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Pceh-2dsRed-expressing

By Frederick Robinson,2014-12-25 23:40
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Pceh-2dsRed-expressing

SUPPLEMENTARY DATA

Supplementary tables

    Table S1. Ectopic expression of icd-1 does not suppress programmed cell death during development

    A. The expression of icd-1 under the control of a heat-inducible promoter does not block he programmed death of cells during the development of the anterior pharynx t

     Average number of extra cells in

    Strain Heat shock anterior pharynx ? STDEV n Range

    JR2028 - 0.0 ? 0.2 25 0-1

    JR2028 0.1 ? 0.4 48 0-1 +

    JR2028 animals carry an integrated array of the transgene Picd-1, which allows the HS1heat-inducible expression of icd-1. Heat shock was applied during embryogenesis as 1described by Bloss et al. The number of extra cells in the anterior pharynx was determined in animals at the fourth larval stage of development using DIC.

    B. The expression of icd-1 in a ced-1(e1735) background under the control of a heat-inducible promoter does not result in fewer cell corpses at the comma stage of embryogenesis

     Average number of corpses ? STDEV (n)

    Strain no heat shock heat shock

    ced-1(e1735) 24.2 ? 2.6 (10) 25.0 ? 2.9 (10)

    ced-1(e1735); JR2028 23.0 ? 1.9 (20) 24.6 ? 3.7 (18)

     1The integrated Picd-1 array contained in JR2028 was transferred into a ced-1(e1735) HS21background. Heat shock was applied during embryogenesis as described by Bloss et al.

    The number of cell corpses was determined in animals at the comma stage of embryogenesis using DIC.

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Table S2. Ectopic expression of drp-1(wt) under the control of a heat-inducible promoter

    dsRed-expressing cells results in the loss of Pceh-2

     Average number of Average number of Range sum of

     PdsRed-positive PdsRed-positive PdsRed-positive ceh-2ceh-2ceh-2Genotype living cells cell corpses n cells and corpses bcIs51; Pdrp-1(wt) HS5.0 0 44 5 no heat shock

    bcIs51; Pdrp-1(wt) HS4.0 0.4 37 2-5 heat shock

bcIs51 is an integrated array of the reporter PdsRed, which is expressed in five neurons in the ceh-2

    anterior bulb of the pharynx during the second half of embryogenesis (starting at the 2-fold stage): the 3two NSMs, the two M3s, and the I3. Pdrp-1(wt) is an extrachromosomal array of the transgene HS

    Pdrp-1(wt), which drives the heat-inducible expression of drp-1(wt). Heat shock was applied to HS

    bcIs51; Pdrp-1(wt) embryos as described in Methods, except that adult animals were allowed to lay HS

    eggs for 90’ and that heat shock was applied to embryos 4hr after the adults had been removed from

    the plates. Embryos at the 3?- to 4-fold stage of embryogenesis were analyzed for the presence of Pceh-dsRed- positive cells and corpses using DIC and epi-fluoresence. The complete genotype of the 2

    animals was unc-76(e911); bcIs51; Pmitogfp+Pdrp-1(wt). HS HS

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Table S3. Ectopic expression of drp-1(wt) under the control of a heat-inducible

    promoter causes embryonic lethality, which is at least partially suppressed by ced-

    9(n1950gf)

     no heat shock heat shock

     % transgenic % embryonic % transgenic % embryonic

    Line animals (n) lethality (n) animals (n) lethality (n)

    control - 1 58 (50) 0 (50) 53 (232) 3 (240)

    control - 2 47 (92) 0 (92) 38 (88) 3 (91)

    drp-1(wt) - 2 69 (109) 1 (110) 41 (89) 33 (133) PHS

    Pdrp-1(wt) - 3 69 (156) 0 (156) 54 (95) 35 (147) HS

    Pdrp-1(wt) - 5 76 (98) 0 (98) 52 (42) 38 (68) HS

    ced-9(n1950gf); Pdrp-1(wt) - 9 58 (66) 0 (66) 51 (111) 8 (121) HS

    +/+; P drp-1(wt) - 9* 57 (77) 0 (77) 34 (109) 32 (161) HS

    ced-9(n1950gf); Pdrp-1(wt) - 10 52 (105) 2 (107) 46 (137) 9 (150) HS

    +/+; P drp-1(wt) - 10* 59 (58) 3 (60) 31 (136) 18 (165) HS

Animals carrying extrachromosomal arrays of the Pdrp-1(wt) transgene or control HS

    extrachromosomal arrays were subjected to heat shock during embryogenesis as described in Methods. The percent transgenic animals was determined by counting the total number of adults and the number of non-Unc transgenic adults that developed from the treated embryos. The percent embryonic lethality was determined by counting the total number of embryos treated and the number of embryos that failed to hatch within 24hr of the treatment. The complete genotype of the animals were (from top to bottom): unc-76(e911); Pmitogfp lines 1, 2, unc-76(e911); Pmitogfp+Pdrp-1(wt) lines 2, 3, HS HS HS

    5, ced-9(n1950gf); unc-76(e911); Pmitogfp+Pdrp-1(wt) line 9, unc-76(e911); PHS HS HS

    mitogfp+Pdrp-1(wt) line 9, ced-9(n1950gf); unc-76(e911); Pmitogfp+Pdrp-1(wt) HS HS HS

    line 10, unc-76(e911); Pmitogfp+Pdrp-1(wt) line 10. Lines marked with * were HS HS

    crossed out of the ced-9(n1950gf) background to confirm that the transgenes were active in a wild-type background.

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    Table S4. Animals overexpressing drp-1(wt) contain larger numbers of cell corpses than animals severely defective in engulfment

     Average number of

    Genotype Heat shock corpses ? STDEV n Range

    +/+ - 7.3 ? 1.7 16 4-10

    ced-7(n1892); ced-5(n1812) - 43.4 ? 4.9 15 38-51

    egl-1 + 49.3 ? 13.4 10 25-76 PHS

    Pdrp-1(wt) + 55.4 ? 16.1 10 31-74 HS

    The strain ced-7(n1892); ced-5(n1812) carries a mutation in each of the two parallel, partially redundant C. elegans engulfment pathways and is therefore severely defective in 4-67engulfment. Pegl-1 and Pdrp-1(wt) animals carry extrachromosomal arrays of a HS HS

    Pegl-1 or Pdrp-1(wt) transgene, respectively, which allow the heat-inducible HS HS

    expression of egl-1 or drp-1(wt). Pegl-1 and Pdrp-1(wt) animals were subjected to HS HS

    heat shock as described in Methods except that transgenic embryos were analyzed 3-4hr after the heat shock rather than 2hr after the heat shock. The number of cell corpses was

    determined in embryos at the 1?-fold stage of embryogenesis using DIC. The complete genotypes of the animals were (from top to bottom): wild-type (N2), ced-7(n1892); ced-

    5(n1812), unc-76(e911); Pmito-gfp+Pegl-1; unc-76(e911); Pmito-gfp+Pdrp-HS HS HS HS

    1(wt).

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Supplementary figure legends

Figure S1. egl-1(n1084 n3082), ced-9(n1950gf), ced-9(n2812lf), ced-4(n1162), and

    ced-3(n717) do not affect mitochondrial morphology in general. Representative

    (n1084 n3082), confocal images of rhodamine (bottom) and DIC (top) of wild-type, egl-1

    ced-9(n1950gf), ced-9(n2812lf); ced-3(n717), ced-4(n1162), and ced-3(n717) animals at

    the comma to 1 ?-fold stage of embryonic development. Mitochondria were stained with rhodamine B hexyl ester as described in Methods. Images of rhodamine-stained embryos represent single confocal planes.

    Figure S2. Reducing icd-1 function induces the appearance of small refractile structures, which are not suppressed by mutations in egl-1, ced-9, ced-4, or ced-3.

    1,8 was reduced by RNA-mediated interference (RNAi) using feeding as the icd-1 function

    8method for delivering dsRNA. icd-1 RNAi resulted in embryonic lethality and the

    appearance of small refractile structures in 50-75% (n=20 or more) of arrested embryos in a wild-type background as well as in an egl-1(n1084 n3082), ced-9(n1950gf), ced-

    4(n1162), or ced-3(n717) background. Representative DIC images of affected embryos.

Figure S3. Most refractile structures induced by icd-1 RNAi differ in their

    appearance from cell corpses in wild-type animals and from ectopic cell corpses induced by overexpression of egl-1 or drp-1(wt). Refractile structures in wild-type

    embryos, in wild-type embryos ectopically expressing egl-1 or drp-1(wt) from a heat-

    7inducible transgene (Pegl-1 and Pdrp-1(wt)), or in wild-type embryos, in which icd-HSHS

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    1 was reduced by RNAi (icd-1(RNAi)). Arrows point to refractile structures. 1 function

    Representative DIC images of embryos at the comma to 1?-fold stage of embryonic

    development. The complete genotypes of the animals analyzed were: N2 (‘wild-type’),

    unc-76(e911); Pmito-gfp+Pegl-1 (‘egl-1 expression’); unc-76(e911); Pmito-HS HS HS

    gfp+Pdrp-1(wt) (‘drp-1(wt) expression’); icd-1(RNAi) (‘icd-1(RNAi)’). HS

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Supplementary references

1. Bloss, T. A., Witze, E. S. & Rothman, J. H. Suppression of CED-3-independent

    apoptosis by mitochondrial betaNAC in Caenorhabditis elegans. Nature 424,

    1066-71 (2003).

    2. Hedgecock, E. M., Sulston, J. E. & Thomson, J. N. Mutations affecting

    programmed cell deaths in the nematode Caenorhabditis elegans. Science 220,

    1277-9. (1983).

    3. Aspock, G., Ruvkun, G. & Burglin, T. R. The Caenorhabditis elegans ems class

    homeobox gene ceh-2 is required for M3 pharynx motoneuron function.

    Development 130, 3369-78 (2003).

    4. Wu, Y. C. & Horvitz, H. R. C. elegans phagocytosis and cell-migration protein

    CED-5 is similar to human DOCK180. Nature 392, 501-4 (1998).

    5. Wu, Y. C. & Horvitz, H. R. The C. elegans cell corpse engulfment gene ced-7

    encodes a protein similar to ABC transporters. Cell 93, 951-60 (1998).

    6. Stanfield, G. M. & Horvitz, H. R. The ced-8 gene controls the timing of

    , 423-33 (2000). programmed cell deaths in C. elegans. Mol Cell 5

    7. Conradt, B. & Horvitz, H. R. The C. elegans protein EGL-1 is required for

    programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93,

    519-29 (1998).

    8. Timmons, L., Court, D. L. & Fire, A. Ingestion of bacterially expressed dsRNAs

    can produce specific and potent genetic interference in Caenorhabditis elegans.

    Gene 263, 103-12. (2001).

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