anti p acly  (Santa Cruz Biotechnology)

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Study design; DEN-injected animals fed Control Chow (Control-DEN) or Western Diet (WD-DEN). b , Liver mass, c , Liver mass/BW, and d , plasma AFP levels; mean ± SEM, WD-DEN (n = 10) vs Control-DEN (n = 8); unpaired two-tailed t-test: P = 3.86 × 10 −6 (b), P = 1.41 × 10 −5 (c). e , Representative H and E-stained liver sections. f , histological scores for steatosis, ballooning degeneration, inflammation, and NAS; mean ± SEM, WD-DEN (n = 10) vs Control-DEN (n = 8); unpaired Mann-Whitney two-tailed test: P = 2.29 × 10 −5 . g , Dorsal and ventral liver images. h , Tumor counts; mean ± SEM, WD-DEN (n = 10) vs Control-DEN (n = 8) mice; unpaired two-tailed t-test: P = 9.47 × 10 −8 . i , Tumor count distribution and j , tumor surface area; mean ± SEM, WD-DEN (n = 10) vs Control-DEN (n = 8); unpaired two-tailed t-test: P = 2.18 × 10 −5 . k , Distribution of tumor surface area per group. l , Histological feature of lesions (blue star: hepatic parenchyma, red star: Non-neoplastic proliferative lesion (FAH), orange arrow: slightly atypical hepatocyte (enlarged nuclei, higher N/C ratio, basophilic cytoplasm), black arrow: mitosis, blue arrows: atypical hepatocytes with enlarged and occasional binucleated nuclei, irregular nuclear membrane, prominent nucleoli and cytoplasmic alterations, red arrow: balloon cells with Mallory Denk bodies, yellow arrow: inflammatory infiltration). m , Number of neoplastic lesions in Control-DEN (n = 8) and WD-DEN (n = 10); mean ± SEM; unpaired two-tailed t-test. n , Representative H and E-stained liver images showing similar macro/microvesicular steatosis in hepatocytes and HCC cells and ballooning (arrowhead) in livers from WD-DEN mice and MASH HCC samples from humans. Scale bar = 100 µm. o , Average pairwise correlation on a per-model basis for mouse samples (column) and per-tissue or tissue molecular subtype basis for human samples (row). p , Proportion of mouse and human tumor tissue classification based on Hoshida molecular subtypes. q , Pairwise Pearson correlation between mouse models (columns) for cell types of interest (rows), using normalized gene expressions for all 1-to-1 orthologous genes between the mouse models (WD-DEN, WD-CCl 4 ) and humans with MASH-HCC. r , Individual sample pairwise mouse to human correlation matrix. s - t , Mean Acly mRNA expression by cell types in livers from WD-DEN mice ( s ) and human MASH-HCC ( t ).

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Injection, Control, Western Blot, Clinical Proteomics, Two Tailed Test, Staining, MANN-WHITNEY, Membrane, Expressing

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Experimental scheme of the Acly -KO model. TN, thermoneutral. b , Representative images of ACLY protein expression in WT and hepatocyte-specific Acly -KO liver and tumours. c , d , The H -score of ACLY protein expression in tumour ( c ) and tumour-adjacent liver ( d ). Data are presented as mean ± s.e.m. n = 5 Acly -KO versus n = 5 WT livers. P values were determined by two-tailed, unpaired Student’s t -test: P = 3.54 × 10 −5 ( c ) and P = 1.23 × 10 −6 ( d ). e , Representative Acly -KO mouse liver stained with ACLY antibody. The asterisk indicates inflammatory cell aggregation-positive ACLY staining; the blue arrows denote mesenchymal cell (endothelial or Kupffer cells)-positive ACLY staining; and the orange arrows show hepatocyte-negative ACLY staining. f , Representative dorsal and ventral images of WT and Acly -KO liver with tumours. g , Visible number of tumours on the liver surface. Data are presented as mean ± s.e.m. n = 11 WT and n = 11 Acly -KO mice. P values were determined by unpaired, two-tailed Student’s t -test. h , Percentage distribution graph of tumour numbers from livers of WT ( n = 11) and Acly -KO ( n = 11) mice. i , Number of neoplastic lesions in livers from WT ( n = 11) and Acly -KO ( n = 11) mice. Data are presented as mean ± s.e.m. P values by unpaired, two-tailed Student’s t -test. j , Representative images of neoplastic lesions from the livers of WT and Acly -KO mice. k , Representative images showing tumour with lipid (left) and calculation of the percentage lipid area of WT ( n = 5) and Acly -KO ( n = 5) livers (right). Scale bars, 600 µm. Data are presented as mean ± s.e.m. P values were determined by unpaired, two-tailed Student’s t -test. l , Fatty acids (FAs) in tumours from WT ( n = 11) and Acly -KO ( n = 10) mice. C:D, the total number of carbon atoms to the number of carbon–carbon double bonds. Data are presented as mean ± s.e.m. P values were determined by unpaired, two-tailed Student’s t -test.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Expressing, Two Tailed Test, Staining

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Baseline serum AFP levels prior to AAV injection in WD-DEN treated mice. Data are presented as mean ± SEM. WT (n = 8) and Acly KO (n = 8) mice and statistical comparison was analyzed by two-tailed unpaired t-test. b , Representative diagram of genetic deletion of ACLY using hepatocyte-specific AAV. The diagram was created using BioRender ( https://biorender.com ). c , Pathological scoring of livers for steatosis, ballooning, inflammation, and NAFLD activity in WT (n = 8) and Acly KO (n = 8) mice. Data are presented as mean ± SEM. Statistical comparison for histological scores was analyzed by an unpaired Mann-Whitney two-tailed test. d , Fatty acid levels in tumor adjacent liver sections from WT and Acly KO mice (FA, Fatty acid; C:D, the total number of carbon atoms to the number of carbon-carbon double bonds). Data are presented as mean ± SEM, Acly KO (n = 10) vs WT (n = 11) mice by unpaired two-tailed t-test. Significant differences observed for FA(18:0): P = 3.7 × 10 −5 , FA(20:1): P = 5.09 × 10 −5 , FA(20:2): P = 8.35 × 10 −5 , FA(22:1): P = 9.46 × 10 −7 , FA(22:5): P = 4.39 × 10 −7 and FA(22:6): P = 1.08 × 10 −7 . e , Percentage of total liver area covered by tumors. Data are presented as mean ± SEM. WT (n = 5) and Acly KO (n = 5) mice analyzed by unpaired two-tailed t-test. f , Non-neoplastic proliferative lesions in livers from WT (n = 11) and Acly KO (n = 11) mice. Data are presented as mean ± SEM. P-values by unpaired two-tailed t-test. g , Average lipid droplet size in liver tumors from WT and Acly KO mice. Data are presented as mean ± SEM, n = 5/group. Statistical significance within each genotype condition was analyzed by an unpaired two-tailed t-test.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Injection, Comparison, Two Tailed Test, Activity Assay, MANN-WHITNEY

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , 14 C-acetate incorporation into fatty acids and cholesterol in mouse primary hepatocytes treated with varying doses of EVT0185 (0.1 and 0.5 µM ( n = 4 samples per group), 0.3, 1 and 3 µM ( n = 10 samples per group), and 10, 30 and 100 µM ( n = 6 samples per group)). Data are presented as mean ± s.e.m. b , Effect of EVT0185 and EVT0185-CoA on ACLY activity. Data are presented as mean ± s.e.m. Inhibition of ACLY activity by EVT0185 ( n = 3 samples) and EVT0185-CoA ( n = 3 samples). c , d , Michaelis–Menten ( c ) and Lineweaver–Burk ( d ) plots for ACLY with EVT0185-CoA at three distinct concentrations. Data are presented as mean ± s.e.m. n = 3 independent experiments. e , Domain architecture of ACLY. f , 2D cryo-EM class averages for the ACLY–( R , S )-EVT0185-CoA complex. ess, effective sample size; ptcls, particles. g , Cryo-EM reconstruction without symmetry applied. The sharpened map is contoured at 8.74 σ (absolute level of 0.176) and coloured by the different structural domains as in panel e . The red dashed line indicates the region used for local refinement. h , i , Sharpened cryo-EM map contoured at 9.94 σ ( h ; absolute level of 0.12) and real space refinement atomic model ( i ) following local refinement after symmetry expansion and 3D classification. j , Adenosine 3′-phosphate 5′-diphosphate moiety of EVT0185-CoA modelled in the sharpened cryo-EM map at the CoA-binding pocket located at the interface between the CCS and CSH modules of ACLY. The map is carved around the ligand with a carve radius of 2 Å. The interacting residues of ACLY are labelled. The disordered pantothenyl arm is tentatively indicated.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Activity Assay, Inhibition, Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Global fit of Michaelis-Menten Plots by GraFit software for EVT0185-CoA effects on ACLY activity. b , Motion-corrected micrograph with picked particles encircled. The bottom scale bar is 20 nm. c , Cryo-EM data processing workflow in cryoSPARC for the ACLY:(R,S)-EVT0185-CoA complex. d , Sharpened cryo-EM map for ACLY:(R, S)-EVT0185-CoA complex following refinement without symmetry applied illustrating the ligand density at the four CoA-binding binding pockets of ACLY. The density is overlayed with the final real-space refined model molecular model for adenosine 3’-phosphate 5’-diphosphate (shown in Fig. ) based on the structural superposition of the CSH domains. e , Segment of the sharpened cryo-EM map following symmetry expansion, 3D classification, and local refinement and carved around residues ACLY residues 1054–1077 (carve radius = 2 Å). f-g , Comparison between the binding modes of (R, S)-EVT0185-CoA (panel f) and CoA bound to human ACLY in pdb 6hxh (panel g).

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Software, Activity Assay, Cryo-EM Sample Prep, Binding Assay, Comparison

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Graphical representation showing metabolic enzymes in the regulation of sterol and fatty acid synthesis. The graphical representation was created using BioRender ( https://biorender.com ). ( b-c ), Effect of BA (Bempedoic acid) and EVT0185 on b , lactate and c , acetate incorporation into fatty acids and sterols in WT and Acly KO mouse hepatocytes. Each stacked bar represents the mean ± SEM, % suppression with BA or EVT0185 in WT (n = 3 ) and Acly KO (n = 3) mice. ( d-g ) Inhibitory effect of EVT0185-CoA on the activity of d , AMPK; e , ACC1; f , ACC2; and g , ACSS2. Each line graph represents the mean ± SEM, enzyme activity inhibition by EVT0185-CoA (n = 3 samples/group). ( h and i ) Effect of Bempedoic acid and EVT0185 on clonogenic survival/colony formation in h , Hep3B human, and i , Hepa1-6 mouse HCC cell lines. Each line graph represents the mean ± SEM colony formation in cells treated with EVT0185 or Bempedoic acid (n = 4 biologically independent samples/treatment groups).

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Activity Assay, Inhibition

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Experimental scheme. b , No. of visible tumors on the liver surface in mice 4 weeks after adenovirus injection. Data are presented as mean ± SEM, WT (n = 7) vs. Acly KO (n = 7). Statistical analysis was performed using a two-tailed unpaired t-test. c , Differential expression analysis of Acly KO using an additive model adjusting for timepoint. Significance was determined by Wald test with a false discovery adjusted threshold of 5% as implemented in DESeq2. d , Modifying effect of time on Acly KO using an interaction model. Significance of interaction was determined by Wald test with a false discovery adjusted threshold of 5% as implemented in DESeq2. e , Comparison between the number of significant genes in each module negatively associated with both Acly expression and surface tumor burden. f , GSEA of decreased tumor growth size. g , TCA cycle showing a percentage change in tumor citrate and succinate levels in Acly KO mice compared to WT mice. The illustration of the TCA cycle was created using BioRender ( https://biorender.com ). h , Tumor Suclg1 and Sucla2 mRNA expression. Each bar represents the mean ± SEM, Acly KO (n = 12) vs WT (n = 9) mice, P values by two-tailed unpaired t-test. i , Hierarchical clustering of upregulated biological processes based on semantic similarity. j , Identification of co-expression modules significantly associated with Acly expression and surface tumor burden. k , Gene ontology analysis of co-expressed genes within module blue, Acly KO (n = 12) vs WT (n = 9) mice.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Injection, Two Tailed Test, Quantitative Proteomics, Comparison, Expressing

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Volcano plot of bulk RNA-seq of tumours showing upregulated and downregulated genes at early or late timepoints from Acly- KO ( n = 12) versus WT ( n = 9) mice. Significance was determined by Wald test with a false discovery-adjusted threshold of 5% as implemented in DESeq2. Horizontal dashed lines demarcate the P value threshold at a 5% false discovery rate (FDR). b , Gene Ontology analysis of selected biological processes involving significantly upregulated genes in tumours from Acly -KO ( n = 12) versus WT ( n = 9) mice. FC, fold change. c , Top 10 Gene Ontology biological processes (GOBP) from clusters identified among significantly upregulated gene sets in tumours from Acly -KO ( n = 12) versus WT ( n = 9) mice. NES, normalized enrichment score. d , Correlation between B cell populations and Acly expression in tumours from WT ( n = 9) and Acly -KO ( n = 12) mice. Confidence bands denote the upper and lower bounds of the 95% confidence interval. Significance of association was determined by a two-sided Student’s t -test of regression coefficients and at a false discovery-adjusted threshold of 5%. e – p , Spatial transcriptomic analysis of livers from WT and Acly -KO mice and vehicle or EVT0185-treated mice. Cluster analysis representing the number of cell types in the liver and tumour ( e , k ). Umapharmony integration analysis showing increased B cells in Acly- KO ( f ) and in EVT0185-treated ( l ) mice. The top upregulated pathways in HCC cells from Acly -KO ( g ) and EVT0185-treated ( m ) mice. Statistical analysis was performed using Fisher’s exact test. Expression level of metabolic genes ( h , n ). Expression of markers of subtypes of B cells ( i , o ). The box-and-whisker plots are defined by the median with the first quartile (Q1), third quartile (Q3), minimum (Q1 − 1.5 × interquartile range (IQR)) and maximum (Q3 + 1.5 × IQR). Cxcl13 expression levels in HCC cells ( j , p ). GC, germinal centre; HSC, hepatic stellate cell; MZ, marginal zone; Tr, regulatory; VSMC, vascular smooth muscle cell.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: RNA Sequencing, Expressing, Whisker Assay

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a-b , Immune cell markers expressed in different cell types in a , WT and Acly KO and b , Vehicle and EVT0185-treated mice. c , Top upregulated pathways in B cells in Acly KO or EVT0185-treated mice (spatial transcriptomics analysis). Statistical analysis was performed using Fisher’s Exact test. d and e , Single seq analysis of d , WD-DEN and e , WD-CCl 4 mouse livers showing top upregulated pathways in B cells. Statistical analysis was performed using a one-sided hypergeometric test (enrichGO); p-values adjusted by Benjamini-Hochberg. f , Cxcl13 mRNA expression analyzed from publicly available RNA-seq dataset in WT and Acly KO DEN tumors cultured in vitro ( GSE223966 ) . Boxplot lines represent the first quartile, median, and third quartile. Whiskers connect the minimum and maximum values. Significance was ascertained by an unpaired two-tailed t-test between Acly KO vs WT (n = 4 hepatocellular carcinoma cell lines derived from DEN-induced tumors in Acly f/f mice).

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Expressing, RNA Sequencing, Cell Culture, In Vitro, Two Tailed Test, Derivative Assay

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , t -distributed stochastic neighbour embedding (tSNE) plots showing cell phenotypes in WT and Acly -KO mice. DC, dentritic cell; endo–meso, endothelial–mesothelial; Mac, macrophage. b , Representative tumour images: 2 × 2 mm 2 montage (25 fields of view (FOVs)), single 400 × 400 μm FOV, and 100 × 100 μm region of interest (ROI). dsDNA, double-stranded DNA. c , d , B cell ( c ) and T cell ( d ) counts at the tumour–liver interface. Data are mean ± s.e.m. n = 4 mice per group. P values were determined by Wilcoxon unpaired, two-sided test. e , Representative image of CD19 + staining (top) and quantification of CD19 + cells per mm 2 tumour area in WT ( n = 12) and Acly -KO ( n = 10) lesions (bottom). Data are mean ± s.e.m. P value was determined by unpaired, two-tailed Student’s t -test. f , Percentage of mice with B cells present or absent in tumour TLSs. g , Pixel images of lymphoid marker levels with cell neighbourhood and phenotype maps overlaid on cell-boundary masks. The tumour interior is marked by a black star. CN1, cellular neighbourhood 1; Hep, hepatocyte; NK cell, natural killer cell. h , Distribution of cells across cellular neighbourhoods. i , CN3 and CN5 (antigen-presenting enriched) represented as ‘antigen-presenting complex’. The box-and-whisker plots show the median as hinge and a box representing the middle 50% of the data, bounded by the lower and upper quartiles. The whiskers extend to data within 1.5 × IQR. P value was determined by Wilcoxon non-parametric unpaired, two-sided test. n = 4 WT and n = 4 Acly -KO tumour border regions. j , Schematic of B cell depletion protocol in the Acly f/f WD-DEN model. k , Representative liver images. l , m , Tumour count ( l ) and surface area ( m ). Data are mean ± s.e.m. n = 12 WT isotype-injected, n = 4 WT anti-CD20-injected, n = 7 Acly -KO isotype-injected and n = 7 Acly -KO anti-CD20-injected mice. P values were determined by one-way ANOVA with Fisher’s LSD: P = 7.51 × 10 −5 ( Acly -KO isotype versus WT isotype). n , ACLY upregulation in human MASH-HCC ( n = 53) relative to non-tumour adjacent tissue ( n = 29), MASH liver ( n = 74), cirrhotic liver ( n = 8) and healthy liver ( n = 6). The box-and-whisker plot lines represent the Q1, median and Q3. The whiskers connect the minimum and maximum values. P values were determined by two-tailed Student’s t -test as implemented in limma. o , Hepatocyte-specific ACLY expression in MASH-HCC tumour and adjacent tissues (scRNA-seq). P values were determined by χ 2 test of independence. Error bars represent 95% confidence interval. p , q , Correlation of ACLY expression with B cell-specific markers ( p ), and B cell abundance based on cell-type deconvolution methods ( q ). Confidence bands denote 95% confidence interval bounds; two-tailed Student’s t -test of regression coefficients (FDR < 0.05).

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Staining, Two Tailed Test, Marker, Whisker Assay, Injection, Expressing

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a and b , Quantification of lipid droplet area was extrapolated by converting the PLIN-2 signal into a mask and quantifying the area covered by this mask per 400 × 400 micron field of view (FOV). These calculations were performed in ImageJ using custom scripts. Each dot represents a single FOV, Wilcoxon non-parametric unpaired two-sided test. Acly KO (n = 188) vs WT (n = 170) FOVs from n = 4 mice/group. c , CXCL13 protein level in tumors from WT and Acly KO mice; mean ± SEM, n = 5 mice/group; unpaired two-tailed t-test. d , Representative images showing B cells at tumor centre and periphery (Orange star: Tumor front (border-periphery), Black star: Surrounding non-tumoral hepatic tissue). e , B cell aggregations in Acly KO tumor (Orange star: Tumor, Black star: Non-tumoral liver tissue, Blue star: B Cell aggregations). f , Heatmap of cell neighborhoods hierarchically sorted by cell phenotype and showing relative cell abundances. ( g-j, l-n ) WD-DEN mice treated with Vehicle or EVT0185. g , Representative MIBI images of liver tumors. h , B cell, and i , T cell counts from regions representing the tumor lesion interface with the liver; mean ± SEM, n = 4 mice/group; unpaired Wilcoxon non-parametric unpaired two-sided test. j , CD19+ positive cells count/mm 2 tumor area; mean ± SEM, Vehicle (n = 29) and EVT0185 (n = 21) lesions; unpaired two-tailed t-test. k , CD3+ positive cells count/mm 2 tumor area; mean ± SEM, WT (n = 12) and Acly KO (n = 10) lesions; unpaired two-tailed t-test. l , CD3+ positive cells count/mm 2 tumor area; mean ± SEM, Vehicle (n = 29) and EVT0185 (n = 21) lesions; unpaired two-tailed t-test. m , Percentage of mice with/without B cells in TLS. n , TLS in EVT-treated mouse compared with vehicle control. o , GO enrichment analysis showing upregulated pathways related to complement activation in B cells in the EVT0185-treated group relative to Vehicle. Statistical analysis was performed using one-sided hypergeometric test (enrichGO); p-values adjusted by Benjamini-Hochberg.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Two Tailed Test, Control, Activation Assay

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a-c, a , Representative images with percentage area fluorescence level of b , CCP3; mean ± SEM, Acly KO (n = 11) vs WT (n = 13) tumors by unpaired two-tailed t-test, and c , Ki67 protein expression in tumor leading edge; mean ± SEM, Acly KO (n = 12) vs WT (n = 15) tumors by unpaired two-tailed t-test: P = 8.74 × 10 −8 . d-f, d , Representative images and percentage area fluorescence level of e , CCP3 ( P = 1.34 × 10 −8 ) and f , Ki67 ( P = 1.27 × 10 −6 ) protein expression in the tumor leading edge. Data are presented as mean ± SEM. EVT0185 vs Vehicle control by unpaired two-tailed t-test, (n = 18 tumors per group). g , DOX inducible shRNA suppresses ACLY expression and phosphorylation in Hep3B cells. For gel source data, see Supplementary Fig. . ( h-j ) Inducible knockdown led to h , reduced [14C] glucose-mediated DNL and i , increased [3H] acetate mediated DNL in Hep3B cells and j , increased fatty acid oxidation based on the sampling of [ 14 C] incorporation in gaseous carbon dioxide obtained from cell culture media. Data are presented as mean ± SEM. Number of cell culture replicates per condition were WT (n = 3), shNTC (n = 3), shACLY #1 (n = 3), and shACLY #2 (n = 3). Significance was determined by an unpaired one-tailed t-test. k , Outline of in vivo orthotopic experiment. The illustration of the mouse was created using BioRender ( https://biorender.com ). l , Bioluminescence at 6 weeks was not significantly different between control and ACLY-deficient tumor. Each bar represents the mean ± SEM. Number of mice per condition at the time of sacrifice were shNTC -Dox (n = 3), shNTC +Dox (n = 5), shACLY -Dox (n = 7), shACLY +Dox (n = 9). Significance was determined by unpaired one-tailed t-test.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Fluorescence, Two Tailed Test, Expressing, Control, shRNA, Phospho-proteomics, Knockdown, Sampling, Cell Culture, One-tailed Test, In Vivo

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Gating strategy to identify B220 + CD19 + cells in Isotype control, and Anti-CD20 injected WT and Acly KO mice. Briefly, cells were gated as follows: SSC vs FSC plot of lymphocytes population, FSC-H vs FSC-A to identify single cell population, FSC vs 7-AAD to identify live cells population, B220 vs CD19 to identify B220 + CD19 + B cell population. b . B220 + CD19 + cells in Iso (Isotype control) and Anti-CD20 (B cell-depleted)-injected WT or Acly KO mice. Data are presented as mean ± SEM, Significant reductions were observed in WT-Anti-CD20 (n = 4; P = 2.37 × 10 –6 ) and in Acly KO-Anti-CD20 (n = 7; P = 9.76 × 10 –8 ), compared to WT-Isotype control (n = 11)-injected mice. The Acly KO-Isotype control group included n = 8 mice. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD test.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Control, Injection

Journal: Nature

Article Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer

doi: 10.1038/s41586-025-09297-0

Figure Lengend Snippet: a , Differential expression analysis comparing MASH-HCC (n = 53) and non-tumor adjacent liver (n = 29), MASH liver (n = 74), cirrhotic liver (n = 8), and healthy liver tissue (n = 6); two-tailed t-test; 5% FDR as implemented in limma. b , Overlap between significantly upregulated and downregulated genes in each pairwise comparison. c , Gene expression of lipogenic enzymes acetyl-CoA carboxylase ( ACACA or ACACB ), ACSS2 , and FASN across disease states (MASH-HCC, n = 53; non-tumor adjacent, n = 29; MASH liver, n = 74; cirrhotic liver, n = 8; healthy liver, n = 6). Boxplot lines represent the first quartile, median, and third quartile. Whiskers connect the minimum and maximum values. d , ACLY upregulation in human MASH-HCC compared to all other tissue types using scRNAseq samples. Significance assessed using Wilcoxon rank-sum tests comparing each condition to MASH-HCC, with p-values adjusted using the Benjamini-Hochberg method. Exact p-adj values: vs healthy P adj = 6.9 × 10 −247 , vs MALSD P adj = 1.0 × 10 −16 , vs non-tumor adjacent P adj = 1.6 × 10 −18 . e , Differential expression analysis of genes associated with ACLY expression in MASH-HCC; two-tailed t-test; 5% FDR as implemented in limma. f , Principal component embeddings of genes differentially expressed with respect to ACLY expression validate stratification of MASH-HCC tissues by high, medium, and low ACLY expression levels. g , Upregulation of CXCL13 among human MASH-HCC samples with reduced (bottom tertile, n = 18) relative to elevated (top tertile, n = 18) ACLY expression. Significance determined by two-tailed t-test with a false discovery adjusted threshold of 5% as implemented in limma. Boxplot lines represent the first quartile, median, and third quartile. Whiskers connect the minimum and maximum values. h , Identification of co-expression modules associated with ACLY expression and immune features. i , Significant biological processes associated with the immune and ACLY associated gene co-expression module.

Article Snippet: Primary antibodies (ACLY (13390; 1:1,000 dilution); p-ACLY S455 (4331; 1:1,000 dilution); and β-actin HRP conjugate (12620; 1:5,000 dilution); all Cell Signaling Technology) were diluted at 1:1,000 in TBST-BSA solution.

Techniques: Quantitative Proteomics, Two Tailed Test, Comparison, Gene Expression, Expressing