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: Nanomedicine : nanotechnology, biology, and medicine

Article Title: Chemosensitization of tumors via simultaneous delivery of STAT3 inhibitor and doxorubicin through HPMA copolymer-based nanotherapeutics with pH-sensitive activation.

doi: 10.1016/j.nano.2023.102730

Figure Lengend Snippet: Fig. 6 S3i and its derivatives S3iD2 and S3iD3 inhibit STAT3 phosphorylation. CT26

Article Snippet: Determination of STAT3 and pSTAT3 levels in cell lysates (50 μg of protein) was carried out using a PathScan Total STAT3 Sandwich ELISA kit and PathScan Phospho-STAT3 (Y705) Sandwich ELISA kit (both Cell Signaling Technology, USA), respectively, according to the manufacturer’s instructions.

Techniques: Phospho-proteomics

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