Journal: Nature Communications

Article Title: Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes

doi: 10.1038/s41467-023-38541-2

Figure Lengend Snippet: a Left: Crystal structure of N -terminal domains 1–3 of IL23R (orange) in complex with IL-23 (IL23p19 = blue, IL12p40 = green) and the N -terminal domain of IL12Rβ1 (PDB: 6WDQ ). Right: The interface between IL23R and IL23p19 with C115 coloured cyan. b The phospho-STAT3 signal generated when cells expressing different constructs were treated with 5 nM IL-23. Data are mean ± SEM from five or six (untagged) or three (NanoLuc tagged) independent experiments conducted in triplicate. For the IL23R + IL12Rβ1 data set, one outlier (11108.7) was detected using the Grubbs test (with alpha = 0.001) and removed from the statistical analysis. Statistical significance of the C115Y mutation on the alphaLISA data was assessed using a 2-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. The p value for the comparison of NL tagged construct response was 0.0001 and the p value for the comparison of untagged receptor was 0.951. c Luminescence signal measured when IL12Rβ1 was expressed with either NL-IL23R or the C115Y mutant. Data are mean ± SEM generated from the mean luminescence values of seven (C115Y) or eight independent experiments performed in pentuplicate. d The proportion of luminescence from c that emanated from extracellular protein (as defined by the reduction in luminescence after treatment with 60 μM NanoLuc extracellular inhibitor). Data are mean ± SEM generated from the mean luminescence values of five (C115Y) or six independent experiments performed in triplicate. e The BRET ratio generated from HaloTag-618 labelled cells expressing NL tagged wildtype or C115Y IL23R with HT-IL12Rβ1 or unlabelled IL12Rβ1. Data are mean ± SEM generated from the mean BRET values of three independent experiments performed in pentuplicate. Statistical significance of differences in mean values shown in c – e were measured using a 2-sided, non-paired t test with ** indicating a p value of <0.005 and *** indicating a p value of <0.0005. The p values for the results shown in c , d and e were p = 0.148, 0.0005 and 0.0013 respectively. Source data are provided as a Source Data file.

Article Snippet: The following day media was replaced with serum free DMEM and the cells incubated for 3 h. Media was then replaced with HBSS containing 1 mg/ml BSA and increasing concentrations of IL-23 and the cells incubated for 30 min. An AlphaLISA SureFire Ultra assay kit (Perkin Elmer #ALSU-PST3) was then used to measure STAT3 phosphorylation at residue Tyr705.

Techniques: Generated, Expressing, Construct, Mutagenesis, Comparison

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: International Journal of Molecular Sciences

Article Title: Gallotannin from Bouea macrophylla Seed Extract Suppresses Cancer Stem-like Cells and Radiosensitizes Head and Neck Cancer

doi: 10.3390/ijms22179253

Figure Lengend Snippet: MPSE and PGG suppressed cancer stem-like cell phenotypes in HNSCC. ( a ) Left: representative images of spheroids derived from CAL27 and FaDu cells. Right: the protein expression of stem-cell markers in adherent CAL27 and FaDu cells and their CSC-rich spheroids. The number represents the relative band intensity of p-STAT3, STAT3, Oct4, Sox2, and CD44 was normalized to the band intensity of GAPDH. ( b ) AldeRed ALDH assay by flow cytometry showed decreased ALDH+ population after MPSE treatment. Data was expressed as percent of ALDH+ cells and shown as mean ± SD ( n = 3). Different letters (abc—adherent; abc —spheroid) at the top of columns indicate significant differences at p < 0.05 using one-way ANOVA and Tukey’s multiple comparison test. Student’s t -test was used to compare the differences between the adherent and spheroid groups, * p < 0.05. ( c , d ) Tumor sphere formation capacity in CAL27 and FaDu cells. Left: representative images of spheroids treated with either MPSE or PGG. Right: quantification of spheroid numbers derived from CAL27 and FaDu cells after treatment. ( e ) Protein expression of p-STAT3, STAT3, and stem cell markers including, Oct4, Sox2, and CD44 in adherent CAL27 and FaDu cells and the spheroid-derived CSC-rich cells after being treated with 20 µg/mL of either MPSE or PGG, were determined using Western blotting. ( f ) The immunoblot signal intensities were quantified by densitometry. Relative band intensity was normalized to the band intensity of GAPDH. All data show the mean ± SD ( n = 3). Statistical significance was evaluated by one-way ANOVA, followed by post hoc Tukey’s multiple comparison test. * p < 0.05. MPSE, Maprang seed extract. PGG, pentagalloyl glucose. HNSCC, head and neck squamous cell carcinoma.

Article Snippet: Primary antibodies against total Akt, phosphorylated Akt (Ser473), total ERK1/2, phosphorylated ERK1/2(Thr202/Tyr204, Thr185/Tyr187)) total STAT3, phosphorylated STAT3 (Tyr705), cleaved caspase 3, Bcl2, cleaved PARP, CD44 and GAPDH, as well as horseradish-peroxidase-labeled secondary antibodies, were purchased from Merck (Merck KGaA, Darmstadt, Germany).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Gallotannin from Bouea macrophylla Seed Extract Suppresses Cancer Stem-like Cells and Radiosensitizes Head and Neck Cancer

doi: 10.3390/ijms22179253

Figure Lengend Snippet: Pretreatment with MPSE or PGG before combining with irradiation suppressed the radiation-induced CSC phenotype in the HNSCC cell line. ( a ) Western blot analysis was performed to determine STAT3 phosphorylation in the indicated groups and ( b ) their band intensity was quantified. ( c , d ) CAL27 and FaDu cells were treated with either IR alone or the combination of 15 µg/mL MPSE or PGG with 6 Gy IR X-ray for 24 h and then were resuspended and cultured to form spheroids. Tumorsphere growth was quantified by determining the spheroid area using ImageJ software on day 10 (bar, 100 µm). ( e , f ) The percentages of head and neck cancer cell populations that expressed CSC markers (ALDH+). Data are presented as the mean ± SD of three independent experiments. ( g ) Immunofluorescence staining of the CSC marker (CD44) was determined in tumor spheroids. Statistical significance was evaluated by one-way ANOVA, followed by post hoc Tukey’s multiple comparison test (* p < 0.05). MPSE, Maprang seed extract. PGG, pentagalloyl glucose. HNSCC, head and neck squamous cell carcinoma. IR, irradiation. CSCs, cancer stem cells. ALDH, Aldehyde dehydrogenases.

Article Snippet: Primary antibodies against total Akt, phosphorylated Akt (Ser473), total ERK1/2, phosphorylated ERK1/2(Thr202/Tyr204, Thr185/Tyr187)) total STAT3, phosphorylated STAT3 (Tyr705), cleaved caspase 3, Bcl2, cleaved PARP, CD44 and GAPDH, as well as horseradish-peroxidase-labeled secondary antibodies, were purchased from Merck (Merck KGaA, Darmstadt, Germany).

Techniques: Irradiation, Western Blot, Cell Culture, Software, Immunofluorescence, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: B-Cell-Activating Factor Depletion Ameliorates Aging-Dependent Insulin Resistance via Enhancement of Thermogenesis in Adipose Tissues

doi: 10.3390/ijms21145121

Figure Lengend Snippet: BAFF depletion enhances expression of leptin and FGF21 in subcutaneous and brown adipose tissues. Effect of BAFF deficiency on leptin and FGF21 mRNA expression in ( A ) BAT and ( B ) SAT ( n = 9–12). Gene expression level is normalized with mRNA expression level of Arbp. ( C ) Effect of BAFF deficiency on serum protein levels of leptin and FGF21 ( n = 6–8). ( D ) Effect of BAFF deficiency on leptin-dependent STAT3 phosphorylation in brown adipose tissue of 10-month-old mice. Proteins were extracted from BAT for SDS-PAGE-immunoblot analysis ( n = 4–5). Data represent means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 between wild-type and BAFF −/− mice.

Article Snippet: Antibodies against GAPDH, phospho-IKKα/β (Ser176+Ser180), IKKα (Bioss Antibodies, Woburn, MA, USA), NF-κB p100/p52, NIK, RelB (Cell Signaling Technology, Berverly, MA, USA), phospho-STAT3 (Tyr705) (Cambridge Bioscience, Cambridge, UK) and UCP1 (Abcam, Cambridge, UK) were used as primary antibodies, followed by the appropriate IgG-HRP conjugated secondary antibody (Cell Signaling Technology).

Techniques: Expressing, Gene Expression, Phospho-proteomics, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Novel role of lncRNA CHRF in cisplatin resistance of ovarian cancer is mediated by miR-10b induced EMT and STAT3 signaling

doi: 10.1038/s41598-020-71153-0

Figure Lengend Snippet: EMT and STAT3 phosphorylation in CR cells and role of CHRF-miR-10b. EMT markers E-cadherin and Vimentin were quantitated using quantitative RT-PCR in ( A ) CR and ( B ) miR-10b transfected ES2 cells, relative to parental cells (control). For each EMT marker, the expression in control parental cells was set to ‘1’ and the relative expression in CR/miR-10b transfected ES2 cells is shown. ( C ) miR-10b attenuates the effects of CHRF down-regulation on EMT markers in cisplatin resistant ES2 cells. CR ES2 cells (control) and CR ES2 cells with down-regulated CHRF (without and with miR-10b transfections: CHRF-Down and CHRF-down + miR-10b, respectively) were subjected to quantitative RT-PCR for the evaluation of EMT markers E-cadherin and Vimentin. The expression levels of EMT markers in control group were set as ‘1’ and the relative expressions in other groups are reported. STAT3 phosphorylation was quantitated using ELISA in ( D ) CR and ( E ) miR-10b transfected ES2 cells, relative to parental cells (control), as described in “ ” ( F ) miR-10b attenuates the effects of CHRF down-regulation on STAT3 phosphorylation in cisplatin resistant ES2 cells. CR ES2 cells (control) and CR ES2 cells with down-regulated CHRF (without and with miR-10b transfections: CHRF-Down and CHRF-Down + miR-10b, respectively) were subjected to ELISA for the evaluation of STAT3 phosphorylation. *p < 0.01, relative to control, # p < 0.01, relative to CHRF-Down.

Article Snippet: We used HTRF ® (Homogeneous Time Resolved Fluorescence) cell based phospho and total STAT3 assay kit (Cisbio, China) for quantitation of phosphorylated STAT3.

Techniques: Quantitative RT-PCR, Transfection, Marker, Expressing, Enzyme-linked Immunosorbent Assay