human fgf-basic standard abts enzyme-linked immunosorbent assay (elisa) development kit  (PeproTech)

Journal: Cell Death Discovery

Article Title: Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway

doi: 10.1038/s41420-020-0252-9

Figure Lengend Snippet: a , b Atorvastatin suppressed IL-6 secretion in HepG2 and Hep3B cells. Cells were treated with various concentrations of atorvastatin for 72 h. Culture supernatants were collected during treatment, and IL-6 concentrations were measured by ELISA. c , d Pretreatment with IL-6 and the addition of exogenous IL-6 every 24 h rescued HCC cells from atorvastatin-induced senescence. e Cell growth, which was arrested after atorvastatin treatment, was restored in HCC cells pretreated with IL-6 and administered exogenous IL-6. IL-6 (50 ng/ml) was administered to HCC cells before and during atorvastatin treatment ( c , d ) or for 3 or 5 days ( e ), and cells were then subjected to SA-β-gal staining ( c , d ) and counted to determine the viable population ( e ).

Article Snippet: Recombinant human IL-6 and a human IL-6 Standard ABTS enzyme-linked immunosorbent assay (ELISA) Development Kit were purchased from Peprotech (Rocky Hill, NJ, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell Death Discovery

Article Title: Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway

doi: 10.1038/s41420-020-0252-9

Figure Lengend Snippet: a The levels of TERT and p-STAT3 proteins were restored upon the addition of exogenous IL-6 to atorvastatin-treated HepG2 cells. Then 50 ng/ml IL-6 was added back to atorvastatin-treated cells for 72 h, and the cells were then subjected to immunoblotting. b Overexpression of constitutively active STAT3 protected HepG2 cells from atorvastatin-induced cellular senescence. HepG2 cells overexpressing a constitutively active mutant of STAT3 (STAT3C) and control HepG2 cells were cultured in the presence or absence of atorvastatin (20 μg/ml) for 72 h, and then senescence was evaluated by SA-β-gal staining. c , d The TERT mRNA ( c ) and protein expression levels ( d ) in STAT3C-overexpressing HepG2 cells were not decreased compared with those in control HepG2 cells after atorvastatin treatment. STAT3C-overexpressing and control HepG2 cells were treated with atorvastatin (0, 5, 10, 20, and 40 μg/ml) for 72 h, and mRNA and protein were then isolated and analyzed by RT-PCR ( c ) and immunoblotting ( d ), respectively. e Overexpression of STAT3C in HepG2 cells reversed atorvastatin-mediated downregulation of telomerase activity. Telomerase activity was measured in STAT3C-overexpressing and control HepG2 cells treated with 20 μg/ml atorvastatin for 72 h. f , g The levels of IL-6 mRNA ( f ) and secreted IL-6 ( g ) were restored in STAT3C-overexpressing HepG2 cells treated with atorvastatin. STAT3C-overexpressing and control HepG2 cells were treated with atorvastatin (0, 5, 10, 20, and 40 μg/ml) for 72 h. Then, after the cell culture supernatant was collected, mRNA was extracted, and RT-PCR and IL-6 ELISA assays were performed.

Article Snippet: Recombinant human IL-6 and a human IL-6 Standard ABTS enzyme-linked immunosorbent assay (ELISA) Development Kit were purchased from Peprotech (Rocky Hill, NJ, USA).

Techniques: Western Blot, Over Expression, Mutagenesis, Cell Culture, Staining, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death Discovery

Article Title: Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway

doi: 10.1038/s41420-020-0252-9

Figure Lengend Snippet: A tumor-bearing mouse model was established by subcutaneously inoculating a suspension of HepG2 cells into the flanks of BALB/c nude mice. HepG2 tumor-bearing mice were treated with vehicle or atorvastatin (20 mg/kg) twice daily by intraperitoneal injection. a Mice in the vehicle and atorvastatin treatment groups were sacrificed 17 days after the last atorvastatin injection. Tumor tissues were harvested from each mouse. b Tumor weights were measured after sacrifice. c Growth curves of tumors excised from vehicle- and atorvastatin-treated mice. Tumor volumes were calculated twice daily for 17 days. The results are shown as the means ± S.E.M; vehicle-treated mice were compared to atorvastatin-treated mice. * p < 0.05, ** p < 0.01, *** p < 0.001. d , e IHC analysis using antibodies against human IL-6, p-STAT3, TERT, and β-gal ( d ) and measurement of SA-β-gal activity ( e ) in tumor tissues from vehicle- and atorvastatin-treated mice. All scale bars represent 50 μm. f Increased IL-6 and TERT mRNA expression was significantly associated with poor overall survival in patients with HCC. Clinical data for patients with liver cancer were obtained from a TCGA dataset in The Human Protein Atlas ( https://www.proteinatlas.org/ ).

Article Snippet: Recombinant human IL-6 and a human IL-6 Standard ABTS enzyme-linked immunosorbent assay (ELISA) Development Kit were purchased from Peprotech (Rocky Hill, NJ, USA).

Techniques: Injection, Activity Assay, Expressing

Journal: Cell Death Discovery

Article Title: Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway

doi: 10.1038/s41420-020-0252-9

Figure Lengend Snippet: Atorvastatin inhibited STAT3 phosphorylation to decrease IL-6 production and TERT expression. The decrease in IL-6 production attenuated the STAT3/IL-6 positive feedback loop and decreased TERT expression, inducing senescence in HCC cells.

Article Snippet: Recombinant human IL-6 and a human IL-6 Standard ABTS enzyme-linked immunosorbent assay (ELISA) Development Kit were purchased from Peprotech (Rocky Hill, NJ, USA).

Techniques: Expressing

Journal: Journal of Immunology Research

Article Title: Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

doi: 10.1155/2020/7375947

Figure Lengend Snippet: PD-1 expression and effector function capacity of T-cells isolated from healthy controls (HC) and epithelial ovarian cancer (EOC) patients. (a) Proportion of PD-1+ (%) CD4+ and CD8+ T-cells isolated from the peripheral blood of HC ( n = 10) and the blood ( n = 12), ascites ( n = 16), and tumor ( n = 8) of EOC patients. Gating strategy included singlets, followed by viable cells (being 7AAD-), lymphocyte gate (by forward/side scatter), and T-cells (CD3+), followed by expression of CD4+ and CD8+ and lastly by expression of PD-1 on these subsets. (b) Representative histograms of PD-1 expression on CD4+ and CD8+ T-cells from one patient. Cells from the tumor were used for the isotype control. (c) Absolute concentrations of IFN- γ (normalized to reflect per 500,000 T-cells) after stimulation with α -CD3 (OKT-3) for 48 h. (d) The median release of IFN- γ in HC after stimulation with α -CD3 was set to 100% and defined full response (effector function capacity). The release measured in the samples from EOC patient blood, ascites, and tumor was compared to the release observed in HC blood samples. Median values with interquartile ranges are presented. IFN- γ was measured using ELISA, and the data was normalized to reflect the same cell number (per 500,000 T-cells). Unpaired Mann-Whitney was performed as statistical analysis. Significance levels were set to ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: After 6 days, IFN- γ secretion in culture supernatants was analyzed using the Human IFN- γ standard ABTS enzyme-linked immunosorbent assay (ELISA) development kit (PeproTech).

Techniques: Expressing, Isolation, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of Immunology Research

Article Title: Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

doi: 10.1155/2020/7375947

Figure Lengend Snippet: Overview of PD-1-blocking monoclonal antibodies and DARPin® proteins. (a) Schematic of the PD-1-binding reagents: two conventional monoclonal antibodies (mAbs) of IgG4 subtype (nivolumab and pembrolizumab) and two DARPin® proteins with monovalent (DARPin-1) or bivalent (DARPin-2) PD-1 binding. (b) Assessment of PD-1 binding using a titration of PD-1-targeting reagents with a PD-1-expressing cell line (transfected HEK293). PD-1 binding was measured by mean fluorescence intensity (MFI) using flow cytometry. Half-effective concentrations (EC50 values) are presented for each PD-1-binding reagent—nivolumab (nivo) in circles, pembrolizumab (pembro) in reversed triangles, DARPin-1 in squares, and DARPin-2 in diamonds. (c) PD-1/PD-L1 blockade bioassay (Promega) ( n = 2) was performed using PD-1+ effector T-cells and PD-L1-expressing aAPC/CHO-K1 cells in the presence of dose titrations of PD-1-targeting reagents. Increased luminescence indicates recovered T-cell activation as PD-1 engagement with PD-L1 interferes with the T-cell receptor-mediated transcription of the reporter gene luciferase. (d) Mixed lymphocyte reaction was performed using CD4+ T-cells and allogeneic dendritic cells generated from healthy donors in the presence of PD-1-targeting mAbs (nivolumab, n = 3; pembrolizumab, n = 1), DARPin® proteins ( n = 2 for both), or corresponding controls (human IgG4 and negative control DARPin® protein, NCD, respectively, n = 2 for both). The levels of IFN- γ in the supernatants were analyzed using ELISA, and results are presented for indicated concentrations. (e) Fold induction of IFN- γ release from T-cells isolated from the ascites or tumor from ovarian cancer patients ( n = 7) after 48 h incubation with α -CD3 (OKT-3) and different concentrations of PD-1-targeting mAbs ( n = 8), DARPin® proteins ( n = 4), or corresponding controls (IgG4 and NCD, n = 8 and n = 4, respectively, both used at 100 nM). The response was normalized based on the response with only α -CD3, which was set to 1 (dashed line). The results are presented as a median with interquartile range, and concentrations are indicated in the figure.

Article Snippet: After 6 days, IFN- γ secretion in culture supernatants was analyzed using the Human IFN- γ standard ABTS enzyme-linked immunosorbent assay (ELISA) development kit (PeproTech).

Techniques: Blocking Assay, Bioprocessing, Binding Assay, Titration, Expressing, Transfection, Fluorescence, Flow Cytometry, Bioassay, Activation Assay, Luciferase, Generated, Negative Control, Enzyme-linked Immunosorbent Assay, Isolation, Incubation

Journal: Journal of Immunology Research

Article Title: Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

doi: 10.1155/2020/7375947

Figure Lengend Snippet: Secretion of IFN- γ by T-cells in the presence of α -CD3 and PD-1-targeting reagents. T-cells isolated from the ascites ( n = 16) and tumor ( n = 8) were activated and cultured with α -CD3 (OKT-3) and 100 nM of PD-1-directed reagents (nivolumab, Nivo; pembrolizumab, Pembro; DARPin-1; and DARPin-2) or controls (IgG4 and negative control DARPin® protein, NCD) for 48 h. (a) The relative fold increase of IFN- γ when adding PD-1 blockers or control was compared to the release caused by α -CD3 alone (represented by a dashed line set to 1). The results are presented for T-cells isolated from the ascites (circles) or (b) tumor (squares) separately. The number of samples is indicated in each bar ( n ). Corresponding controls for anti-PD-1 reagents are presented next to each anti-PD-1. (c) Significant differences among PD-1 blockers in ascites samples are presented separately and also (d) grouped together with tumor samples. Lines represent paired comparisons in which presented reagents have been assessed in parallel (in the same sample). (e) Comparing fold increase in paired samples of the ascites and tumor from the same patient ( n = 7). Absolute concentrations of IFN- γ released by T-cells isolated from (f) the ascites or (g) the tumor. (h) Comparing absolute IFN- γ concentrations in paired samples of the ascites and tumor ( n = 7). All data have been normalized to reflect the same number of T-cells (per 500,000 T-cells). Wilcoxon signed rank test was used, and median values and interquartile ranges are plotted. Significance levels were set to ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: After 6 days, IFN- γ secretion in culture supernatants was analyzed using the Human IFN- γ standard ABTS enzyme-linked immunosorbent assay (ELISA) development kit (PeproTech).

Techniques: Isolation, Cell Culture, Negative Control, Control

Journal: Journal of Immunology Research

Article Title: Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

doi: 10.1155/2020/7375947

Figure Lengend Snippet: Fold induction of secreted soluble factors from T-cells isolated in the presence of α -CD3 and 100 nM of PD-1-directed reagents. T-cells from the ascites ( n = 14) and tumor ( n = 5) were activated and cultured with α -CD3 (OKT-3) and 100 nM of PD-1-directed reagents or controls for 48 h. Results were generated using a multiplex immunoassay (Luminex) for (a) IFN- γ , (b) granzyme B, (c) IL-2, (d) TNF- α , (e) IL-10, and (f) soluble 4-1BB (s4-1BB)/sCD137 in samples of ascites and tumor (presented together). Fold induction was calculated based on the release when adding only α -CD3 with no presence of anti-PD-1 reagent or control (represented by a dashed line at 1). Several ascites samples reached the upper detection limit in all conditions (including α -CD3 alone) and were excluded for several cytokines. The total number of samples evaluated is presented in each bar ( n ). (g) The response by different anti-PD-1 reagents was compared with each other, and significant findings are presented. Although data from the ascites and tumor is pooled together, ascites samples are presented as circles and tumor samples as squares. Lines represent paired comparisons in which presented reagents have been assessed in parallel (in the same sample). Wilcoxon signed rank test was used for all statistical comparisons. Median values and interquartile ranges are plotted. Significance levels were set to ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: After 6 days, IFN- γ secretion in culture supernatants was analyzed using the Human IFN- γ standard ABTS enzyme-linked immunosorbent assay (ELISA) development kit (PeproTech).

Techniques: Isolation, Cell Culture, Generated, Multiplex Assay, Luminex, Control