Figure 2
Figure 2. GS-1101 blocks secretion of the CCL5 and induces apoptosis in HL cell lines. (A) HL cells were cultured for 24 hours at 37°C with or without GS-1101. The concentrations of CCL5 in supernatants were assayed by ELISA. Values represent the means (± SEM) for 5 experiments. GS-1101 inhibits induction of CCL5 secretion by HL cells in cocultures with HS-5 cells. The bars represent the mean (± SEM) CCL5 concentration in HL cell supernatants (N = 4). L1236 and L591 cells were incubated for 48 hours with HS-5 cells in the presence or absence of GS-1101, as indicated on the horizontal axis. Treatment with GS-1101 significantly inhibited the induction of CCL5 secretion by the HL cells. No significant CCL5 concentrations were detected in supernatants of HS-5 cells alone (data not shown). *P = .02 (t test). **P = .004 (t test). (B) GS-1101 induces cell cycle arrest and apoptosis in HL cell lines. HL cells were cultured for 24 hours at 37°C with or without GS-1101. For cell-cycle analysis, cells were permeabilized, stained with propidium iodide, and subjected to FACS evaluation of labeled DNA. **P = .03 (t test). *P < .05 (t test). The percentage of apoptotic cells was determined by annexin V–FITC/7-AAD staining followed by 2-color flow cytometric analysis. Percentages represent cells that were annexin V–FITC/7-AAD double-positive. Values for both the cell-cycle analysis and apoptosis analysis represent the means (± SEM) for 3 experiments. (C) GS-1101 enhances the antiproliferative and proapoptotic activities of everolimus in HL cell lines. Cells were cultured for 48 hours at 37°C with or without GS-1101, everolimus, or GS-1101 and everolimus. Cell viability was determined using the Cell Titer Aqueous One Solution Cell Proliferation assay (Promega). The percentage of apoptotic cells was determined by annexin V–FITC/7-AAD staining followed by 2-color flow cytometric analysis. Percentages represent cells that were annexin V–FITC/7-AAD double-positive. Values for both the cell-viability and apoptosis analysis represent the means (± SEM) for 4 experiments.

GS-1101 blocks secretion of the CCL5 and induces apoptosis in HL cell lines. (A) HL cells were cultured for 24 hours at 37°C with or without GS-1101. The concentrations of CCL5 in supernatants were assayed by ELISA. Values represent the means (± SEM) for 5 experiments. GS-1101 inhibits induction of CCL5 secretion by HL cells in cocultures with HS-5 cells. The bars represent the mean (± SEM) CCL5 concentration in HL cell supernatants (N = 4). L1236 and L591 cells were incubated for 48 hours with HS-5 cells in the presence or absence of GS-1101, as indicated on the horizontal axis. Treatment with GS-1101 significantly inhibited the induction of CCL5 secretion by the HL cells. No significant CCL5 concentrations were detected in supernatants of HS-5 cells alone (data not shown). *P = .02 (t test). **P = .004 (t test). (B) GS-1101 induces cell cycle arrest and apoptosis in HL cell lines. HL cells were cultured for 24 hours at 37°C with or without GS-1101. For cell-cycle analysis, cells were permeabilized, stained with propidium iodide, and subjected to FACS evaluation of labeled DNA. **P = .03 (t test). *P < .05 (t test). The percentage of apoptotic cells was determined by annexin V–FITC/7-AAD staining followed by 2-color flow cytometric analysis. Percentages represent cells that were annexin V–FITC/7-AAD double-positive. Values for both the cell-cycle analysis and apoptosis analysis represent the means (± SEM) for 3 experiments. (C) GS-1101 enhances the antiproliferative and proapoptotic activities of everolimus in HL cell lines. Cells were cultured for 48 hours at 37°C with or without GS-1101, everolimus, or GS-1101 and everolimus. Cell viability was determined using the Cell Titer Aqueous One Solution Cell Proliferation assay (Promega). The percentage of apoptotic cells was determined by annexin V–FITC/7-AAD staining followed by 2-color flow cytometric analysis. Percentages represent cells that were annexin V–FITC/7-AAD double-positive. Values for both the cell-viability and apoptosis analysis represent the means (± SEM) for 4 experiments.

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