DEV016295 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Geminin staining in the embryo and larval Malpighian tubules. Wild-type (A,A′) stage 5 embryo, and (B,B′) larval Malpighian tubules stained with (A,B, white; A′,B′, inset, green) α-Geminin and (A′,B′, blue) DAPI. (A,A′) Geminin is expressed at high levels during cellularization. (B,B′) Geminin staining is absent in the larval Malpighian tubules, except in the mitotically cycling stelate cells (insets).

• Supplemental Figure S2 -

  Fig. S2. Expressing individual Apc^RNAi constructs results in APC/C target accumulation during endocycles. The Flipout/Gal4 technique was used to clonally express UAS-Apc^RNAi constructs with GFP. Salivary glands containing (A1-A2′′′) Apc8^RNAi, (B-B′′′ ,D-D′′′ ) Apc10^RNAi or (C-C′′′ ,E-E′′′ ) Apc6^RNAi Flipout/Gal4 clones stained with (A1,A2,B,C,D,E, white; A1′′′,A2′′′,B′′,C′′′,D′′′,E′′′, green) α-GFP, (A1′,A2′, white; A1′′′,A2′′′, red) α-Cyclin B, (B′,C′, white; B′′,C′′′, red) α-Geminin and (D′,E′, white; D′′′,E′′′, red) α-Cyclin A antibodies, and (A1′′,A2′′,C′′,D′′,E′′, white; A1′′′,A2′′′,B′′,C′′′,D′′′,E′′′, blue) DAPI. A1 and A2 correspond to two different focal planes of the same cells. Because the majority of the α-Cyclin B staining is observed on the membrane, whereas the α-GFP and DAPI staining is observed in the nucleus, we show two panels representing different focal plans for this experiment. (A2′) Cyclin B, (B′,C′) Geminin and (D′,E′) Cyclin A are ectopically expressed in cells that express the Apc6^RNAi, the Apc8^RNAi or the Apc10^RNAi constructs.

• Supplemental Figure S3 -

  ,C-C′′ ,D-D′′ ) pOrc1-Orc1Oboxmut-GFP and (B-B′′ ) pOrc1-Orc1-GFP (A-C′′) egg chambers and (D-D′′) salivary gland stained with (A,B,C,D, white; A′′,B′′,C′′,D′′, green) α-GFP and (A′,B′,C′,D′, white; A′′,B′′,C′′,D′′, red) α-Mpm2 antibodies and (A′′,B′′,C′′,D′′, blue) DAPI. (A-A′′) The nurse cells marked with an asterisk correspond to the Mpm2-positive cells. There is correlation between the α-Mpm2 staining and the α-GFP staining in (B-B′′) pPrc1-Orc1-GFP follicle cells, and there is an absence of correlation between α-GFP and α-Mpm2 in (C-C′′) pOrc1-Orc1Oboxmut-GFP follicle cells.

• Supplemental Figure S4 -

  Fig. S4. The transcript levels of Cyclin A, geminin and Orc1 in mitotic versus endocycling cells. (A) Analysis of Cyclin A, geminin and Orc1 and rp49 transcript levels in wild-type embryos and salivary glands by semi-quantitative RT-PCR. (B) Graphic representation of the transcript levels in the salivary gland normalized to rp49 mRNA levels and with 4- to 8-hour-old embryo data.

• Supplemental Figure S5 -

  Fig. S5. The transcript levels of Cyclin A and geminin are slightly increased in endocycling Cyclin E and APC/C ^RNAi overexpressing cells. The Flipout/Gal4 technique was used to clonally overexpress Cyclin E and Apc10^RNAi; Apc8^RNAi. (A) Graphic representation of the Cyclin A, geminin and Orc1 transcript levels in overexpressing Cyclin E (black) and overexpressing Apc10^RNAi; Apc8^RNAi (dark gray) salivary glands, normalized to rp49 mRNA levels and to data from wild-type (light gray) salivary glands. Salivary glands containing (B,B′) UAS-Cyclin E and (C,C′) UAS-Apc10^RNAi; UAS-Apc8^RNAi Flip-out clones labeled with (B,C, white; B′,C′, red) α-Geminin and (B′,C′, green) α-GFP antibodies and (B′,C′, blue) DAPI. After two heat-shocks of 1 hour, the majority of cells express the constructs, as monitored by α-GFP staining. Among GFP-positive cells observed after UAS-CycE expression, almost all the cells also accumulate high levels of Geminin. After the expression of the UAS-Apc10^RNAi; Apc8^RNAi constructs, approximately half of the GFP-positive cells express high levels of Geminin.