Figure S1. Ecdysone–Usp axis regulates... | Rockefeller University Press

Figure S1.

Ecdysone–Usp axis regulates NC removal. (A1–D2) Ecdysone signaling is visualized in egg chambers: stage 10B (A1 and A2); stage 11 (B1 and B2), stage 13 (C1 and C2), late stage 13 (D1 and D2). EcRE-lacz is in green (or in LUT), and DAPI is in red. (E′–G″) Representative image of types of defects of stage 14 egg chambers when ecdysone signaling is downregulated. These are classified into 3 types: only PN (E′ and E″); mild dumping defect (F′ and F″); and dumpless or high dumping defect (G′ and G″). A bright-field image of the egg chamber is shown in the inset (G′). Egg chambers were stained with Arm in green and DAPI in red. (H) Quantification of defects of stage 14 egg chambers. Stage 14 egg chambers were categorized into bins of number of defects: PN only; mild dumping defect; and dumpless or dumping defect. n indicates the number of egg chambers analyzed. (I′ and I″) Representative image of stage 14 egg chambers. Clonal cells are marked in green (GFP) and are also overexpressing EcRRNAi. DAPI is in red. White arrowheads mark the PN in the anterior end. (J) Number of PN in stage 14 egg chambers of indicated genotypes as categorized into various bins. The percentage of stage 14 egg chambers in each bin was calculated. n indicates the number of egg chambers analyzed. (K–N) Activity of the ecdysone pathway decreases upon downmodulation of the ecdysone receptor by EcRRNAi (L–L″) and EcRB1DN (M–M″) with respect to control (K–K″) in motile border cells. Quantification of normalized EcRE-lacz in indicated genotypes (N). Border cells are marked by UAS mCD8GFP in green, EcRE-lacz in magenta, and DAPI in cyan. Border cells are encircled by a white dotted line. The mean value of normalized EcRE-lacz expression is depicted in graph. n indicates the number of egg chambers analyzed. (O1–P) Stage 14 egg chambers. Yellow arrowheads mark persisting NC nuclei. DAPI is in red, and GFP is in green. Egg chambers of genotype: slbo Gal4/UAS GFP (O1), slbo-Gal4/EcRB1DN (O2), cy2 Gal4/EcRB1DN (P). (Q) Quantification of persisting NC nuclei in stage 14 egg chambers of indicated genotypes. n indicates the number of egg chambers analyzed. The mean number of PN is provided in the graph. (R) Alternative quantification of PN. The number of PN in stage 14 egg chambers as categorized into various bins, and the percentage of stage 14 egg chambers in each bin was calculated. (S) Number of PN in stage 14 egg chambers as categorized into various bins t, and the percentage of stage 14 egg chambers in each bin was calculated. Error bars represent the SEM. ****P ≤ 0.0001 represents the level of significance.

Ecdysone–Usp axis regulates NC removal. (A ¹ –D ² ) Ecdysone signaling is visualized in egg chambers: stage 10B (A¹ and A²); stage 11 (B¹ and B²), stage 13 (C¹ and C²), late stage 13 (D¹ and D²). EcRE-lacz is in green (or in LUT), and DAPI is in red. (E′–G″) Representative image of types of defects of stage 14 egg chambers when ecdysone signaling is downregulated. These are classified into 3 types: only PN (E′ and E″); mild dumping defect (F′ and F″); and dumpless or high dumping defect (G′ and G″). A bright-field image of the egg chamber is shown in the inset (G′). Egg chambers were stained with Arm in green and DAPI in red. (H) Quantification of defects of stage 14 egg chambers. Stage 14 egg chambers were categorized into bins of number of defects: PN only; mild dumping defect; and dumpless or dumping defect. n indicates the number of egg chambers analyzed. (I′ and I″) Representative image of stage 14 egg chambers. Clonal cells are marked in green (GFP) and are also overexpressing EcR^RNAi. DAPI is in red. White arrowheads mark the PN in the anterior end. (J) Number of PN in stage 14 egg chambers of indicated genotypes as categorized into various bins. The percentage of stage 14 egg chambers in each bin was calculated. n indicates the number of egg chambers analyzed. (K–N) Activity of the ecdysone pathway decreases upon downmodulation of the ecdysone receptor by EcR^RNAi (L–L″) and EcRB1^DN (M–M″) with respect to control (K–K″) in motile border cells. Quantification of normalized EcRE-lacz in indicated genotypes (N). Border cells are marked by UAS mCD8GFP in green, EcRE-lacz in magenta, and DAPI in cyan. Border cells are encircled by a white dotted line. The mean value of normalized EcRE-lacz expression is depicted in graph. n indicates the number of egg chambers analyzed. (O¹–P) Stage 14 egg chambers. Yellow arrowheads mark persisting NC nuclei. DAPI is in red, and GFP is in green. Egg chambers of genotype: slbo Gal4/UAS GFP (O¹), slbo-Gal4/EcRB1^DN (O²), cy2 Gal4/EcRB1^DN (P). (Q) Quantification of persisting NC nuclei in stage 14 egg chambers of indicated genotypes. n indicates the number of egg chambers analyzed. The mean number of PN is provided in the graph. (R) Alternative quantification of PN. The number of PN in stage 14 egg chambers as categorized into various bins, and the percentage of stage 14 egg chambers in each bin was calculated. (S) Number of PN in stage 14 egg chambers as categorized into various bins t, and the percentage of stage 14 egg chambers in each bin was calculated. Error bars represent the SEM. ****P ≤ 0.0001 represents the level of significance.

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