from Cell Signaling Technology.

Techniques: Gene Expression, Phospho-proteomics, Expressing, Whisker Assay, Western Blot

Journal: ACS Omega

Article Title: Humanin Ameliorates Free Fatty Acid-Induced Endothelial Inflammation by Suppressing the NLRP3 Inflammasome

doi: 10.1021/acsomega.0c01778

Figure Lengend Snippet: Humanin prevented FFA-induced inactivation of the AMPK/ACC signaling pathway in HAECs. Cells were treated with high FFA (1 mM) with or without humanin (25, 50 μM) for 2 h. (A) Phosphorylated and total levels of AMPKα; (B) phosphorylated and total levels of ACC (****, P < 0.0001 vs vehicle control; ##, p < 0.01 vs FFA treatment group; $$$$, P < 0.0001 vs FFA + 25 μM humanin group, n = 4–5).

Article Snippet: The following primary antibodies were used: NOX4 (1:2000, Cat#GTX12206, Gene Tax), TxNIP (1:2000, Cat#ab188865, Abcam), NLRP3 (1:2000, Cat#19771-1-AP, Protein Tech), p10 (1:2000, Cat#sc-514, Santa Cruz Biotechnology), phospho-AMPKα (1:2000, Cat #50081, Cell Signaling), AMPKα (1: 2000, Cat#2532, Cell Signaling), phospho-ACC (1:2000 Cat# 3661, Cell Signaling), ACC (1:2000, Cat#3662, Cell Signaling), and β-actin (1:5000, Cat#sc-130656, Santa Cruz Biotechnology).

Techniques: Control