FIGURE 2:

Rab11 is required for amphisome formation. (A) Fat body cells of Rab11-depleted fed L3 larvae clonally expressing Lamp1-GFP (green) were stained with LTR (red). Nuclei were stained using DAPI (blue). The LTR-positive dot per cell area ratio was calculated and compared between clone and neighboring control cells using Wilcoxon's signed-rank test (n = 21, p < 0.0001). Lamp1-GFP–positive dot per cell area ratio was also calculated and compared between control (see Figure S2C) and Rab11-depleted groups using the Mann-Whitney test (n_ctrl = 34, n_RNAi = 27, p < 0.0001). (B) Control and green, Rab11-depleted fat body cells of fed L3 larvae were stained with anti-Rab7 antibody (red). Nuclei were stained using DAPI (blue). Dot per cell area ratio was calculated and compared between the groups using Wilcoxon's signed-rank test (Rab7: n = 28, p < 0.0001; Rbsn5: n = 15, p = 0.8647). (C–C′) Colocalization between GFP-Atg8a (green) and TRA (red) decreased due to Rab11 depletion. Nuclei were stained with DAPI (blue). Colocalization between GFP-Atg8a and TRA, mCh-Atg8a and anti-Rab7, and mCh-Atg8a and Lamp1-GFP was quantified and compared between the groups using the Mann-Whitney test (Atg8-TRA: n_ctrl = 100, n_RNAi = 190, p = 0.0002; Atg8-Rab7: n_ctrl = 41, n_RNAi = 39, p = 0.0008; Atg8-Lamp1: n_ctrl = 57, n_RNAi = 57, p < 0.0001). On box-and-whisker plots, bars (gray, control; green, Rab11 RNAi) show the data lying between the upper and lower quartiles; the median is indicated as a horizontal line within the box. Whiskers plot the smallest and largest observations. NS, p > 0.05; ***, p < 0.001. Scale bars: 10 μm.