Figure 5
Figure 5. Apoptosis pathway driven by IL-23 in 697 cells. (A) Relative quantification of miR15a in wild-type 697 cells and 697 cells transfected with anti-miR15a or irrelevant (irr) anti-miR cultured 48 hours with or without IL-23, as assessed by real-time PCR. MiR15a expression was normalized to an endogenous reference (miRU6) and expressed relative to a calibrator sample (primary B-ALL sample). (B) Left panel: flow cytometric analysis of apoptosis in wild-type 697 and in 697 cells transfected with anti-miR15a or irrelevant anti-miR cultured 48 hours with or without IL-23 (med). Whisker lines represent highest and lowest values, horizontal lines represent median values (n = 3). Dot plots on the right: annexin V/PI double staining in one representative experiment is shown. (C) Left panel: flow cytometric analysis of BCL-2 expression in wild-type 697 cells and 697 cells transfected with anti-miR15a or irrelevant (irr) anti-miR cultured with medium (med) or IL-23 (48 hours). Whisker lines represent highest and lowest values; horizontal lines represent median values (n = 3). Histograms on the right: one representative experiment of BCL-2 staining is shown. (D) Western blot of BCL-2 expression in wild-type 697 cells and 697 cells transfected with anti-miR15a or irrelevant (irr) anti-miR upon incubation with medium (med) or IL-23 (48 hours). (E) Flow cytometric analysis of caspase activation and AKT, ERK/1, and p38MAPK phosphorylation in 697 cells upon incubation for 48 hours with medium or IL-23 (bottom panels). Positive controls are shown in top panels. Open profile: caspase-3, -7, -8, and -10, and phosphorylated AKT, ERK1/2, and p38MAPK staining in stimulated cells; dark profile: caspase-3, -7, -8, and -10 and phosphorylated AKT, ERK1/2, and p38MAPK staining in untreated cells. (F) Flow cytometric analysis of apoptosis of 697 cells pretreated 1 hour with the Z-VAD-FMK pancaspase inhibitor and then cultured for an additional 48 hours with medium (Z-VAD-FMK) or with IL-23 (Z-VAD-FMK+IL23). One representative experiment is shown. (G) PARP cleavage in 697 cells cultured 48 hours with medium (med) or with IL-23, as assessed by flow cytometry (histograms on the left) and by Western blot (right panel). Histogram plots: staining with anti-cleaved PARP Ab of cells cultured with IL-23 (open profile) or with medium (dark profile). Jurkat cells treated 5 hours with etoposide represent the positive control.

Apoptosis pathway driven by IL-23 in 697 cells. (A) Relative quantification of miR15a in wild-type 697 cells and 697 cells transfected with anti-miR15a or irrelevant (irr) anti-miR cultured 48 hours with or without IL-23, as assessed by real-time PCR. MiR15a expression was normalized to an endogenous reference (miRU6) and expressed relative to a calibrator sample (primary B-ALL sample). (B) Left panel: flow cytometric analysis of apoptosis in wild-type 697 and in 697 cells transfected with anti-miR15a or irrelevant anti-miR cultured 48 hours with or without IL-23 (med). Whisker lines represent highest and lowest values, horizontal lines represent median values (n = 3). Dot plots on the right: annexin V/PI double staining in one representative experiment is shown. (C) Left panel: flow cytometric analysis of BCL-2 expression in wild-type 697 cells and 697 cells transfected with anti-miR15a or irrelevant (irr) anti-miR cultured with medium (med) or IL-23 (48 hours). Whisker lines represent highest and lowest values; horizontal lines represent median values (n = 3). Histograms on the right: one representative experiment of BCL-2 staining is shown. (D) Western blot of BCL-2 expression in wild-type 697 cells and 697 cells transfected with anti-miR15a or irrelevant (irr) anti-miR upon incubation with medium (med) or IL-23 (48 hours). (E) Flow cytometric analysis of caspase activation and AKT, ERK/1, and p38MAPK phosphorylation in 697 cells upon incubation for 48 hours with medium or IL-23 (bottom panels). Positive controls are shown in top panels. Open profile: caspase-3, -7, -8, and -10, and phosphorylated AKT, ERK1/2, and p38MAPK staining in stimulated cells; dark profile: caspase-3, -7, -8, and -10 and phosphorylated AKT, ERK1/2, and p38MAPK staining in untreated cells. (F) Flow cytometric analysis of apoptosis of 697 cells pretreated 1 hour with the Z-VAD-FMK pancaspase inhibitor and then cultured for an additional 48 hours with medium (Z-VAD-FMK) or with IL-23 (Z-VAD-FMK+IL23). One representative experiment is shown. (G) PARP cleavage in 697 cells cultured 48 hours with medium (med) or with IL-23, as assessed by flow cytometry (histograms on the left) and by Western blot (right panel). Histogram plots: staining with anti-cleaved PARP Ab of cells cultured with IL-23 (open profile) or with medium (dark profile). Jurkat cells treated 5 hours with etoposide represent the positive control.

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