Fig 2: Pre-primed LC3B is lipidated, localizes to autophagosomes, and can restore endogenous SQSTM1 turnover when expressed at high levels in ATG4B KO cells. (a) HAP1 control and ATG4B KO cells transfected with 3xFLAG-LC3B-G120 using PEI were treated with combinations of DMSO or 250 nM Torin1 with or without 10 nM baf A1 for 3 h prior to lysis and western blot analysis. (b) Immunocytochemistry of endogenous SQSTM1 and LAMP1 in HeLa control and ATG4B KO cells stably expressing GFP-LC3B-G120 under control of the CMV promoter. Cells were treated for 3 h with combinations of DMSO or 250 nM Torin1 with or without 10 nM baf A1 for 3 h prior to fixation and staining. Yellow boxes show region-of-interest in the Torin1 + baf A1 treated condition that is enlarged in the panels below. Scale bar: 10 µm. (c) Assessment of colocalization between endogenous SQSTM1 and LAMP1 in HeLa control and ATG4B KO cells stably expressing CMV promoter-driven GFP-LC3B-G120 using single-plane confocal images as represented in Figure 6(b). Bars indicate mean and error bars show standard deviation (n = 10 randomly-selected cells per condition). **** P = 0.0001, n.s. (not significant) P > 0.05 (Sidak’s multiple comparison test). (d) CLEM of CMV-driven GFP-LC3B-G120 stably expressed in HAP1 control and ATG4B KO cells. Cells were treated for 3 h with 250 nM Torin1 + 10 nM baf A1 prior to fixation. Upper panels show low magnification images with a boxed region-of-interest (ROI); scale bar: 10 µm. Lower panels show enlarged ROI, with yellow arrowheads indicating GFP-positive autophagic structures; scale bar: 1 µm. Confocal microscopy image corresponds to a single confocal slice.

Fig 3: Identification and characterization of LC3/GABARAP conjugates in ATG4-deficient cells. A, expression of pre-primed 3xFLAG-tagged LC3/GABARAP isoforms (LC3A G120, LC3C G126, GABARAP G116, GABARAPL1 G116, and GABARAPL2 G116) and LC3B mutants in control, ATG4B KO, and ATG4A/B DKO HeLa cells using transient transfection prior to Western blot analysis. Panels are vertically divided for presentation; all results are from the same membrane and displayed at the same exposure. B, digestion of 3xFLAG–LC3B conjugates from HeLa ATG4B KO cells. Lysates were prepared in IP buffer and subsequently incubated on ice (untreated samples) or at 37 °C for 1 h with purified recombinant GST–ATG4B WT or inactive (C74S) mutant at a final concentration of 0.02 mg/ml, prior to Western blotting. ATG4B antibody was used to detect GST–ATG4B. C, assessment of GFP–LC3B conjugates following autophagy modulation. HeLa control and ATG4B KO cells stably expressing GFP–LC3B G120 were treated with combinations of EBSS, DMSO, 250 nm Torin1, and 10 nm baf A1 for 3 h prior to lysis and Western blot analysis. Endogenous LC3B was detected using anti-LC3B antibody, and GFP–LC3B conjugates were detected using anti-GFP. D, cell fractionation and GST–ATG4B protease protection assessment of 3xFLAG–LC3B conjugates. HAP1 ATG4B KO cells transiently transfected with 3xFLAG–LC3B G120 were treated for 3 h with 250 nm Torin1 and 10 nm baf A1 prior to sample preparation, processing, and analysis by Western blotting (left). 3xFLAG–LC3B G120 was detected using anti-FLAG antibody, and endogenous LC3B was detected using anti-LC3B (consisting of pro-LC3B in untreated samples). On right is a schematic highlighting how the presence of detergent (Triton) disrupts protease protection as a positive control.

Fig 4: Development of deconjugation-resistant LC3B mutant reveals novel LC3B conjugates in cells. A, LC3B priming assay in HeLa cells using transiently transfected 3xFLAG–LC3B–GFP construct and point mutants. Western blotting of lysates using anti-FLAG antibody is shown on the left-hand side (asterisk indicates nonspecific cleavage product) and schematic of construct processing by endogenous ATG4 in cells shown on right. B, confocal microscopy of GFP localization in HAP1 control cells transiently transfected with GFP–LC3B constructs and treated with DMSO or 250 nm Torin1 + 10 nm baf A1 for 3 h prior to fixation. Point mutations were introduced into the pre-primed G120 form of GFP–LC3B where indicated. Scale bar, 10 µm. C, location of LC3B Gln-116 residue within the binding pocket of ATG4B visualized using co-crystal structure PDB code 2Z0E (42), with LC3B displayed as a ribbon in yellow and ATG4B as surface model in green (left-hand side). Boxes show close up of H-bond interactions between LC3B Gln-116 and ATG4B residues Asn-146, Ile-317, and Leu-228 for WT (middle) and modeled Q116P mutation (right). D, 3xFLAG–LC3B constructs (schematics shown on left) were transiently expressed in HeLa control cells prior to lysis and Western blotting detection using anti-FLAG antibody (right).

Fig 5: MST4-induced p-ATG4B Enhances Autophagic Activity and GBM Tumorigenicity(A) IP-IB or IB analysis of MST4, ATG4B, p-ATG4B, LC3B, p62, and ß-actin in GSC 1123 and JK83 expressing sh-control (C), sh-MST4 (M), and sh-ATG4B (A).(B) Measurements of ATG4B activity in GSC 1123 or JK83 with or without knockdown of MST4 or ATB4B using an FRET-LC3B fusion protein as a substrate (left), and quantification of cleaved FRET-LC3B substrates (right). Red arrow, FRET-LC3B fusion protein; black arrows, cleaved CFP-LC3B and YFP.(C) IF analyses (left) and quantification (right) for LC3B in GSC 1123 and JK83 without or with knockdown of MST4 or ATG4B. Arrows point to LC3B puncta.(D) Transmission electron microscopy analyses for autophagic vacuoles in GSC 1123 with or without knockdown of MST4 or ATG4B (left), and quantification of autophagosomes in GSCs (right). Arrows point to autophagic vacuoles.(E) IB of GSC 1123 transduced with sh-C or sh-MST4, reconstituted with vector (Vec), WT ATG4B, or the S383D ATG4B mutant.(F) Limiting dilution neurosphere-forming assay in GSC 1123 with indicated modifications.(G) In vivo BLI of GBM xenografts derived from luciferase-expressing GSC 1123 with indicated modifications. Colored scale bar represents photons/s/cm2/steradian.(H) Bar graph for tumor volume formed by indicated GSC 1123 cells in mouse brains. n = 5.(I) IB analysis of MST4, ATG4B, p-ATG4B, LC3B, p62, and ß-actin in GSC JK16 with or without MST4 knockdown and transfected with a control vector, WT ATG4B, or the S383D ATG4B mutant.(J) IF analyses (left) and quantification (right) for LC3B in GSC JK16 with indicated modification.(K) Limiting dilution neurosphere-forming assay in GSC JK16 expressing indicated plasmid.Data are representative of two to three independent experiments with similar results. All bar plot data are means ± SEM. **p < 0.01, ***p < 0.001. Scale bars, 100 µm (C), 1.0 µm (D), and 1 mm (J). See also Figure S4.