Fig. 1: AAV.ULK1.DN-mediated overexpression of dominant-negative ULK1 in vitro alters the levels of the autophagic proteins ULK1, LC3-II, and p62.

a Scheme of experimental setup. DOP day of preparation of embryonic day 18 (E18) rat cortical neurons. DIV day in vitro, AAV transduction with adeno-associated viral vectors, MC medium change, RAP addition of rapamycin (750 nM) 24 h before lysis. b Vector maps of the AAV used to express the mCherry fluorophore as well as the untranslated 9(5) fragment (AAV.CTRL) or the C-terminal domain (CTD) of ULK1, which has dominant-negative properties (AAV.ULK1.DN), both of them expressed under the control of a human synapsin promoter (hSYN). ITR AAV-2 inverted terminal repeat, Int intron, SV40-pA SV40-polyadenylation site, WPRE Woodchuck hepatitis virus posttranscriptional regulatory element. bGH-pA bovine growth hormone-polyadenylation site. c Photomicrographs (DIV 8) of E18 rat cortical neurons transduced with AAV.CTRL or AAV.ULK1.DN. Top: mCherry fluorescence, bottom: overlay of mCherry fluorescence with brightfield images. Scale bar: 50 µm. d Representative immunoblot of cell lysates transduced with given AAV. Expression of ULK1.DN was validated for all samples by blots against its myc-tag. e–j Top: Representative immunoblots of ULK1, LC3 (high exposure to detect LC3-II), p62, ATG7, and the 56 and 30 kDa bands of ATG5 as well as the corresponding bands of the loading control GAPDH are displayed. Bottom: Quantifications of band intensities of ULK1, LC3-II, p62, ATG7 (all n = 6 independent cultures), and the 56 and 30 kDa bands of ATG5 (n = 4–5 independent cultures) normalized to GAPDH as loading control. CTR control, RAP treated with rapamycin. Data are presented as single data points and means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, N.S. no significant difference, according to one-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test.