Expression and activation of caspase-3/CPP32 in CD34+ cord blood cells is linked to apoptosis after growth factor withdrawal  Li-Sheng Wang, Hong-Jun Liu, Zhen-Biao Xia, Hal E. Broxmeyer, Li Lu  Experimental Hematology  Volume 28, Issue 8, Pages 907-915 (August 2000) DOI: 10.1016/S0301-472X(00)00485-9

Figure 1 Expansion of hematopoietic cells in suspension culture. Nucleated cells (NC) and MACS separated CD34+ CB cells were cultured in suspension culture with 10% FCS in the presence of SLF (50 ng/mL), IL-3 (200 U/mL), GM-CSF (200 U/mL), and Epo (1 U/mL). Results are expressed as mean ±SEM from three separate experiments for absolute number of NC and CD34+ cells (I) and CFU-GM and BFU-E colonies (II). Percent of CD34+ cells in freshly isolated CD34+ cells (III) and 3-day–expanded (IV) cells is shown for one of three representative experiments Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 1 Expansion of hematopoietic cells in suspension culture. Nucleated cells (NC) and MACS separated CD34+ CB cells were cultured in suspension culture with 10% FCS in the presence of SLF (50 ng/mL), IL-3 (200 U/mL), GM-CSF (200 U/mL), and Epo (1 U/mL). Results are expressed as mean ±SEM from three separate experiments for absolute number of NC and CD34+ cells (I) and CFU-GM and BFU-E colonies (II). Percent of CD34+ cells in freshly isolated CD34+ cells (III) and 3-day–expanded (IV) cells is shown for one of three representative experiments Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 2 Expression of caspase-3/CPP32 in CD34+ cells by RT-PCR analysis. Total RNA was extracted from 104 freshly isolated CD34+ cells and CD34+ cells expanded with SLF, GM-CSF, IL-3, and Epo for 3 days. RT-PCR products were separate in 1% agarose gel and hybridized as described in the Materials and methods section. A 540 bp product was generated for caspase-3/CPP32, and a 600 bp product was generated for GAPDH, which was used as an internal control. S1, S2, and S3 represent three different CB samples Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 3 Expression of caspase-3/CPP32 protein in CD34+ cells by Western blot analysis. Cell lysates from freshly isolated CD34+ and 3-day–expanded cells were separated in 15% SDS-PAGE. Each lane containing 20 μg of total protein was used for Western blot analysis with anti–caspase-3/CPP32 antibody. A 32 KDa band represents the pro-enzyme of caspase-3/CPP32 (A). The relative expression of caspase-3/CPP32 (B) was from one of three CB samples. Tubulin was used as an internal protein control Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 3 Expression of caspase-3/CPP32 protein in CD34+ cells by Western blot analysis. Cell lysates from freshly isolated CD34+ and 3-day–expanded cells were separated in 15% SDS-PAGE. Each lane containing 20 μg of total protein was used for Western blot analysis with anti–caspase-3/CPP32 antibody. A 32 KDa band represents the pro-enzyme of caspase-3/CPP32 (A). The relative expression of caspase-3/CPP32 (B) was from one of three CB samples. Tubulin was used as an internal protein control Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 4 Representative analysis of expression of caspase-3/CPP32 in CD34+ cells by flow cytometry analysis. CD34+ cells were cultured with SLF, IL-3, GM-CSF, and Epo at the concentrations as mentioned in the legend to Figure 1 for 3 days. Day 0 and day 3 cells were stained with CD34/FITC and caspase-3/CPP32/PE antibodies Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 5 Cleavage of caspase-3/CPP32 in apoptotic CD34+ cells. Withdrawal of cytokines for 12 and 24 hours was done for 3-day–expanded CD34+ cells. A total of 20 μg of protein from cell lysate was separated in 15% SDS-PAGE and transferred to membrane. A 20 kDa (p20) fragment was detected by using a polyclonal rabbit anti-human caspase-3/CPP32 antibody Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 6 Influence of caspase inhibitors on caspase-3/CPP32 activity in apoptotic CD34+ cells. The 3-day–expanded CD34+ cells were withdrawn of cytokines and incubated with caspase inhibitors, z-VAD-fmk and DEVD-CHO, at 50 μmol/mL and DMSO as control for 12 hours. Caspase-3/CPP32 activity was determined by a caspase-3 activity assay kit. Results are expressed as mean pmolρNA/min/μg protein ± range from two separate experiments. Significant differences for expanded cells withdrawn of cytokines for 12 hours compared with control cells, ap < 0.05. Significant differences for expended cells treated with inhibitors compared with medium control, bp < 0.05 Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)

Figure 7 Apoptotic cells in CD34+ cells by TUNEL assay. CD34+ cells were expanded for 3 days and withdrawn of cytokines for 24 hours in the absence (A) and presence of caspase inhibitors, Z-VAD-fmk (B) and DEVD-CHO (C), each used at 50 μmol/mL. Results are from one of two representative experiments Experimental Hematology 2000 28, 907-915DOI: (10.1016/S0301-472X(00)00485-9)