Figure 2. Prospective losers display defective autophagic flux.

(a-b) Apoptotic cell death, as detected by anti-cleaved Caspase-3 reactivity (green), in wing discs of a p62 ^+/- heterozygote (a, left), RpS3 ^+/- heterozygote (a, middle), or p62 ^+/-, RpS3 ^+/- transheterozygote (a, right) and corresponding quantification (n=10, 7, and 11 respectively, two-sided Mann-Whitney U test without p-adjustment for multiple comparisons) (b). (c) Staining of autophagosomes and autolysosomes, as detected by atg8-GFP-mCherry expression (red) in the P-compartment of wild type (c, left), or Rps3 ^+/- (c, right) wing discs. (d-e) Immunostaining for p62 in wing discs harboring RpS3 ^+/- A cells and wild-type P cells (d) and corresponding fluorescence intensity quantification (n=9, two-sided paired t-test) (e). (f) Immunostaining of p62 in a wing disc with mahj ^-/- clones (GFP-negative) induced in a mahj ^+/- heterozygous background (1XGFP). Wild-type twin spots are 2XGFP. (g) Immunostaining for p62 in wing discs harboring wild-type A cells and 4E-BP^TA-expressing P cells (labelled by the absence of Ci, magenta). (h) Schematic representation of ReFLUX: the autophagy cargo p62 is fused to GFP and driven by a hs promoter for pulse-chase expression. (i-k) GFP-p62 ReFlux signal (green) in wing discs harboring RpS3 ^+/- A cells (dsRed-positive) and wildtype P cells (dsRed-negative) immediately after heat shock (i), or three hours later (j) and corresponding signal quantifications (n= 7 and 8 respectively, two-sided student’s t-test) (k). (l-n) GFP-p62 ReFlux signal (green) in wing discs expressing mahj-RNAi in the P compartment (RFP-positive), immediately after heat shock (l) or three hours later (m) and corresponding signal quantifications (n=8 and 7 respectively, two-sided student’s t-test) (n). For all micrographs, scale bars correspond to 50μm. For all quantification, the horizontal line represents the mean and whiskers indicate 95% confidence intervals. All n numbers refer to the number of individual wing discs.