Figure 1
Figure 1. MePDN-mediated effects on NKp30 and NKp44 surface expression and on NK-cell survival and proliferation in the presence of IL-2 or IL-15. (A) NK cells were cultured for 5 days in the presence of IL-2 (100 U/mL) or IL-15 (20 ng/mL) either with MePDN at the final concentration of 0.5 μg/mL (gray profiles) or without MePDN (empty profiles). Cytofluorimetric analysis revealed that MePDN selectively inhibited NKp30 and NKp44 surface expression. This experiment is representative of 15 independent experiments performed using different donors. (B) In order to analyze whether MePDN could affect NK-cell survival, we performed, at different time intervals (day 3 and day 5), cytofluorimetric analysis of annexin-V surface expression on NK cells cultured in the presence of different concentrations of IL-2 (200 U/mL, 100 U/mL, 20 U/mL) or IL-15 (50 ng/mL, 25 ng/mL, 5 ng/mL, corresponding to 100 U/mL, 50 U/mL, and 10 U/mL, respectively). (left) An experiment representative of 11 independent analyses (performed at day 3) is shown. (right) Analysis of annexin V–binding NK cells cultured with MePDN (0.5 μg/mL) in the presence of IL-2 (hatched bars) or IL-15 (gray bars) versus control NK cells in IL-2 or IL-15 alone. Data are expressed as the mean percentage (± SEM) of 7 independent experiments. Statistical significance was checked by nonparametric Wilcoxon signed rank test. (C) NK cells cultured for 5 days in different concentrations of IL-2 (hatched bars) or IL-15 (gray bars) in the presence or absence of MePDN (0.5 μg/mL) were counted with trypan blue to exclude dead cells. Results are expressed as the mean percentage (± SEM) of NK-cell proliferation in response to IL-2 or IL-15 with MePDN (0.5 μg/mL) versus control NK cells (ie, without MePDN), evaluated in 11 independent experiments. Bars indicate standard error. Statistical significance was checked by a nonparametric Wilcoxon signed rank test. (D) NK cells were cultured for 2 days in the presence of IL-2 (100 U/mL) or IL-15 (20 ng/mL) in the presence or absence of MePDN at the final concentration of 0.5 μg/mL. Cell extracts were analyzed by WB in order to assess the content of CD132. The expression of α-tubulin was analyzed to assess comparable amounts of protein. This experiment is representative of 3 performed.

MePDN-mediated effects on NKp30 and NKp44 surface expression and on NK-cell survival and proliferation in the presence of IL-2 or IL-15. (A) NK cells were cultured for 5 days in the presence of IL-2 (100 U/mL) or IL-15 (20 ng/mL) either with MePDN at the final concentration of 0.5 μg/mL (gray profiles) or without MePDN (empty profiles). Cytofluorimetric analysis revealed that MePDN selectively inhibited NKp30 and NKp44 surface expression. This experiment is representative of 15 independent experiments performed using different donors. (B) In order to analyze whether MePDN could affect NK-cell survival, we performed, at different time intervals (day 3 and day 5), cytofluorimetric analysis of annexin-V surface expression on NK cells cultured in the presence of different concentrations of IL-2 (200 U/mL, 100 U/mL, 20 U/mL) or IL-15 (50 ng/mL, 25 ng/mL, 5 ng/mL, corresponding to 100 U/mL, 50 U/mL, and 10 U/mL, respectively). (left) An experiment representative of 11 independent analyses (performed at day 3) is shown. (right) Analysis of annexin V–binding NK cells cultured with MePDN (0.5 μg/mL) in the presence of IL-2 (hatched bars) or IL-15 (gray bars) versus control NK cells in IL-2 or IL-15 alone. Data are expressed as the mean percentage (± SEM) of 7 independent experiments. Statistical significance was checked by nonparametric Wilcoxon signed rank test. (C) NK cells cultured for 5 days in different concentrations of IL-2 (hatched bars) or IL-15 (gray bars) in the presence or absence of MePDN (0.5 μg/mL) were counted with trypan blue to exclude dead cells. Results are expressed as the mean percentage (± SEM) of NK-cell proliferation in response to IL-2 or IL-15 with MePDN (0.5 μg/mL) versus control NK cells (ie, without MePDN), evaluated in 11 independent experiments. Bars indicate standard error. Statistical significance was checked by a nonparametric Wilcoxon signed rank test. (D) NK cells were cultured for 2 days in the presence of IL-2 (100 U/mL) or IL-15 (20 ng/mL) in the presence or absence of MePDN at the final concentration of 0.5 μg/mL. Cell extracts were analyzed by WB in order to assess the content of CD132. The expression of α-tubulin was analyzed to assess comparable amounts of protein. This experiment is representative of 3 performed.

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