Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Ascites in peritoneal carcinomatosis (PC) is biologically active (A) Kaplan-Meier curve showing survival of PC patients who underwent cytoreductive surgery (n = 121), stratified by the presence or absence of ascites. (B and C) Proliferation curves of (B) Colo-205 and (C) SNU-C1 cells treated with varying concentrations of ascites, measured by CellTitre-Glo assay. Data are represented as cell viability at day 5 relative to day 0. Dotted line represents cell viability of Colo-205 treated with 10% fetal bovine serum (FBS) at day 5 relative to day 0. Graph shows mean ± SD. (D and E) Migration of (D) Colo-205 and (E) SNU-C1 cells pre-treated with serum-free media (SFM), 10% FBS medium or 5% cell-free ascites (CFA) medium, assessed by transwell migration assay. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with SFM and 10% FBS or 5% CFA was denoted by ∗. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with 10% FBS and SFM or 5% CFA was denoted by # . # p < 0.05, ## p < 0.01, ### p < 0.001. Graph shows mean ± SD. (F) Enriched signaling pathways in colorectal PC cell lines upon treatment with CFA, identified by comparing gene expressions of cells treated with 5% versus 0.1% CFA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G and H) Treatment of (G) Colo-205 and (H) SNU-C1 cells with 5% CFA activated STAT3 signaling pathway through phosphorylation at Tyr705.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Glo Assay, Migration, Transwell Migration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Identification and validation of prognostic putative activators of STAT3 signaling in ascites (A) Workflow to identify clinically significant putative secreted STAT3 activators. (B) Kaplan-Meier survival curve illustrating poorer survival in patients expressing three biomarkers as compared to patients expressing 0–2 biomarkers. (C) Characterization of cytokine profiles in colorectal PC ascites and benign ascites. Top 25% most highly abundant cytokines in colorectal PC ascites (by mean abundance) are shown. Heatmap displays Z score of normalized mean pixel density of duplicate cytokine spots on the array. (D) Downregulated signaling pathways upon PAI-1 inhibition in Colo-205 cells exposed to CFA with high PAI-1 levels (PC085), CFA with low PAI-1 levels (PC249) and no PAI-1 (FBS control), identified using RNA microarray. Only signaling pathways with differential downregulation are presented. IL6-JAK-STAT3 signaling pathway was significantly downregulated in high PAI-1 CFA-treated cells upon PAI-1 inhibition. Normalized enrichment scores <0 indicate pathway suppression and scores >0 indicate pathway activation. (D) is representative of two independent biological experiments. ∗p < 0.05.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Expressing, Inhibition, Microarray, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Correlating PAI-1 level in ascites and intracellular STAT3 activation in cancer cells revealed distinct subgroups associated with differing susceptibility to PAI-1 inhibition (A) PAI-1 prevalence in colorectal PC ascites (n = 54). (B) Correlation between colorectal PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.4691, p = 0.0003). (C) PAI-1 prevalence in ascites of various histological PC subtypes (n = 156). † indicates benign ascites. (D) Correlation between various histological PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.6476, p < 0.0001). (A–D) PAI-1 concentrations are plotted on a log 2 scale to transform skewed data to normal distribution. p-STAT3(Y705) level was shown as optical density reading at 450 nm (OD450). (E) Untransformed values of PAI-1 and p-STAT3(Y705) levels from (D) were used for stratification strategy to identify patient subpopulations who might benefit from PAI-1 inhibition. Using 20 ng/mL PAI-1 level and 0.2 OD450 p-STAT3(Y705) level as cut-off values, three distinct subgroups of samples were observed: (i) high PAI-1 and high p-STAT3 levels, termed PAI-1 paracrine addicted (PPA) group (yellow region), (ii) low PAI-1 and high p-STAT3 levels, termed co-activators predominant (CAP) group (pink region) and (iii) low PAI-1 and low p-STAT3 levels, termed alternative pathways activation (APA) group (blue region). Each dot represents one patient ascites. Colors in each panel represent the various histological PC subtypes. All histological subtypes with less than five samples are grouped into other histological subtypes.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Activation Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Cells dependent on PAI-1 to activate STAT3 are most susceptible to PAI-1 inhibition (A) Effect of TM5441 (PAI-1 inhibitor) on CFA-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow), CAP group (pink), APA group (blue) and FBS (control, black) demonstrated a left shift, indicating responsiveness to PAI-1 inhibition. (B) Differential sensitivity to TM5441 corresponding to the three subgroups, with PPA (n = 18) being the most sensitive to PAI-1 inhibition, followed by CAP (n = 59) and APA (n = 17). Graph shows mean ± SD. (C–E) Effect of (C) Napabucasin (STAT3 inhibitor), (D) BEZ235 (dual PI3K/mTOR inhibitor) and (E) Mitomycin C (conventional chemotherapeutic agent – DNA crosslinker) on the three subgroups of ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow; n = 3), CAP group (pink; n = 3), APA group (blue; n = 1) and FBS (control, black). Targeting PAI-1, a dominant paracrine factor in ascites, was more effective than targeting downstream signaling pathway activated by ascites, proliferation pathway or DNA synthesis. (F) Evaluation of STAT3 suppression in Colo-205 cells treated with PPA CFA (PC085 and PC383), CAP CFA (PC249), APA CFA (PC010) and various concentrations of TM5441 by ELISA. STAT3 activation was shown as p-STAT3(Y705) and total STAT3 ratio at the indicated concentration relative to DMSO vehicle. Cells exposed to PPA CFA relied on PAI-1 to activate STAT3 as they required lower concentrations of TM5441 to suppress STAT3 activation. Graph shows mean ± SEM. Data in (A-E) are representative of at least three independent biological experiments and (F) is representative of two independent biological experiments. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Inhibition, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet:

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Sequencing, Microarray, Cell Culture, Software

Journal: Molecular cell

Article Title: Heat Shock Factor 1 Is a Direct Antagonist of AMP-Activated Protein Kinase

doi: 10.1016/j.molcel.2019.08.021

Figure Lengend Snippet: Key Resources Table

Article Snippet: NEB® 5-α Competent E. coli (High Efficiency) , New England Biolabs , Cat# C2987.

Techniques: FLAG-tag, Amplification, Staining, Plasmid Preparation, Recombinant, Transfection, Viability Assay, In Situ, Amplex Red Cholesterol Assay, Protease Inhibitor, Sandwich ELISA, Mutagenesis, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Silver Staining, Mass Spectrometry, Western Blot, Sequencing, Luciferase, Software, Magnetic Beads