Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Structures of six reactivity probes. MSD-DTB, maleimide-sulfonate-dibenzocyclooctyne-DTB. MS-DTB, maleimide-sulfonate-DTB. DBIA, desthiobiotin iodoacetamide. IA-DTB, iodoacetamide-PEG-desthiobiotin. CA-DTB, chloroacetamide-PEG-desthiobiotin. IA-DTB-COOH, iodoacetamide-carboxylate-PEG-desthiobiotin. b MSD-DTB and MS-DTB probes exhibited greater cell surface labeling compared to the others in BV173 and MT2 cells. The result is a representative of three experiments ( n = 3 independent replicates). Fluorescein isothiocyanate (FITC)-A represents the fluorescence intensity of FITC. c . LC-MS analyses revealed that MSD-DTB and MS-DTB probes modify cysteine, but not histidine, serine, and N-terminal amine. The result is a representative of two experiments ( n = 2 independent replicates). d Structures of MD (maleimide-dibenzocyclooctyne), MSD (maleimide-sulfonate-dibenzocyclooctyne), and MSD4 (maleimide-sulfonate-dibenzocyclooctyne-PEG4). e In-gel fluorescence analyses revealed that sulfonated maleimide probes minimally labeled proteins in live cells, while yielding comparable levels of proteome labeling to its cell-permeable counterpart probe, MD, in cell lysates. The result is a representative of two experiments ( n = 2 independent replicates). f Cysteine-directed activity-based protein profiling (ABPP) analyses revealed that MS-DTB modified five cysteines with >20% engagement out of 15,310 quantified cysteines in live cells, while it modified 1730 cysteines with >20% engagement in cell lysates. Data represent mean values ( n = 2 independent replicates). Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Labeling, Fluorescence, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Modification

Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Quantitative global proteomics and flow cytometry studies confirmed the knockout (KO) of HLA class I genes in MT2 cells. Global proteomics data represent mean values ( n = 3 independent replicates). The result of flow cytometry analysis is a representative of two experiments ( n = 2 independent replicates). b Flow cytometry analyses measuring Streptavidin-FITC staining on HLA wildtype (WT) and KO cells treated with the MSD-DTB or MS-DTB probe. Data represent mean values ( n = 2 independent replicates in MT2 and MDA-MB-231 cells treated with MSD-DTB, and MDA-MB-231 cells treated with MS-DTB) and mean values ± SEM ( n = 3 independent replicates in BV173 cells treated with MSD-DTB and MS-DTB, and n = 4 independent replicates in MT2 cells treated with MS-DTB). The statistical significance was assessed using unpaired two-tailed Student’s t-tests. P values were 0.037, 0.0029, and 0.048. c Quantitative global proteomics and flow cytometry studies confirmed the complete knockout of HLA class I genes in MT2 cells. Global proteomics data represent mean values ( n = 3 independent replicates). P values were calculated by two-sided t test and adjusted using Benjamini-Hochberg correction for multiple comparisons. The result of flow cytometry analysis is a representative of two experiments ( n = 2 independent replicates). d Flow cytometry analyses measuring Streptavidin-FITC staining on MT2 parental and HLA knockout cells treated with MS-DTB. Data represent mean values ± SEM ( n = 4 independent replicates). The statistical significance was assessed using unpaired two-tailed Student’s t -tests. P values were 0.0029 and 9.2 × 10 − 6 . e Structure of NHS-sulfonate-biotin and flow cytometry analyses measuring Streptavidin-FITC staining on BV173 HLA wildtype and knockout cells treated with NHS-sulfonate-biotin. Data represent mean values ( n = 2 independent replicates). NHS, N-hydroxysuccinimide. f ELISA assay measuring MS-DTB in the pMHC-I complex. Data represent mean values ± SEM ( n = 3 independent replicates). The statistical significance was assessed using unpaired two-tailed Student’s t-tests. P values were 8.1 × 10 − 5 , 0.00028, 6.2 × 10 − 6 , and 8.7 × 10 − 6 . HRP, horseradish peroxidase. g ELISA assay measuring MHC-I and β2-microglobulin (β2M) assembly. Data represent mean values ± SEM ( n = 3 independent replicates). The statistical significance was assessed using unpaired two-tailed Student’s t -tests. Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Flow Cytometry, Knock-Out, Staining, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Schematic representation of the immunopeptidomics workflow. b The number of 8-13-mer MHC-I-associated peptides in MT2 and BV173 cells treated with 50 µM of MS-DTB. The result is a representative of two experiments ( n = 2 independent replicates). c Motif analysis of all 9-mer MHC-I-bound antigens, cysteine-containing 9-mer MHC-I-bound antigens, cysteinylated 9-mer MHC-I-bound antigens, and MS-DTB-modified 9-mer MHC-I-bound antigens. d Distribution of all cysteines, cysteinylated cysteines, and MS-DTB-modified cysteines on 9-mer peptide antigens in MT2 and BV173 cells. e Venn diagram of MHC-I-bound antigens with unmodified, cysteinylated, and MS-DTB-modified cysteines. Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Modification

Journal: Nature Communications

Article Title: A platform for mapping reactive cysteines within the immunopeptidome

doi: 10.1038/s41467-024-54139-8

Figure Lengend Snippet: a Schematic representation of comparative analysis in interferon gamma (IFNγ)-stimulated versus non-stimulated BV173 cells. b Western blot analysis of MHC-I and PSMB9 expression in BV173 cells stimulated with IFNγ (50 ng/mL, 24 h). The result is a representative of two experiments ( n = 2 independent replicates). c ELISA assay measuring the presence of MS-DTB in the pMHC-I complex with or without IFNγ stimulation in BV173 cells. Data represent mean values ± SEM ( n = 3 independent replicates). The statistical significance was evaluated through unpaired two-tailed Student’s t -tests. P value was 0.025. d The number of 8-13-mer MHC-I-associated peptides in IFNγ-stimulated versus non-stimulated cells. e . Motif analysis of all 9-mer MHC-I-bound antigens and MS-DTB-modified MHC-I-bound antigens. f Distribution of MS-DTB-modified cysteines on 9-mer MHC-I-bound antigens in IFNγ-stimulated versus non-stimulated cells. g Heatmap showing the ratio values of amino acid (aa) frequency in IFNγ-stimulated versus non-stimulated cells. The percentage of each aa relative to all amino acids at each position was calculated to derive the ratio values between IFNγ-stimulated and non-stimulated cells. A gray shade indicates aa with a percentage below 2%. Source data are provided as a Source Data file.

Article Snippet: BV173 cells were obtained from CLS Cell Lines Service.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Modification

Journal: Journal for immunotherapy of cancer

Article Title: Systemic and intravesical adoptive cell therapy of tumor-reactive T cells can decrease bladder tumor growth in vivo.

doi: 10.1136/jitc-2020-001673

Figure Lengend Snippet: Figure 5 CXCR3 mediates T cell infiltration into orthotopic ovalbumin expressing MB49 tumor cell line (MB49OVA) bladder tumor from intravesical space. (A) RNA was isolated from MB49 and MB49OVA tumor cells and analyzed for expression of chemokines CXCL9, CXCL10 and CXCL11 as determined by PCR. (B) OT-I T cells were isolated from spleen of transgenic mice and tumor-infiltrating lymphocytes (TILs) were isolated from MB49 orthotopic tumors. Expression of CXCR3 was determined on these cell populations by flow cytometry. (C) TIL samples expanded from patient bladder tumors were analyzed for CXCR3 expression by flow cytometry. In vivo, mice were treated with OT-I T cells coated with a CXCR3 blocking antibody or rat IgG (rIgG). (D) Tumors were collected 3 and 24 hours after infusion and analyzed for CD45.1+ T cell infiltration by flow cytometry. (E) Tumor volume was measured by ultrasound. Precoating OT-I T cells with αCXCR3 decreased T cell infiltration into the tumor and abrogated the decrease in tumor growth seen with OT-I T cells coated with a control rIgG antibody. N=5 per group. Repeated 2 times. ns, not significant.

Article Snippet: OT- I T cells were isolated as described and incubated for 30 min on ice with either normal rat IgG isotype control or CXCR3 blocking antibody (500 μg, catalog# BE0249, BioXcell).

Techniques: Expressing, Isolation, Transgenic Assay, Flow Cytometry, In Vivo, Blocking Assay, Control

Journal: Journal of medicinal chemistry

Article Title: A Potent and Highly Efficacious Bcl-2/Bcl-xL Inhibitor

doi: 10.1021/jm4001105

Figure Lengend Snippet: Antitumor activity of compounds 31 and 32 in the H146 small-cell lung cancer xenograft model in SCID mice. Tumors were grown to an average size of 126 mm3 and compound 31 or 32 was administered at 15 mg/kg intravenously, daily, 5 days a week for 2 weeks. (a). Tumor growth. (b). Animal body weight.

Article Snippet: 7 Human small cell lung cancer cell lines H146, H1963, H187, and H1417 were purchased from American Type Culture Collection (ATCC) and were maintained in RPMI-1640 medium containing 10% FBS.

Techniques: Activity Assay