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Figure 1. Expression levels of Bid transcripts in Xenopus. A–D: Bid mRNA detection by reverse transcriptase-polymerase chain reaction analysis on embryos of stages 1 to 35 (A; Niewkoop and Faber,1994), on several tissues throughout metamorphosis (B,C), and adult tissues (C, right column and D). L8 ribosomal protein (rpl8) or ornithine decarboxylase (odc) transcripts were used for normalization. Ad, adult; Te, testis; Br, brain; Lu, lung; Bl, blood; In, intestine; Ov, ovary; Sk, skin; St, stomach; Li, liver; Mu, muscle.Download figure to PowerPoint
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Figure 2. Xenopus laevis tBID localizes to mitochondria and triggers a drop of δψm, which is inhibited by Bcl-2. Fluorescence analysis of HeLa-Bcl-2 cells transfected with CMV-BIDGFP (upper panel) and CMV-tBIDGFP vectors (lower panel). A: Green fluorescent protein (GFP) fluorescence of BID and tBID fusion proteins, immunostaining of the mitochondrial marker Cox VIc and morphology of nuclei stained with Hoechst examined under a fluorescent microscope (original magnification, ×630). Right panel shows a merged picture (original magnification, ×2,500) of the boxed cells of the left panel. The arrow in the lower Hoechst panel indicates the nuclear condensation and fragmentation of the cells. B: Mitochondrial membrane potential (δψm) of GFP-positive transfected cells assessed by flow cytometry, using the CMX-Ros fluorochrome, 24 hr after beginning of transfection. Low δψm corresponds to low fluorescence (L area), and high δψm corresponds to high fluorescence (H area). C: Mitochondrial membrane potential (δψm) variations in HeLa cells overexpressing or not bcl-2 after transfection of plasmids expressing GFP alone (control), BIDGFP, tBIDGFP, or their BH3 mutated relatives (G87A).Download figure to PowerPoint
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Figure 3. Xenopus laevis BID accelerates the drop of δψm during TNF-α-induced apoptosis but not during etoposide-induced apoptosis. Fluorescence analysis (original magnification, ×1,000) of HeLa-Bcl-2 cells transfected with CMV-BIDGFP in the presence of etoposide: eto (upper line)or Emetine + TNF-α: E/TNF (lower line). A: Arrow in Hoechst panel (upper line) shows nuclear condensation in the presence of etoposide. Boxed cells at a magnification of ×1,000 (left panel) are merged (original magnification, ×3,500) to show the cytosolic (BID + eto) or mitochondrial (BID + ET) distribution of BIDGFP (right panel). B: Mitochondrial membrane potential (δψm) of Bcl2-Hela cells transfected by green fluorescent protein (GFP) or BIDGFP-expressing vectors assessed by flow cytometry.Download figure to PowerPoint
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Figure 4. Validation of the protective effect of xR11 against tBID-induced apoptosis by somatic gene transfer in tadpole muscle cells. Cell survival measured by the luciferase activity (RLU) in muscle extracts 72 hr after transfection into the dorsal muscle of stage 56 tadpoles. Each group was injected with 200 ng of pcDNA3-LUC. In the Ct group, 1 ng of CMV-GFP and 1 μg of empty pCDNA3 were coinjected. In the tBID group, 1 ng of cmv-gfptbid and 1 μg of empty pCDNA3 were coinjected. In tBid/xR11 group, 1 ng of cmv-gfptbid and 1 μg of cmv-xr11 were coinjected. In the BID group, 500 ng of cmv-gfpbid and 500 ng of empty pCDNA3 were coinjected. Means ± SEM are given; n > 4 in each group. *P < 0.05; **P < 0.01; ***P < 0.001. The experiment was repeated three times providing similar results.Download figure to PowerPoint
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Figure 5. BIDGFP cleavage during induced or spontaneous metamorphosis. Western blot analysis on total proteins extracted from metamorphic CMV-BIDGFP transgenic tadpoles tissues using a GFP antibody (molecular probes). A: Detection of the BID 45-kDa truncated form (tBIDGFP) during natural metamorphosis. Stage 54, premetamorphosis; stages 61 to 63, climax of metamorphosis. B: Detection of tBIDGFP in heads and tails of transgenic CMV-BIDGFP tadpoles in premetamorphosis (stage 42) maintained in water supplemented with 10 nM T3 for a week. For the heads, autoradiography exposure was 2 min for the BIDGFP band and increased to 120 min to enhance the faint tBIDGFP band (lower lane). C: tBIDGFP detection in ex vivo cultured tails of transgenic CMV-BIDGFP tadpoles (stage 57) 2 days and 4 days after continuous 10 nM T3 incubation.Download figure to PowerPoint
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Figure 6. mRNA Expression and activity levels of Xenopus caspases, calpain, and calpastatin during tail regression. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis performed on metamorphic tails at the indicated developmental stages (stage 56, premetamorphosis; stages 61 to 63, climax of metamorphosis). L8 ribosomal protein transcript was used to normalize the amplification products. Positives controls (C+) are analyzed by RT-PCR on laid eggs with the following exception: for Cl2-Calpain and Caspase 8, RT-PCR was performed on adult stomach, and for Calpastatin-3, RT-PCR was performed on stage 63 metamorphic tails. B: Fluorometric analysis of calpain and caspases-2 and -8 activities in tadpoles tails before (stage 54) and during metamorphosis (stages 58, 62, 63). Means ± SEM are given; n > 5 in each group. *P < 0.05; **P < 0.01; ***P < 0.001, compared with stage 54. The figures represent one of two independent experiments that gave similar results.Download figure to PowerPoint
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Figure 7. Caspases-2 and -8 inhibitors inhibit tail regression. A: Effect of T3 (10 nM) during 24 hr or 48 hr on caspases activities in ex vivo cultured tails from wild-type siblings tadpoles killed at stage NF55. Caspase activities were determined using fluorometric dosage. B,C: Effect of T3 (10 nM) with or without caspase inhibitors (100 μM) during 24 hr or 48 hr on tail regression of ex vivo cultured tails from wild-type siblings tadpoles killed at stage NF55. Tails were photographed at different times of T3 treatments (B), and the regression of the area of each tail was calculated (C). Means and SEM are shown, *P < 0.05; **P < 0.01; *P < 0.001; n = 3 for caspase dosage, n = 5 for tail regression. The figures represent one of two independent experiments with similar results.Download figure to PowerPoint
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Figure 8. BID overexpression accelerates tail regression. Ex vivo cultured tails in the presence of T3 (10 nM) from BIDGFP-positive and -negative siblings tadpoles killed at stage NF55. Tails were photographed at different times of T3 treatment (A) and the area of each tail was determined (B). Histogram represents quantification (Means ± SEM) of the regression of the area of eight BIDGFP positive and negative tails. *P < 0.05 **P < 0.01.Download figure to PowerPoint
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Figure 9. Caspase 8 activity is required for BIDGFP cleavage during tail regression. Effect of caspase inhibitors (100 μM) on tail regression of ex vivo cultured tails from wild-type sibling tadpoles killed at stage NF55, in the presence of T3 (10 nM) during 24 hr or 48 hr. Western blot analysis was performed on total proteins extracted from tails. Green fluorescent protein (GFP) -tagged proteins were detected using a GFP antibody (Roche). The asterisk indicates an unspecific band. The percentage of cleaved BIDGFP was determined by densitometry using Image J (Wayne Rasband, NIH). Caspase-2 inhibitor-treated sample and the control on the right were run on a separate gel. The data represent one of two independent experiments that gave similar results.Download figure to PowerPoint
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