JCS032904 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Mitochondrial morphology observed with either MitoTracker Red CMXRos or mitoGFP. (a) To test whether CED-9 is required for mitochondrial homeostasis, we stained young adult animals with MitoTracker Red CMXRos to examine mitochondrial morphology in body wall muscles. Shown are images from wild-type (N2), ced-9(n2812lf); ced-3(n717lf), ced-3(n717lf) and maternally rescued ced-9(n1950n2161lf) animals. (b) Increased CED-9 expression in ced-9(n2812lf); ced-3(n717lf) animals results in dilated, interconnected mitochondria. (c) Expression of CED-9(G169E) in wild-type animals resulted in dilated, interconnected mitochondria. n, nucleus. Bar, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. CED-9 lacking the sixth α-helix localizes to mitochondria. To determine the localization of CED-9 lacking the sixth α-helix (residues 199-209), a construct that is not functional in regulating apoptosis (Tan et al., 2007), ced-9Δα6 transgenes with GFP at the N terminus, were expressed by a copy of the ced-9 promoter in ced-9(n2812lf); ced-3(n717lf) animals as previously described (Tan et al., 2007). Both the punctate GFP localization indicative of mitochondrial localization, and the mosaic expression pattern, was similar to that observed for wild-type ced-9 transgenes. Shown are GFP and DIC images of embryos. Bar, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Increased FZO-1 expression or fzo-1(tm1133) deletion result in aberrant mitochondria. The mitofusin proteins Mfn-1 and Mfn-2 regulate mitochondrial fusion in mammalian cells (Santel and Fuller, 2001). In C. elegans, one putative mitofusin protein has been identified: FZO-1. (a) Increased expression of FZO-1 using the myo-3 promoter in adult body wall muscle cells resulted in disconnected vesicular mitochondria in both ced-9(+) and ced-9(n2812lf); ced-3(n717lf) animals. (b) A similar morphology was observed in animals homozygous for the fzo-1(tm1133) deletion allele. n, nucleus. Bar, 10 µm. (c) Mitochondrial ultrastructure in ten adult fzo-1(tm1133) animals was examined by transmission electron microscopy. Shown are representative images from hypodermis (left) and body wall muscles (right). The inset image is an enlargement of the area marked with a dashed box. Bar, 500 nm.

• Supplemental Figure S4 -

  Fig. S4. Analysis of CED-9 expression in C. elegans L4 larval stage animals by western blotting. To estimate the relative expression of CED-9 between various lines, we harvested L4 larval stage animals for western blot analysis using a polyclonal antibody against the N terminus of CED-9 (#sc-9201; Santa Cruz Biotech, Inc.). Transgenic animals were identified by the Rol phenotype (see Materials and Methods), individually picked into M9 buffer salt solution, spun down, resuspended with 1×SDS-PAGE loading buffer, freeze-thawed, and boiled for 5 minutes. Approximately five animals were loaded for separation on a 10% SDS-PAGE, and transferred onto a PVDF membrane at 120 mAmp, 20 V for 2 hours. The membrane was blocked with 5% non-fat milk (+ 0.1% Tween 20), blotted with 1:200 α-CED-9 and 1:1000 α-goat(HRP) (#sc-2020, Santa Cruz Biotech, Inc.), and exposed on film using SuperSignal West Pico Chemiluminescent (Pierce). These results suggest that lines that co-express DRP-1 and either CED-9 or CED-9(G169E) express CED-9 at approximately similar levels. However, some line-to-line expression variability exists among CED-9(G169E) transgenes. Importantly, we note that a quantitative comparison between lines is not possible, as each L4 animal contains a variable number of cells that express a given transgene.

• Supplemental Table S1 - Adobe PDF