Extended Data Figure 4. Verification of reverse MARCM clonal technique for atg13 and atg14.

a, Cartoon depicting the genetic basis of the reverse MARCM clonal technique to induce labelled tumour cells confronted with autophagy-deficient neighbours. Experimental animals are heterozygous for atg13 and the tumour suppressor scrib on homologous sister chromatid arms. Mitotic (G2 phase) site-specific recombination at FLP recombination target sites (FRT) is induced between sister chromatids by transient tissue-specific expression of the dsDNA processing enzyme flipase. Severed chromosome arms can be stoichiometrically re-annealed with the sister chromatid, leading to simultaneous generation of cells carrying either two copies of scrib or atg13 loss-of-function alleles. Upon mitotic recombination, scrib^−/− mutant cells simultaneously express GFP and oncogenic Ras^V12 under the control of an upstream activating sequence (UAS), driven by tissue-specific expression of the GAL4 yeast transcription factor. Non-recombined cells and atg13^−/− cells do not express GFP or Ras^V12 as GAL4 activity is repressed by the presence of the yeast GAL4 repressor GAL80. b, c, Confocal images showing eye antennal discs with GFP-labelled control clones and reverse MARCM atg13^−/− (b) or atg14^−/− (c) clones detected by immunolabelling against the accumulating autophagy cargo protein Ref(2)P. Images are representative of more than 25 discs for each genotype. Scale bars, 50 μm (b, c).