American Society of Hematology

Figure 3

$Figure 3. PU-H71 induces PEL cell death by apoptosis and autophagy. (A) BC3 cells treated with 1 µM PU-H71 for 72 hours were examined by flow cytometry after staining for 7-AAD and Annexin V. Results were quantified into percentages of live and apoptotic cells (Annexin V positive). ***Statistical significance of P < .001 as compared with the control (untreated) group. (B) Top: Immunoblot analysis using extracts from BC3 cells treated at the indicated time points with 2 µM PU-H71 for the indicated time points shows PARP cleavage by 24 hours as indication of apoptosis. Lower: PARP cleavage is apparent after PU-H71 treatment (2 μM for 24 hours) in additional PEL cell lines. (C) BC3 cells treated with 1 µM PU-H71 for 72 hours were examined by flow cytometry for DNA content cell-cycle analysis. Only the Sub-G0 fraction, indicative of apoptosis, was found to increase in treated cells. (D) Autophagy induction was examined by immunoblot of PU-H71–treated cells. Appearance of the cleavage product of LC3B is indicative of autophagy. (E) BC3 cells treated with 1 µM PU-H71 were examined for the appearance of autophagosomes containing degraded cellular organelles using electron microscopy. Autophagosomes are clearly visible in the right panel at higher magnification (arrows).$

PU-H71 induces PEL cell death by apoptosis and autophagy. (A) BC3 cells treated with 1 µM PU-H71 for 72 hours were examined by flow cytometry after staining for 7-AAD and Annexin V. Results were quantified into percentages of live and apoptotic cells (Annexin V positive). ***Statistical significance of P < .001 as compared with the control (untreated) group. (B) Top: Immunoblot analysis using extracts from BC3 cells treated at the indicated time points with 2 µM PU-H71 for the indicated time points shows PARP cleavage by 24 hours as indication of apoptosis. Lower: PARP cleavage is apparent after PU-H71 treatment (2 μM for 24 hours) in additional PEL cell lines. (C) BC3 cells treated with 1 µM PU-H71 for 72 hours were examined by flow cytometry for DNA content cell-cycle analysis. Only the Sub-G0 fraction, indicative of apoptosis, was found to increase in treated cells. (D) Autophagy induction was examined by immunoblot of PU-H71–treated cells. Appearance of the cleavage product of LC3B is indicative of autophagy. (E) BC3 cells treated with 1 µM PU-H71 were examined for the appearance of autophagosomes containing degraded cellular organelles using electron microscopy. Autophagosomes are clearly visible in the right panel at higher magnification (arrows).

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