Fig. 4

Quantification of LC3-positive autophagosomes in ATG4B knockout cells rescued with the phosphomimetic ATG4B S316D mutant. a Validation of CRISPR-mediated modification to the genomic locus of ATG4B exon 4 in the HAP1 ATG4B KO cell line. The gene structure of the WT human ATG4B locus on chromosome 2 is shown, and the region in exon 4 targeted for modification is revealed at the sequence level. The DNA sequence targeted by the custom sgRNA is shown in bold. The 16 bp deletion in the ATG4B KO clone results in a frameshift and early stop codon, as shown by the DNA sequence alignment. b Protein-level validation of ATG4B knockout. Lysates from untreated HAP1 control and ATG4B KO cells were run on a 4–20% polyacrylamide gel and immunoblotted with antibodies against ATG4B and beta-actin. The ATG4B KO cells showed a total absence of the specific band representing ATG4B. c ATG4B KO HAP1 cells were transfected with the indicated Halo constructs. After 24 h, cells were simultaneously stained live with a Halo ligand dye and treated with 250 nM Torin1 and 10 nM bafilomycin A1 for 3 h, prior to fixation and staining for endogenous LC3. Arrowheads indicate high Halo construct-expressing cells, and arrows indicate cells expressing low levels of these constructs. Scale bar 10 µm. d Quantification of the average number of autophagosome per cell in the high expressing cells is shown (Tukey’s plot, n = 10 fields, with an average of 5 quantified cells per field). ****p < 0.0001, NS not significant (Sidak’s multiple comparison test)