Fig 2: CYR61 is a direct target gene of TGF-β in liver cancer cells.A, four different HCC cell lines were treated with or without 2.5 ng/ml of TGF-β1 for the indicated time periods, and then total RNAs were extracted and assessed by quantitative PCR. B and C, liver cancer cells treated with 2.5 ng/ml of TGF-β1 (B) or 5 μM of SB431542 (C) were harvested at different time intervals for Western blotting analyses. D, HCCLM3 or HLE cells transfected with a control siRNA (NS) or those targeting Smad2/3/4 were treated with 2.5 ng/ml of TGF-β1 for 24 h, followed by western blotting. E, schematic diagrams showing different CYR61 promoter regions that were cloned to generate a series of luciferase reporter constructs. Predicted Smad-binding elements (SBEs), TATA box and the transcription start site (TSS) were indicated. F, HEK293FT cells transfected with the above CYR61 promoter luciferase reporter constructs (CYR61-pro-luciferase reporters, 200 ng for each) and Renilla-luciferase plasmid (50 ng) were treated with or without 2.5 ng/ml of TGF-β1 for 24 h, followed by luciferase activity measurement. G, HEK293FT cells transfected with CYR61-pro-luciferase plasmid (−222/+15, 200 ng), Renilla-luciferase (50 ng) and constructs expressing Smad2, Smad3, or Smad4 for 24 h were subjected to luciferase activity determination. H, HEK293FT cells transfected with CYR61-pro-luciferase (−222/+15, with WT or mutated SBE1/2 sites, 200 ng each) were treated with 2.5 ng/ml of TGF-β1 for 24 h before luciferase activity determination. I, HCCLM3 cells treated with 2.5 ng/ml of TGF-β1 for 24 h were lysed for ChIP assay using anti-Smad2/3 antibody or the control rabbit IgG, followed by quantitative PCR analysis with a pair of primers as shown in Figure 2I. J, HCCLM3 cells treated with 2.5 ng/ml of TGF-β1 for 2 h were lysed for Smad2/3-DNA binding assays using biotin-labeled CYR61 promoter oligos. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 were calculated using two-tailed student t test. Data were shown as mean ± SD. ChIP, chromatin immunoprecipitation; HCC, hepatocellular carcinoma; IgG, immunoglobulin G; TGF-β, transforming growth factor-β.

Fig 3: Effect of CYR61 on endothelial cells and monocytes/macrophages(A) Heatmap showing HUVEC genes whose expression is dysregulated after 24-h incubation with CYR61 (adjusted p value < 0.05; n = 5 wells/group).(B) Heatmap showing THP1 gene expression changes after treatment with CYR61 (24 h; n = 5 wells/group). Genes shown are those with adjusted p value < 0.05 and |log2FC| > 1.5 in THP1 cells treated with 5 μg/mL.(C and D) Pathway analysis of the 1,110 genes with adjusted p value < 0.05 upregulated in BMDMs following CYR61 treatment (24 h, 0.5 μg/mL). The top 10 pathways, ranked by p value, are shown.(E) Heatmap showing genes dysregulated in BMDMs by CYR61 treatment (24 h, 0.5 μg/mL) with adjusted p value < 0.05 and |log2FC| > 1.5 (n = 4–7 wells/group).(F) Bar graphs showing changes in the expression of three pro-inflammatory and three pro-angiogenic genes in BMDMs following 24-h treatment with CYR61 (0.5 μg/mL). Statistical significance was assessed using Mann-Whitney test. Bars show mean ± SEM for n = 4 wells/group (technical replicates).(G) Venn diagram showing the number of ligand-receptor interactions with a ligand dysregulated when BMDMs are treated with CYR61 and/or a receptor that is dysregulated in HUVECs when co-incubated with CYR61-activated BMDMs. These ligand-receptor interactions were from the LRdb database downloaded from https://github.com/SCA-IRCM/LRdb.(H) Fifty-eight macrophage-to-endothelial cell ligand-receptor interactions that are dysregulated when macrophages are activated by CYR61.(I) Macrophage ligands dysregulated by CYR61, which can signal to endothelial cells (n = 4–7 wells/group). ∗p < 0.05. See also Figure S7.

Fig 4: Prognostic value of CYR61 expression in BRCA. (A, B) Kaplan-Meier survival curve analyses illustrating the OS (A) and DSS (B) rates of high or low CYR61-expressing BRCA patients in the TCGA database. (C, D) Kaplan-Meier survival curve analyses illustrating the OS (C) or DSS (D) rates of high or low CYR61-expressing ER-positive BRCA patients in the TCGA database. (E, F) Kaplan-Meier survival curve analyses illustrating the OS (E) or RFS (F) rates of high or low CYR61-expressing BRCA patients in the METABRIC database. (G, H) Kaplan-Meier survival curve analyses illustrating the OS (E) or RFS (F) rates of high or low CYR61-expressing ER-positive BRCA patients in the METABRIC database.

Fig 5: Hepatic lipid accumulation and insulin resistance are induced in Cyr61 overexpression (AAV‐Cyr61) mice. Blood glucose levels in AAV‐GFP or AAV‐Cyr61 during (A) ITT and (B) GTT assays after 6 h of fasting. Relative mRNA expression levels of key genes involved in (C) lipogenesis and (D) fatty acid oxidation in the liver of AAV‐GFP mice or AAV‐Cyr61 mice. (E) Quantitative analysis of Sirtuin genes determined by quantitative polymerase chain reaction. Data shown represent the mean ± standard error of mean. Statistical significance was determined using the t‐test. *p < .05, **p < .01, ***p < .001. AAV, adeno‐associated virus; AUC, area under the curve; Cyr61, cysteine‐rich 61; GTT, glucose tolerance test; ITT, insulin tolerance test.