Figure S1. WT (A, E, I), AP-2 (B, F, J), Chc (C, G, K) and shi (D, H, L) mutant tissue stained to assess hallmarks of neoplastic transformation; F-actin staining is red in all images. A-D: Neuronal differentiation (anti-Elav, green) is lost in mutant eye
discs. E-H: Mmp1 (cyan) is elevated in mutant eye discs. I-L: mutant follicle cell clones (lacking GFP) are round and form
multilayers. Crb activity is not required for neoplastic transformation of Scrib module or endocytic mutants in follicle cells (MP) or imaginal discs (Q-T). Removing Crb does not suppress Vps45 or scrib phenotypes. F-actin (red); Mmp1 (cyan), GFP
represents WT cells. Double mutant discs still show ectopic apical polarity, demonstrated by aPKC staining (violet) (U-X).
Scale bars: A, I 50 µm, U 10 µm.
Development | Supplementary Material
Figure S2. Scrib module mutant cells exhibit AP-2-dependent endocytosis. Notch localization (green) from live trafficking
experiments in mutant eye discs (A-D, F) and wing discs expressing aPKCCA (E). Notch at the cell surface labeled at time
point zero (A-F) is no longer seen after 300 minutes (A-F’) except in AP-2. 20’ time point shows comparable numbers of
Notch-containing endosomes in WT (G) and scrib (H) cells. (I) Quantitation of endocytosed Notch at 20’ time point in WT
(blue points) and scrib (red points). (J, K) Dextran is endocytosed by both WT and dlg cells. Scale bar: 5 µm.
Development | Supplementary Material
Figure S3. Sas (A, B) and FasIII (D, E) remain cortical in dlg imaginal discs. Quantitation (C, F) reveals comparable cortical localization. Scale bar: 10 µm.
Development | Supplementary Material
Figure S4. Crb does not colocalize well with Hrs (A, B) and Rab5 (C, D) in dig discs. Scale bar: 10 µm.
Figure S5. Wls is found in lysosomal puncta in dlg discs treated with a lysosomal inhibitor. Scale bar: 5 µm.
Development | Supplementary Material
Figure S6. Though polarization is lost, the organization of many membrane-bound subcellular compartments are not grossly altered in dlg mutant wing discs (phalloidin in red marks cortical actin). Scale bar: 10 µm
Development | Supplementary Material
Table S1. P-values for figures
Fig. 1 R
P-value
Fig. 2 K
P-value
Fig. 3 N
P-value
WT vs AP2 KD
2.16E-05
Crb: WT vs dlg
3.19E-08
Rab9YFP WT vs dlg
4.00E-01
WT vs lgl KD
3.84E-13
Crb: WT vs aPKC act
1.04E-07
Vps29GFP WT vs dlg
7.66E-01
WT vs AP2 lgl KD
1.13E-04
Crb: dlg vs aPKC act
7.40E-01
Rab5YFP WT vs dlg
4.43E-01
AP2 KD vs lgl KD
1.60E-09
Wls: WT vs dlg
3.21E-14
AP2 KD vs AP2 lgl KD
9.81E-01
Wls: WT vs aPKC act
1.45E-09
lgl KD vs AP2 lgl KD
2.35E-09
Wls: dlg vs aPKC act
7.77E-02
Ecad: WT vs dlg
7.12E-01
p<0.05
CD8: WT vs dlg
6.21E-01
p>0.05
Development | Supplementary Material