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Blood, Vol. 105, Issue 8, 3278-3285, April 15, 2005
Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrafish
Blood Langenau et al.
105: 3278
Supplemental materials for: Langenau, Vol 105, Issue 8, 3278-3285
Files in this Data Supplement:
- Figure S1. Amino acid alignment and phylogenetic analysis of vertebrate Bcl-2 proteins. (JPG, 144 KB)
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(A) Alignment of Bcl-2 and Bcl-xL proteins from zebrafish (z), Xenopus (x), chicken (c), and human (h). Amino acid residues conserved among both Bcl-2 and Bcl-xL family members are indicated (#) while amino acid residues conserved among only Bcl-2 proteins are noted (*). Conserved BH1, BH2, BH3, and BH4 domains are denoted by a single line above the alignment. Dashes denote gaps introduced to maximize alignment. (B) Diagram showing conserved domain homologies when zebrafish bcl-2 is compared to the human BCL-2 protein. (C) Phylogenetic analysis of vertebrate Bcl-2 and Bcl-xL proteins confirms that zebrafish bcl-2 clusters with other known Bcl-2 homologues.
- Figure S2. The EGFP-bcl-2 fusion protein blocks irradiation-induced apoptosis. (JPG, 61 KB)
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(A, B) TUNEL staining of uninjected embryos or (C) embryos injected with GFP or (D) EGFP-bcl-2 RNA. (A) Embryos were either not exposed or (B-D) exposed to 15G -irradiation at 14 hpf. All embryos were analyzed at 20 hpf for apoptotic cells by TUNEL assay. (A-D) Lateral view of embryos at 20 hpf, dorsal to the right. TUNEL-positive apoptotic cells are stained black. (E) TUNEL positive cells within the tails of embryos were quantified. Cells residing in the tail region below the yolk (as indicated by a dashed line in A-D) were counted. Asterisks denote significant differences between uninjected, unirradiated embryos (n = 10) and both GFP-injected (n = 12, p = 0.0007) and uninjected irradiated fish (n = 12, p = 0.0003), and significant differences between EGFP-bcl-2-injected (n = 12) and both GFP-injected (n = 12, p = 0.0006) and uninjected irradiated fish (n = 12, p = 0.0002). There were no differences in the mean number of apoptotic cells between the uninjected, irradiated embryos and GFP-injected, irradiated embryos (p = 0.93) or between uninjected, unirradiated embryos and EGFP-bcl-2 injected, irradiated embryos (p = 0.99). E-bcl-2; EGFP-bcl-2.
- Figure S3. Thymocytes from 10-week-old rag2-EGFP-bcl-2 transgenic fish do not undergo programmed cell death following irradiation treatment. (JPG, 78 KB)
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(A, C) FACS analysis of AnnexinV-stained cells in the absence of irradiation or 24 hours post-irradiation treatment (20 Gy, B, D). Percentage of GFP-labeled cells that are annexinV-positive are noted (+/- 1 SD, n = 3). (A, B) Top panels are of FACS-sorted rag2-GFP thymocytes and (C, D) bottom panels are rag2-EGFP-bcl-2 thymocytes.
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