Figure 3
Figure 3. Autophagy in CLL cells treated with the CDK inhibitor flavopiridol. (A) Confocal fluorescence microscopy for CLL cells untreated or treated 4 hours with flavopiridol (2μM) or flavopiridol + CQ (0.5μM). Samples were visualized as in Figures 1 and 2. (B) Quantification of LC3 immunofluorescence in CLL cells from panel A. LC3 fluorescence was increased by flavopiridol and further by the addition of CQ (P < .0001 for the increase with flavopiridol vs vehicle, the increase with flavopiridol + CQ vs vehicle, and the increase of flavopiridol + CQ vs flavopiridol). (C) Quantification of LC3-Lamp2 colocalization by average correlation index in 6 CLL samples treated with flavopiridol or flavopiridol + CQ as in panel A. The decreased colocalization with CQ was significant (P < .0001). (D) Immunoblot for LC3B I and II and p62, representative of 3 experiments. (E) Quantification of LC3 immunofluorescence in a time course with flavopiridol. After incubations, CLL cells (n = 3) were collected by cytospin at the specified time points. Samples collected after the 4-hour time point were washed and resuspended in media without flavopiridol for the remainder of the incubation. The increase in LC3 fluorescence flavopiridol compared with vehicle was significant (P = .001) at 3, 4, and 8 hours and decreased to levels comparable with vehicle alone at 12 and 24 hours. (F) Confocal fluorescence microscopy of CLL cells obtained from patients treated with flavopiridol. Samples were collected before treatment and at the end of flavopiridol infusion (4.5 hours) and visualized as in previous figures. (G) Average of LC3 fluorescence in CLL cells (n = 16) collected from patients treated with flavopiridol, showing LC3 intensity before treatment (PRE) and at the end of flavopiridol infusion (4.5 hours). Overall, LC3 intensity was 1.44 times higher in nonresponders compared with responders (95% CI, 1.07-1.95; P = .0183). Although the increase in intensity 4.5 hours versus before treatment was slightly higher for nonresponders compared with responders (fold change of 2.51 vs 1.82), it was not significantly higher (P = .3445). (H) Immunoblot for p62 in samples from CLL patients treated with flavopiridol. Samples were collected before treatment, at the end of flavopiridol infusion (4.5 hours), and at 24 hours from beginning of infusion. Data shown are representative of samples from 8 patients.

Autophagy in CLL cells treated with the CDK inhibitor flavopiridol. (A) Confocal fluorescence microscopy for CLL cells untreated or treated 4 hours with flavopiridol (2μM) or flavopiridol + CQ (0.5μM). Samples were visualized as in Figures 1 and 2. (B) Quantification of LC3 immunofluorescence in CLL cells from panel A. LC3 fluorescence was increased by flavopiridol and further by the addition of CQ (P < .0001 for the increase with flavopiridol vs vehicle, the increase with flavopiridol + CQ vs vehicle, and the increase of flavopiridol + CQ vs flavopiridol). (C) Quantification of LC3-Lamp2 colocalization by average correlation index in 6 CLL samples treated with flavopiridol or flavopiridol + CQ as in panel A. The decreased colocalization with CQ was significant (P < .0001). (D) Immunoblot for LC3B I and II and p62, representative of 3 experiments. (E) Quantification of LC3 immunofluorescence in a time course with flavopiridol. After incubations, CLL cells (n = 3) were collected by cytospin at the specified time points. Samples collected after the 4-hour time point were washed and resuspended in media without flavopiridol for the remainder of the incubation. The increase in LC3 fluorescence flavopiridol compared with vehicle was significant (P = .001) at 3, 4, and 8 hours and decreased to levels comparable with vehicle alone at 12 and 24 hours. (F) Confocal fluorescence microscopy of CLL cells obtained from patients treated with flavopiridol. Samples were collected before treatment and at the end of flavopiridol infusion (4.5 hours) and visualized as in previous figures. (G) Average of LC3 fluorescence in CLL cells (n = 16) collected from patients treated with flavopiridol, showing LC3 intensity before treatment (PRE) and at the end of flavopiridol infusion (4.5 hours). Overall, LC3 intensity was 1.44 times higher in nonresponders compared with responders (95% CI, 1.07-1.95; P = .0183). Although the increase in intensity 4.5 hours versus before treatment was slightly higher for nonresponders compared with responders (fold change of 2.51 vs 1.82), it was not significantly higher (P = .3445). (H) Immunoblot for p62 in samples from CLL patients treated with flavopiridol. Samples were collected before treatment, at the end of flavopiridol infusion (4.5 hours), and at 24 hours from beginning of infusion. Data shown are representative of samples from 8 patients.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal