Validation and functional characterization of genome-edited clones expressing HaloTagged autophagy factors. (A and B) PCR analysis from genomic DNA of genome-edited clones for verifying the correct insertion of the HaloTag at the autophagy loci. For amplifying the insertion, primers outside the homology arms region were designed. The edited clones show an expected shift of ∼2 kb on the PCR product, corresponding to the 3xFlag-HaloTag insert. (C) PCR analysis from genomic DNA of genome-edited clones for verifying the correct insertion of the HaloTag in the high GC-rich ULK1 and WIPI2 gene loci. For amplifying the insertion, primers outside the homology arms and inside the 3xFlag-HaloTag regions were designed. The edited clones show a PCR product, which is absent in the parental U2OS cell line. (D) Western blots for determining the expression levels of the HaloTagged autophagy proteins relative to the wildtype protein before and after removal of HaloTag using the TEV protease. (E) Quantification of the Western blots (A), showing the ratio between HaloTag and parental cell line (N = 3, mean ± SD). (F) Western blot analysis of LC3 levels in parental and genome-edited cell lines in control (Ctr) and upon treatment with rapamycin (R, 100 nM for 2 h), or rapamycin + bafilomycin (R+B, 100 nM each, for 2 h). Source data are available for this figure: SourceData FS1.