Journal: Frontiers in Immunology

Article Title: Establishment and validation of evaluation models for post-inflammatory pigmentation abnormalities

doi: 10.3389/fimmu.2022.991594

Figure Lengend Snippet: The role of IL-37 in melanogenesis. MNT1 cells were treated with different concentrations of IL-37. (A) qRT-PCR was used to examine the mRNA levels of melanogenesis-related genes. (B) The protein levels of MITF, TYR, TYRP1, DCT, PMEL17 and MLANA were analyzed by Western blotting. (C) Tyrosinase activity was measured by L-DOPA assay. (D) representative immunofluorescence images showing the distribution of melanosomes. (N = 3, ANOVA, error bar represents mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Primary antibodies specific for GAPDH (#AP0066, Bioworld), TYR (BS1484, Bioworld), MITF (STJ94134, St. John’s Laboratory), TYRP1 (ab235447, Abcam), DCT (NBP1-56058, Novusbio), PMEL17(#H1219, Santa Cruz and bs-17478R, Bioss) and MLANA (bs-0051R, Bioss and ET1610-47, HUABIO) were purchased as indicated.

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay, Immunofluorescence

Journal:

Article Title: Engagement of CD83 ligand induces prolonged expansion of CD8 + T cells and preferential enrichment for antigen specificity

doi: 10.1182/blood-2005-05-2073

Figure Lengend Snippet: CD83L signaling enhances the expansion of antigen-specific CD8+ T cells. HLA-A2+ CD8+ T cells were stimulated by MART1 (A) or Flu (B) peptide-pulsed APCs (APC/A2, APC/A2/CD80, APC/A2/CD83, or APC/A2/CD80/CD83). After 3 rounds of antigen stimulation and IL-2/IL-15 addition in between the stimulations, the cultures were stained with the relevant tetramer to determine percentage of peptide-specific T cells. The number of peptide-specific T cells was determined by calculating the product of the total number of T cells and the percentage of tetramer-staining cells. Representative results from 4 different donors are presented. (A) Compared with APC/A2, large increases in the percentage and number of antigen-specific CD8+ T cells were observed when T cells were stimulated by MART1-pulsed APC/A2/CD80 (percentage, P = .017; number, P = .002) and APC/A2/CD80/CD83 (percentage, P = .009; number, P < .001), but not by APC/A2/CD83 (percentage, P = .48; number, P = .30). When compared with APC/A2/CD80, APC/A2/CD80/CD83 generates a significant increase in the percentage and number of MART1-specific T cells (percentage, P = .012; number, P = .003). indicates A2; □, A2/CD80; , A2/CD83; ▪, A2/CD80/CD83. (B) Likewise, when APCs are pulsed with Flu peptide, the percentage and total number of antigen-specific T cells was increased when T cells were stimulated by APC/A2/CD80 (percentage, P = .009; number, P = .01) and APC/A2/CD80/CD83 (percentage, P = .009; number, P = .023), but not by or APC/A2/CD83 (percentage, P = .32; number, P = .32). When compared with APC/A2/CD80, APC/A2/CD80/CD83 generates a significant increase in the percentage and number of Flu-specific T cells (percentage, P = .01; number, P = .035). indicates A2; □, A2/CD80; , A2/CD83; ▪, A2/CD80/CD83.

Article Snippet: K562-derived cells were pulsed with synthetic peptides, 27 AAGIGILTV 35 of MART1, 58 GILGFVFTL 66 of the influenza virus matrix antigen, or 476 ILKEPVHGV 485 peptide of the RTpol (Pol) of HIV (New England Peptides, Fitchburg, MA) for 6 to 10 hours at room temperature.

Techniques: Staining

Journal:

Article Title: Engagement of CD83 ligand induces prolonged expansion of CD8 + T cells and preferential enrichment for antigen specificity

doi: 10.1182/blood-2005-05-2073

Figure Lengend Snippet: Effector function of MART1-specific CD8+ T cells. (A) MART1-specific cytotoxic CD8+ T cells generated by APC/A2/CD80/CD83 killed target cells in an HLA-A2-specific manner. Target cells (T2 and Jurkat cells transduced with vector or A2) were pulsed with MART1 peptide (▪) or control peptide (•). 11LLFGYPVYV19 peptide of the TAX of HTLV-I was used as a negative control peptide. (B) Blocking experiments were performed using HLA-A2-specific mAb (BB7.2) and isotype control (mIgG2b). Peptide-pulsed T2 cells were used at an E/T ratio of 30:1. (C) MART1-specific cytotoxic CD8+ T cells generated by APC/A2/C80/CD83 secreted IFN-γ in an antigen-specific manner. Error bars represent standard deviation (SD).

Article Snippet: K562-derived cells were pulsed with synthetic peptides, 27 AAGIGILTV 35 of MART1, 58 GILGFVFTL 66 of the influenza virus matrix antigen, or 476 ILKEPVHGV 485 peptide of the RTpol (Pol) of HIV (New England Peptides, Fitchburg, MA) for 6 to 10 hours at room temperature.

Techniques: Generated, Transduction, Plasmid Preparation, Control, Negative Control, Blocking Assay, Standard Deviation

Journal:

Article Title: Engagement of CD83 ligand induces prolonged expansion of CD8 + T cells and preferential enrichment for antigen specificity

doi: 10.1182/blood-2005-05-2073

Figure Lengend Snippet: Stimulation with APCs that express CD83 enhances proliferation and inhibits apoptosis, permitting continued expansion of MART1-specific T cells beyond 6 weeks. (A) Following 5 rounds of stimulation with MART1 peptide-pulsed APC/A2/CD80, T-cell lines noted to cease expansion were split and subsequently stimulated with either peptide-pulsed APC/A2/CD80 (▪) or peptide-pulsed APC/A2/CD80/CD83 (○). Between the stimulations, IL-2 and IL-15 were added to the culture. Those T cells stimulated by peptide-pulsed APC/A2/CD80/CD83 demonstrated significant antigen-specific expansion, while those stimulated by peptide-pulsed APC/A2/CD80 did not (P = .006). (B) An increase in MART1-specific T-cell proliferation was determined by tetramer staining and BrdU incorporation. This was consistently observed in both donors tested at weeks 7 and 8. (C) Apoptosis was inhibited as determined by Annexin V staining. This was consistently observed in both donors tested at weeks 7 and 8. The proliferating (B) and apoptotic fractions (C) are shown as a percentage of MART1 tetramer-positive cells. Two experiments with different donors were performed.

Article Snippet: K562-derived cells were pulsed with synthetic peptides, 27 AAGIGILTV 35 of MART1, 58 GILGFVFTL 66 of the influenza virus matrix antigen, or 476 ILKEPVHGV 485 peptide of the RTpol (Pol) of HIV (New England Peptides, Fitchburg, MA) for 6 to 10 hours at room temperature.

Techniques: Staining, BrdU Incorporation Assay

Journal:

Article Title: Engagement of CD83 ligand induces prolonged expansion of CD8 + T cells and preferential enrichment for antigen specificity

doi: 10.1182/blood-2005-05-2073

Figure Lengend Snippet: CD83L engagement supports sustained growth of antigen-specific CD8+ T cells. MART1-specific CD8+ T cells were generated by stimulation with MART1-pulsed APC/A2/CD80/CD83 every 7 to 14 days. The percentage increase or decrease in the number of cells was determined at each stimulation, and a fraction was subsequently stimulated and maintained in culture. Antigen specificity was demonstrated every 3 or 4 rounds of stimulation by tetramer analysis. (A) The predicted total number of CD8+ T cells generated over a 191-day culture period is shown. Arrow denotes removal of debris by Ficoll density gradient. (B) PE-conjugated MART1 tetramer staining versus CD8 staining is shown at 4 time points during the culture period. (C) Effector function was determined by cytotoxicity assay on day 190 using peptide-pulsed T2 cells as targets (▪ indicates MART1 peptide; •, HIV pol as control peptide).

Article Snippet: K562-derived cells were pulsed with synthetic peptides, 27 AAGIGILTV 35 of MART1, 58 GILGFVFTL 66 of the influenza virus matrix antigen, or 476 ILKEPVHGV 485 peptide of the RTpol (Pol) of HIV (New England Peptides, Fitchburg, MA) for 6 to 10 hours at room temperature.

Techniques: Generated, Staining, Cytotoxicity Assay, Control

Journal: Dose-Response

Article Title: Biochemical Investigation of Inhibitory Activities of Plant-Derived Bioactive Compounds Against Carbohydrate and Glucagon-Like Peptide-1 Metabolizing Enzymes

doi: 10.1177/15593258221093275

Figure Lengend Snippet: Chemical structures of selected ligands for molecular docking analysis. (A) Chemical structures of tested compounds taxifolin (TAX) and resveratrol (RSV). (B) 2D-structural representation of standard compounds acarbose (ACB), miglitol (MGL), and diprotin (DPT). (C) Supposed conformation of selected hits and standard compound in their corresponding molecular targets.

Article Snippet: Resveratrol (CHEM-IMPEX INT’L INC), taxifolin (Sigma aldrich), acarbose (Carbosnyth,USA), diprotin (Sigma aldrich), HPA assay kit (Product code: K-CERA, Megazyme brand), α-glucosidase assay kit (Product code: MAK123, Sigma aldrich), DPP-IV inhibitor screening assay kit (Product code: ab133081 Abcam), starch (Sigma aldrich) and all the other materials of analytical grade were used.

Techniques:

Journal: Dose-Response

Article Title: Biochemical Investigation of Inhibitory Activities of Plant-Derived Bioactive Compounds Against Carbohydrate and Glucagon-Like Peptide-1 Metabolizing Enzymes

doi: 10.1177/15593258221093275

Figure Lengend Snippet: Inhibitory Activity of Bioactive Compounds Against GLU, HPA, and DPP-IV Enzymes.

Article Snippet: Resveratrol (CHEM-IMPEX INT’L INC), taxifolin (Sigma aldrich), acarbose (Carbosnyth,USA), diprotin (Sigma aldrich), HPA assay kit (Product code: K-CERA, Megazyme brand), α-glucosidase assay kit (Product code: MAK123, Sigma aldrich), DPP-IV inhibitor screening assay kit (Product code: ab133081 Abcam), starch (Sigma aldrich) and all the other materials of analytical grade were used.

Techniques: Activity Assay, Positive Control

Journal: Dose-Response

Article Title: Biochemical Investigation of Inhibitory Activities of Plant-Derived Bioactive Compounds Against Carbohydrate and Glucagon-Like Peptide-1 Metabolizing Enzymes

doi: 10.1177/15593258221093275

Figure Lengend Snippet: Surflex Score of Docked Ligands; Taxifolin and Resveratrol for GLU, HPA, and DPP-IV Along With Their Corresponding Standard Molecules Acarbose, Miglitol, and Diprotin A.

Article Snippet: Resveratrol (CHEM-IMPEX INT’L INC), taxifolin (Sigma aldrich), acarbose (Carbosnyth,USA), diprotin (Sigma aldrich), HPA assay kit (Product code: K-CERA, Megazyme brand), α-glucosidase assay kit (Product code: MAK123, Sigma aldrich), DPP-IV inhibitor screening assay kit (Product code: ab133081 Abcam), starch (Sigma aldrich) and all the other materials of analytical grade were used.

Techniques:

Journal: bioRxiv

Article Title: Mitochondrial Signatures Shape Phenotype Switching and Apoptosis in Response to PLK1 and RSK Inhibitors in Melanoma

doi: 10.1101/2024.06.14.599035

Figure Lengend Snippet: (A) CXCL8 transcript levels in MeWo and A-375 cells treated for 72 h with DMSO 0.1 % (v/v) or a panel of inhibitors targeting putative BI-D1870 targets. NA: non-available due to cell death. (B) Viable cell counts of melanoma cells with Presto Blue HS. (C) Representative crystal violet staining of cells treated as in A. (D) Quantification of crystal violet staining, as in C. (E,F) Immunoblotting of cell extracts from cells treated as indicated for 72 h. (G) Densitometric analysis of immunoblotting from 3 independent experiments related to E and F. (H,I) PLK1 and CXCL8 transcript levels in cells 72 h after PLK1 knockdown. (J) Immunoblotting of cell extracts from cells treated as in H-I. Numbers indicate independent experiments. (K) Densitometric analysis of immunoblotting from 3 independent experiments related to J. All data are shown as means ± SEM from 3 independent experiments. A: * p < 0.05, unpaired Student’s t -test (cell lines tested separately). D,K: * p < 0.05, paired Student’s t -test. G-I: * p < 0.05, Two-Way ANOVA with Tukey multiple comparison test.

Article Snippet: Primary antibodies against PARP (#9542), β -actin (#3700), PLK1 (#4535), and Melan-A/MART-1/MLANA (#64718) were purchased from Cell Signaling Technology Inc. (Whitby, ON, CA).

Techniques: Staining, Western Blot, Knockdown, Comparison

Journal: bioRxiv

Article Title: Mitochondrial Signatures Shape Phenotype Switching and Apoptosis in Response to PLK1 and RSK Inhibitors in Melanoma

doi: 10.1101/2024.06.14.599035

Figure Lengend Snippet: (A, B, C) Gene expression in MeWo and A-375 cells treated for 72 h as indicated. NA: Not available due to cell death. (D) Immunoblotting of protein extracts from MeWo cells treated as in A-C. Numbers indicate independent experiments. (E) Gene expression of differentiation markers in cells treated with siRNA against PLK1 versus non-targeting siRNA for 72 h. (F) Heat-map of Log2 fold change of specific transcripts linked to phenotype switching in MeWo cells treated with BI-D1870 (3 µM) for 72 h. Derived from RNA-Seq analyses in . (G,H) Gene expression in MeWo cells treated with BI 6727 (1 µM) or BRD7389 (10 µM) for 72 h. All data are shown as mean ± SEM from 3 independent experiments. A-C, E: * p < 0.05, Two-Way ANOVA with Tukey multiple comparison test. F: * p-adj < 0.05. G-H: * < 0.05, paired Student’s t -test.

Article Snippet: Primary antibodies against PARP (#9542), β -actin (#3700), PLK1 (#4535), and Melan-A/MART-1/MLANA (#64718) were purchased from Cell Signaling Technology Inc. (Whitby, ON, CA).

Techniques: Gene Expression, Western Blot, Derivative Assay, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Mitochondrial Signatures Shape Phenotype Switching and Apoptosis in Response to PLK1 and RSK Inhibitors in Melanoma

doi: 10.1101/2024.06.14.599035

Figure Lengend Snippet: (A) Schematic depicting the strategy to identify transcripts associated with resistance to PLK1 targeting. The six PLK inhibitors, in addition to BI-D1870, are indicated in panel E. (B) Transcripts that significantly correlate (p < 0.05) with 2 or more PLK1-directed treatments were selected for further analyses. (C) Functional gene analysis (g:Profiler, GO:BP) performed on correlating transcripts from B. Bars indicate p-adj values and circles indicate number of transcripts associated with a gene set. (D) Essentiality of transcripts encoding for mitochondrial proteins that significantly correlate with resistance to PLK targeting in melanoma cells (DepMap). (E) Heat-map representation and hierarchical clustering of Spearman correlations between the 124 mitochondrial signature transcripts and respective impact on proliferation dynamics of BI-D1870 and PLK1 targeting approaches (DepMap). (F) Functional classification of 124 mitochondrial signature transcripts associated with resistance to PLK1 targeting, according to MitoPathways3.0. (G) STRING interaction network of the 124 mitochondrial signature transcripts associated with resistance to PLK1 targeting. Nodes indicate proteins, and edges indicate interactions. Spearman’s correlation with proliferation dynamics (DepMap) is indicated by nodes’ colored borders. (H) Gene expression in MeWo cells transfected with the indicated siRNA for 48 h and treated with DMSO 0.1 % (v/v) or BI 6726 (100 nM) for 72 h. (I) Relative viable cell counts of MeWo cells treated as in H but with 10-fold dilutions of BI 6727, using Presto Blue HS. (J) Model depicting the impact of mitochondrial signatures on the response to RSK and PLK inhibitors. All data are shown as means ± SEM from 3 or more independent experiments. * p < 0.05, One-Way ANOVA with Dunnett multiple comparison test.

Article Snippet: Primary antibodies against PARP (#9542), β -actin (#3700), PLK1 (#4535), and Melan-A/MART-1/MLANA (#64718) were purchased from Cell Signaling Technology Inc. (Whitby, ON, CA).

Techniques: Functional Assay, Gene Expression, Transfection, Comparison