Figure 5
Figure 5. Disruption of LAMP1 expression results in decreased association of perforin with lytic granules. YTS or ex vivo isolated NK cells were untransduced (UT; YTS cells), mock-transduced (mock; NK cells), or transduced with control (CTRL) or LAMP1 RNAi. (A) Visualization of perforin in cells. YTS cells were mixed with 721.221 target cells for 30 minutes at 37°C. The cells were fixed and stained with anti-perforin δG9 Ab. Multiple optical sections were acquired every 0.3 μm in order to visualize all the perforin in the cell. The images show two-dimensional reconstruction of overlaid optical sections of the indicated transduced YTS cells interacting with the target cells. Inserts show differential interference contrast (DIC) images of the conjugated cells (YTS cell is depicted above the target cell). Scale bars represent 5 μm. (B) The summary of perforin fluorescence intensity quantification. Perforin in YTS cells was visualized by staining with anti-perforin δG9 Ab. The intensity of main perforin cluster (as defined in the supplemental Methods and supplemental Figure 3) was measured and plotted as mean + SD. The data were determined in 2 experiments by analyzing the following cell numbers: UT, n = 21; CTRL RNAi, n = 12; LAMP1 RNAi, n = 25 cells. (C) Intracellular levels of perforin. YTS or NK cells were fixed, permeabilized, stained with anti-perforin δG9 Ab and analyzed using flow cytometry. Representative histograms of perforin staining by flow cytometry (left; inserts show the protein level of LAMP1), whereas the graphs (right) summarize mean values + SD of the median perforin fluorescence from 4 (NK) or 7 (YTS) experiments. The perforin level in mock-transduced or untransduced cells was regarded as 100%, and the changes in perforin level in relation to the mock-transduced or untransduced cells are indicated for CTRL and LAMP1 RNAi cells. Asterisks in (B) and (C) indicate statistical significance: **P < .01; ***P < .001; 1-way ANOVA. (D) Perforin mRNA levels. Transcripts of perforin in the indicated cells were measured by real-time PCR; relative expression of perforin, normalized to actin, is shown. Data are represented as mean values + SD from 4 or 7 experiments, using NK and YTS cells, respectively. (E) Decreased levels of lysosomal perforin in cells with LAMP1 knockdown. The indicated YTS cells were mixed with 721.221 cells for 30 minutes at 37°C. The cells were fixed, stained with anti-perforin D48 Ab followed by DyLight 549–conjugated isotype specific anti-mouse Ab (red), and then stained with Alexa Fluor 488–conjugated anti-perforin δG9 Ab (green). The inserts show close-ups of perforin polarized to the cell-cell contact area. The plots (right) show profiles of the fluorescence intensity of anti-perforin staining using δG9 or D48 Ab, measured along the lines drawn across the widest area parallel or perpendicular to the cell-cell contact site and indicated in the inserts of the images. Scale bars represent 5 μm. (F) Perforin level in TCL or PLF from YTS cells transduced with CTRL or LAMP1 RNAi was analyzed by western blot (using anti-perforin Pf-344 Ab). Anti–granzyme B immunoblotting was used as a loading control. The result is demonstrative of 3 experiments. The changes in perforin levels, normalized to granzyme B levels, were calculated as the ratio between perforin band intensity from LAMP1 and CTRL RNAi cells in the appropriate sample, as described in Figure 4C.

Disruption of LAMP1 expression results in decreased association of perforin with lytic granules. YTS or ex vivo isolated NK cells were untransduced (UT; YTS cells), mock-transduced (mock; NK cells), or transduced with control (CTRL) or LAMP1 RNAi. (A) Visualization of perforin in cells. YTS cells were mixed with 721.221 target cells for 30 minutes at 37°C. The cells were fixed and stained with anti-perforin δG9 Ab. Multiple optical sections were acquired every 0.3 μm in order to visualize all the perforin in the cell. The images show two-dimensional reconstruction of overlaid optical sections of the indicated transduced YTS cells interacting with the target cells. Inserts show differential interference contrast (DIC) images of the conjugated cells (YTS cell is depicted above the target cell). Scale bars represent 5 μm. (B) The summary of perforin fluorescence intensity quantification. Perforin in YTS cells was visualized by staining with anti-perforin δG9 Ab. The intensity of main perforin cluster (as defined in the supplemental Methods and supplemental Figure 3) was measured and plotted as mean + SD. The data were determined in 2 experiments by analyzing the following cell numbers: UT, n = 21; CTRL RNAi, n = 12; LAMP1 RNAi, n = 25 cells. (C) Intracellular levels of perforin. YTS or NK cells were fixed, permeabilized, stained with anti-perforin δG9 Ab and analyzed using flow cytometry. Representative histograms of perforin staining by flow cytometry (left; inserts show the protein level of LAMP1), whereas the graphs (right) summarize mean values + SD of the median perforin fluorescence from 4 (NK) or 7 (YTS) experiments. The perforin level in mock-transduced or untransduced cells was regarded as 100%, and the changes in perforin level in relation to the mock-transduced or untransduced cells are indicated for CTRL and LAMP1 RNAi cells. Asterisks in (B) and (C) indicate statistical significance: **P < .01; ***P < .001; 1-way ANOVA. (D) Perforin mRNA levels. Transcripts of perforin in the indicated cells were measured by real-time PCR; relative expression of perforin, normalized to actin, is shown. Data are represented as mean values + SD from 4 or 7 experiments, using NK and YTS cells, respectively. (E) Decreased levels of lysosomal perforin in cells with LAMP1 knockdown. The indicated YTS cells were mixed with 721.221 cells for 30 minutes at 37°C. The cells were fixed, stained with anti-perforin D48 Ab followed by DyLight 549–conjugated isotype specific anti-mouse Ab (red), and then stained with Alexa Fluor 488–conjugated anti-perforin δG9 Ab (green). The inserts show close-ups of perforin polarized to the cell-cell contact area. The plots (right) show profiles of the fluorescence intensity of anti-perforin staining using δG9 or D48 Ab, measured along the lines drawn across the widest area parallel or perpendicular to the cell-cell contact site and indicated in the inserts of the images. Scale bars represent 5 μm. (F) Perforin level in TCL or PLF from YTS cells transduced with CTRL or LAMP1 RNAi was analyzed by western blot (using anti-perforin Pf-344 Ab). Anti–granzyme B immunoblotting was used as a loading control. The result is demonstrative of 3 experiments. The changes in perforin levels, normalized to granzyme B levels, were calculated as the ratio between perforin band intensity from LAMP1 and CTRL RNAi cells in the appropriate sample, as described in Figure 4C.

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