Figure 1: HS1BP3 is a negative regulator of autophagy.

(a) HEK GFP-LC3 cells were transfected with four individual siRNA oligonucleotides against HS1BP3. 72 h post transfection the cells were starved or not for 2 h in EBSS, followed by fixation and fluorescence microscopy. Scale bar, 10 μm. (b) The number of GFP-LC3 spots per cell in a was quantified by high-content analysis (mean±s.d. from two independent experiments in triplicates, ∼50,000 cells analysed per condition). (c) Relative expression of HS1BP3 after siRNA knockdown was measured by quantitative PCR with reverse transcription (mean±s.d.). (d) HEK GFP-LC3 cells were transfected with the indicated siRNA oligos and starved or not for 2 h in EBSS in the presence or absence of BafA1. * Indicates an unspecific band in the HS1BP3 immunoblot. (e) The level of LC3-II/actin was quantified from immunoblots and normalized to siControl fed (mean±s.e.m., n=5). (f) HEK GFP-p62 cells were transfected with siRNA against HS1BP3 or ULK1. Expression of GFP-p62 was induced by addition of tetracycline (compare ON versus OFF) for 48 h before expression was shut off and the cells were incubated in EBSS (starved) for 2.5 h to induce autophagic degradation of GFP-p62. GFP-p62 intensity was monitored by flow cytometry and normalized to starved siControl (siCtrl; mean±s.e.m., n=4). (g) The degradation of long-lived proteins in HeLa cells transfected with control siRNA or siRNA against HS1BP3 was quantified as the release of ¹⁴C-valine after 4 h starvation in the absence or presence of 3-methyladenine (3MA) and normalized to the degradation in fed control cells (mean±s.e.m., n=3). *P<0.05, **P<0.01, ***P<0.001, by Student’s t-test.