Figure 6: PLD1 co-localization with ATG16L1 is affected by HS1BP3.
From: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels
(a) HEK cells were transfected with GFP-tagged PLD1 or PLD2. After starvation and fixation the cells were immunostained for ATG16L1 and analysed by confocal microscopy. (b) HEK cells were transfected with GFP-PLD1, starved, fixed and immunostained for ATG16L1 and TfR. (c) HEK cells were first treated with non-targeting or HS1BP3 siRNA, then transfected to express GFP-PLD1, starved, fixed and immunostained for ATG16L1. Yellow arrows indicate ATG16L1 vesicles positive for PLD1 and white arrow heads indicate ATG16L1 vesicles negative for PLD1. Co-localization of GFP-PLD1 to ATG16L1 vesicles was quantified in transfected cells using the ImageJ plugin Squassh, using 10 pictures of each condition from three independent experiments (mean±s.e.m., n=3). (d) HEK cells were transfected with HA-PLD1 together with GFP, GFP-HS1BP3 full-length, -PX or ΔPX constructs, starved and stained for endogenous ATG16L1. Arrows indicate co-localization between ATG16L1 and HA-PLD1. (e) Co-localization of HA-PLD1 with endogenous ATG16L1 vesicles was quantified in transfected cells in d with the Zen software (Zeiss) using 10 pictures of each condition from three independent experiments (mean±s.e.m., n=3). Scale bars, 10 μm. *P<0.05, by Student’s t-test.
