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. 2015 Mar 19;14(10):1496–1506. doi: 10.1080/15384101.2015.1026515

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Figure 2 (See previous page). — Overexpression of Lgl can suppress N-ext, but not N-icd, phenotypes, and does not affect endogenous Notch signaling. (A) Confocal images of β-gal staining (gray, red in merge) of lgl^WT mosaic third instar larval eye discs in the E(spl)m8-lacZ background. Clones are marked by the expression of GFP (green, arrowheads). (B) Quantification of E(spl)m8-lacZ expression in lgl^WT clones compared with wild-type clones, showing that Lgl^WT does not significantly affect the expression of β-gal relative to wild-type tissue. The data is shown relative to the elevated expression of E(spl)m8-lacZ in lgl⁻ clones and control mosaic eye discs. (C) Confocal images of Notch-intra staining (gray, red in merge) of lgl^WT mosaic third instar larval eye discs in the E(spl)m8-lacZ background. Clones are marked by the expression of GFP (green, outlined area). Lgl^WT does not affect the levels or localization of Notch-intra. (D) Control eq-GAL4 adult thorax (D) and eye (D') (E-G) eq > N-ext crossed to UAS-lacZ (B), UAS-mam^DN (C) or UAS-lgl^WT (D) adult thorax and eyes. Note that expression of mam^DN or lgl^WT can suppress the adult eye roughing (outlined areas) and thoracic overgrowth phenotypes (arrowheads) of eq > N-ext, indicating that Lgl can suppress ectopic Notch signaling at the γ-secretase step. (H-J) eq > N-icd crossed to UAS-lacZ (H), UAS-mam^DN (I) or UAS-lgl^WT (J) adult thorax and eyes. Only mam^DN expression is able to rescue the adult eye roughing (outlined areas) and thoracic overgrowth phenotype (arrowheads). Genotypes: (A, C) eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-lgl^WT/ tubGAL80 FRT40; tubGAL4 (D) w;; UAS-myrRFP, eq1-GAL4/TM6B (E) w;; UAS-myrRFP, eq1-GAL4, UAS-N-ext/UAS-lacZ (F) w;; UAS-myrRFP, eq1-GAL4, UAS-N-ext/UAS-mam^DN (G) w;; UAS-myrRFP, eq1-GAL4, UAS-N-ext/UAS-lgl^WT (H) w; +/UAS-N-icd; UAS-myrRFP, eq1-GAL4, UAS-lacZ (I) w; +/UAS-N-icd; UAS-myrRFP, eq1-GAL4, UAS-mam^DN (J) w; +/UAS-N-icd; UAS-myrRFP, eq1-GAL4, UAS-lgl^WT Flystocks: Eq1-Gal4 (H Sun), E(spl)m8-lacZ (A. Bergmann), UAS-N-ext (III) (T. Vaccari), UAS-lacZ-nls (III) (G. Baeg), UAS-mam^DN (BL26672) (Bloomington stock center), UAS-lgl^WT (III) (J. Knoblich), UAS-N-icd (II) (S. Artavanis-Tsakonas). Antibodies: mouse β-galactosidase (Sigma, 1:500), mouse Notch-intra (Developmental Studies Hybridoma Bank, 1:50).