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mad10 clones were induced in a Minute+ background and examined for Dll expression.

Loss-of-function somatic clones were induced using FLP/FRT mediated mitotic recombination [58].

MARCM clones were induced by applying 1 hour heat-shock in 38°C water bath at various developmental stages.

In this genetic background, dTIEG mutant cells were recovered in the wing disc when clones were induced early.

Wild-type and mutant clones were induced using the Mosaic Analysis with a Repressive Marker (MARCM) method [32], and labeled with UAS-mCD8 GFP. UAS-mCD8 GFP

Nevertheless, the presence of twin spots (marked by high levels of GFP) (Figure 1D', encircled) indicated that mutant clones were induced, but not recovered.

When the dTIEGS14 clones were induced later (>60 hours AEL) mutant cells were recovered although clones were smaller than their sister clones (bright green) and showed smooth borders (Fig. 4D).

To study the turnover of the overexpressed optineurin-GFP, the low and the high expresser clones were induced by Dox for 20 h, treated with cycloheximide, and chased for 0, 4, 8, 16, 24 and 40 h.

In addition, when delg homozygous mutant clones were induced, we noted a strong reduction in cell size, yet the nuclear size, shown by the DAPI staining, was not changed (Fig. 2G), demonstrating growth defects, This is surprising, since endoreplication, and thus nuclear size, normally correlates with cell size in this tissue.

To verify that the low rate of recovered clones in the MARCM experiments was not due to the experimental conditions control clones were induced in parallel using FRT40A ubi-GFP to monitor the appearance of twin spots in the wing disc.

Control non-mutant clones were induced in male flies (7).