Journal: Biomedical Reports

Article Title: Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients

doi: 10.3892/br.2022.1527

Figure Lengend Snippet: GNLY expression in the peripheral blood lymphocytes of OA patients and controls. Representative examples of dot plots and histograms showing the results from flow cytometry analyses of the R1 lymphocyte gate for R2 GNLY + cells in the (A) OA patients and (B) controls in comparison to that of the isotype control (n=18). (C) Images show immunohistochemically labeled GNLY in lymphocytes (red color, indicated with arrows) compared with the isotype control. One of three independent samples is shown. Magnification, x1,000. Confocal laser microscopy of GNLY (red fluorescence) and LAMP-1 (green fluorescence) in lymphocytes from (D) OA patients and (E) controls. The colocalization of GNLY and LAMP-1 exhibited yellow fluorescence. The original magnification of the images are 900 x 8. Scale bar, 10 µm. The percentage of GNLY and LAMP-1 colocalization is shown for each of the four independent experiments/samples. (F) The intensities of GNLY fluorescence measured using confocal microscopy in 12 cells from OA patients and controls. (G) Percentage of GNLY + lymphocytes in OA patients and controls measured using flow cytometry (n=18). * P=0.003, ** P=0.000001 (Mann-Whitney U test). GNLY, granulysin; OA, osteoarthritis; R, region; LAMP-1, lysosomal-associated membrane protein-1; FSC-H, forward scatter-height; SSC-H, side scatter-height.

Article Snippet: NK cell samples were untreated, pre-treated with mouse IgG2b anti-human perforin (cat. no. NBP1-45774, δG9 clone; 1 μg/1x10 5 NK cells; Novus Biologicals), anti-human GNLY (cat. no. D185-3, RC8 clone; MBL International, 1 μg/1x10 5 NK cells) or a combination of anti-perforin and anti-GNLY mAbs for 30 min at 4 ̊C The subset of PKH26 labeled K-562 cells (stained red), detected as PI-negative and FITC-Annexin V-positive, were considered early apoptotic cells ( ).

Techniques: Expressing, Flow Cytometry, Comparison, Control, Labeling, Microscopy, Fluorescence, Confocal Microscopy, MANN-WHITNEY, Membrane

Journal: Biomedical Reports

Article Title: Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients

doi: 10.3892/br.2022.1527

Figure Lengend Snippet: Proportion of GNLY+ cells in the peripheral blood subpopulations and analysis of GNLY mediated apoptosis in NK cells. (A) Frequency of GNLY in CD3 - CD56 + NK cells, CD3 + CD56 - T cells and CD3 + CD56 + NKT cells between OA patients and controls. * P=0.03, ** P=0.01, *** P=0.003 (Mann-Whitney U test) in 18 experiments. Quadrants in dot plots illustrate the analysis of lymphocyte subpopulations from (B) OA patients and (C) controls pertaining to CD3/CD56 labeling or isotype control, respectively. GNLY + cells within lymphocyte subpopulations are shown as histograms for the (B) OA patients and (C) controls. Bars in the charts depict the median values of early apoptosis of K-562 cells cultured with NK cells from (D) OA patients or (E) control at the indicated ratios in the medium alone, with an anti-perforin mAb, with an anti-GNLY RC8 mAb, or with a combination of anti-perforin and anti-GNLY RC8 mAbs (n=5-6 per group). * P=0.01, ** P=0.006 (Kruskal-Wallis and post-hoc Dunn's test). GNLY, granulysin; OA, osteoarthritis; anti-P, anti-perforin; PE, phycoerythrin.

Article Snippet: NK cell samples were untreated, pre-treated with mouse IgG2b anti-human perforin (cat. no. NBP1-45774, δG9 clone; 1 μg/1x10 5 NK cells; Novus Biologicals), anti-human GNLY (cat. no. D185-3, RC8 clone; MBL International, 1 μg/1x10 5 NK cells) or a combination of anti-perforin and anti-GNLY mAbs for 30 min at 4 ̊C The subset of PKH26 labeled K-562 cells (stained red), detected as PI-negative and FITC-Annexin V-positive, were considered early apoptotic cells ( ).

Techniques: MANN-WHITNEY, Labeling, Control, Cell Culture

Journal: Biomedical Reports

Article Title: Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients

doi: 10.3892/br.2022.1527

Figure Lengend Snippet: Distribution of GNLY and cytokine expression in the peripheral blood lymphocyte subpopulation. Dot plots show the distribution of GNLY gated cells in lymphocyte subpopulations pertaining to CD3/CD56 labeling or isotype control, respectively in (A) OA patients and (B) controls (n=6). *** P=0.004 (Kruskal-Wallis and post-hoc Dunn's test) (C). Comparison of intracellular IFN-γ and IL-4 expression in CD3 - CD56 + NK, CD3 + CD56 - T, and CD3 + CD56 + NKT cells in the (D) OA patients and (E) controls (n=10-12). * P=0.03, ** P=0.01, *** P=0.004 (Mann-Whitney U test). GNLY, granulysin; OA, osteoarthritis; PE, phycoerythrin; FL3, fluorescence channel 3.

Article Snippet: NK cell samples were untreated, pre-treated with mouse IgG2b anti-human perforin (cat. no. NBP1-45774, δG9 clone; 1 μg/1x10 5 NK cells; Novus Biologicals), anti-human GNLY (cat. no. D185-3, RC8 clone; MBL International, 1 μg/1x10 5 NK cells) or a combination of anti-perforin and anti-GNLY mAbs for 30 min at 4 ̊C The subset of PKH26 labeled K-562 cells (stained red), detected as PI-negative and FITC-Annexin V-positive, were considered early apoptotic cells ( ).

Techniques: Expressing, Labeling, Control, Comparison, MANN-WHITNEY, Fluorescence