Journal: bioRxiv

Article Title: New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics

doi: 10.1101/092536

Figure Lengend Snippet: Comparison of SNAP labeling and nonspecific binding. (A) Wild-type cells were incubated with SNAP-SiR647, washed, and imaged in TIRF as described in the text. (B and C) Cells expressing Pil1p-SNAP (B) or wild-type cells (C) were incubated with SNAP-Alexa647, washed, and imaged as described. Images shown are inverted contrast, Maximum intensity projections of 20-sec movies with median-filter background subtracted. Cell outlines are drawn in orange dash; all image panels are at same scale with scale bar 5 μm.

Article Snippet: To label SNAP-tag protein in live cells, 0.5 mL of cells at OD 595nm 0.5 were incubated at 25 o C on a rotator in liquid EMM5S media containing 0.1, 0.5, or 2.5 μM of the siliconrhodamine benzylguanine derivative SNAP-SiR647 or SNAP-Alexa647 (SNAP-Cell ® 647-SiR, SNAP-Surface ® Alexa Fluor ® 647, New England Biolabs) for 0.5, 5, or 15 hours.

Techniques: Labeling, Binding Assay, Incubation, Expressing

Journal: Nature Communications

Article Title: Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice

doi: 10.1038/s41467-018-08096-8

Figure Lengend Snippet: Functional tolerance of hepatic CD8 + T cells is broken after TIGIT blockade in HBs-tg mice. a–n HBs-tg mice were treated with rat IgG or α-TIGIT mAb for 3 months. a–e Representative flow cytometry graphs showing hepatic CD69 + CD8 + T cells ( a ), CD25 + CD8 + T cells ( b ), CD44 hi CD62L low CD8 + T cells ( c ), CD127 + CD62L + CD8 + T cells ( d ), and Ki67 + CD8 + T cells ( e ) in rat IgG or α-TIGIT-treated HBs-tg mice. f–j Percentages of hepatic and splenic CD69 + CD8 + T cells ( f ), CD25 + CD8 + T cells ( g ), CD44 hi CD62L low CD8 + T cells ( h ), CD127 + CD62L + CD8 + T cells ( i ), and Ki67 + CD8 + T cells ( j ) in rat IgG or α-TIGIT-treated HBs-tg mice ( n = 4–6 in each group). k–n Expression of CD107a ( k , l ) and intracellular IFN-γ ( m , n ) by hepatic and splenic CD8 + T cells of mice after ex vivo stimulation with PMA and ionomycin ( n = 5 in each group). Statistically significant differences between the groups are presented as the mean ± SEM. * P <0.05; ** P < 0.01; and *** P < 0.001 (two-tailed unpaired Student’s t -test). Data are representative of at least two independent experiments

Article Snippet: PerCP-Cy5.5-anti-CD3 (BioLegend), PE-CY7-CD8 (BD), BV510-CD56 (BioLegend), BV605-TIGIT (BioLegend), FITC-anti-IFN-γ(BD), PE-anti-TNF-α(BD), PE-CY7-anti-TIGIT (BioLegend), AF 647-anti-Ki67 (BD), APC-CY7-anti-IL-2 (BioLegend), and BV510-anti-CD107a (BioLegend).

Techniques: Functional Assay, Flow Cytometry, Expressing, Ex Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice

doi: 10.1038/s41467-018-08096-8

Figure Lengend Snippet: TIGIT blockade enhances antiviral responses of CD8 + T cells from CHB patients. a Correlation between the gene expressions of TIGIT and PDCD1 in the HCC tumor tissue. Data are from the TCGA database. Spearman’s correlation coefficients ( r ) and P- values are shown. b TIGIT expression on peripheral blood CD8 + T cells in CHB patients and healthy controls was analyzed by flow cytometry ( n = 31, 35; Healthy controls, HBV patients). Each dot represents one individual. Statistically significant differences between the groups are presented as the mean ± SEM (two-tailed unpaired Student’s t -test). Data are pooled from three independent experiments. c , d The peripheral blood mononuclear cells (PBMCs) from HBV patients were stimulated with HBsAg peptide in the presence of α-TIGIT or control antibody for 10 days and were then tested by intracellular cytokine staining. c Representative flow cytometric graphs showing expression of CD107a, IFN-γ, TNF-α, and Ki67 in CD8 + T cells from samples treated with mouse IgG or α-TIGIT mAb. d The statistical percentages in ( c ) are shown ( n = 7, 7, 8, 6; CD107a, IFN-γ, TNF-α, Ki67). Statistically significant differences are calculated by two-tailed paired Student’s t -test: * P < 0.05 and ** P < 0.01. Data are representative of three independent experiments

Article Snippet: PerCP-Cy5.5-anti-CD3 (BioLegend), PE-CY7-CD8 (BD), BV510-CD56 (BioLegend), BV605-TIGIT (BioLegend), FITC-anti-IFN-γ(BD), PE-anti-TNF-α(BD), PE-CY7-anti-TIGIT (BioLegend), AF 647-anti-Ki67 (BD), APC-CY7-anti-IL-2 (BioLegend), and BV510-anti-CD107a (BioLegend).

Techniques: Expressing, Flow Cytometry, Two Tailed Test, Staining