Fig 2: Truncated ATG4A has impaired functional capacity. (A) HeLa cells were transfected with GFP-LC3 and either wild-type WT-Flag-ATG4A or T-Flag-ATG4A for 24 h. GFP-LC3 puncta was quantified and presented in right panel as percentage of LC3-puncta positive cells (mean ± S.D., n ≥ 30 cells per condition). Scale bar denotes 10 μm. Statistical analysis was performed by unpaired Student’s t test. (B) HEK293T cells were transfected with GFP-GABARAP alone or in combination with WT-Flag-ATG4A, WT-Flag-ATG4B, or T-Flag-ATG4A. Co-immunoprecipitation (IP) was performed with anti-FLAG antibody and subjected to Western blot analysis. IgG light chain was probed as IP loading control. Results in this figure are representative of two to three independent experiments. (C) HeLa cells were transfected with either vector control or constructs encoding WT-3×Flag-ATG4A, truncated 3×Flag-T-ATG4A, or 3×Flag-mut-ATG4A for 24 h prior to CVB3 infection (MOI = 10, 5 h). Lysates were subjected to Western blot analysis for anti-VP1, Flag, and ACTB loading control and densitometry quantified on right panel (mean± S.D., n = 3).

Fig 3: Pre-lysosomal markers subsequent to ACL injury. Protein concentrations of Atg4A, Atg4B, Atg7, and Atg12-Atg5 were measured by Western blot analysis with supportive representative images. (A) Atg4A protein contents were decreased at 14 days compared to 7 days, and this reduction persisted until 56 days. (B) Atg4B protein contents were increased at 14- and 28 days compared to controls and decreased at 56 days. (C) Atg7 protein contents did not show significant change during the recovery period. (D) Atg12-Atg5 protein contents were unchanged. Data are presented as Mean ± SEM. * Significant difference (p < 0.05) versus CON. # Significant difference (p < 0.05) versus 7 days. § Significant difference (p < 0.05) versus 14 days. † Significant difference (p < 0.05) versus 28 days. ¶ Significant main effect of time regardless of sex (p < 0.05). ‡ Significant main effect of sex regardless of time (p < 0.05).

Fig 4: Endogenous Concentration of ATG4A has pro-viral function. (A,B) HeLa cells were transfected with control siRNA (siCON) or siRNA specifically targeting ATG4A for 48 h, followed by CVB3 (MOI = 10) or sham infection for 8 h. Western blot was conducted to analyze VP1 expression and verify knockdown efficacy of ATG4A (A, left). Level of VP1 was quantified by densitometry using Image J and presented as mean ± S.D., n = 3 (A, right). (B) RT-qPCR (left) and TCID50 assessment (right) were performed to determine viral RNA expression and viral titers in siCON- or si-ATG4A-treated cells and presented as mean ± S.D., n = 3. Statistical difference was determined by unpaired Student’s t-test. (C) Cell viability was assessed following 48 h treatment of either siCON or si-ATG4A using MTS assay and presented as relative cell viability (mean ± S.D., n = 6) (D,E) HEK293T cells were transfected with empty vector or Flag-ATG4A for 24 h. Cells were then infected with CVB3 (MOI = 100) or sham-infected for 8 h. Cell lysates were harvested and probed for Flag-ATG4A (with anti-Flag antibody), LC3, and ACTB (E, left). Densitometric analysis of VP1 (D, right) and LC3-II (E) protein levels was conducted via NIH Image J, normalized to ACTB and LC3-I, respectively and presented as mean ± S.D., n = 3. Statistical analysis of VP1 and LC3-II/LC3-I densitometry was performed by student t-test and ANOVA with Tukey post-hoc test respectively (F) HeLa cells transfected with either vector, wildtype (WT)- Flag-ATG4A, or truncated (T)-Flag-ATG4A for 24 h, followed by CVB3 infection (MOI = 10) for the indicated time-points. qPCR was performed with primers targeting VP1, normalized to ACTB gene, and presented as mean ± S.D., n = 3. Results in this figure are representative of three independent biological experiments.

Fig 5: Deficiency of NGLY1 causes inhibition of compensatory autophagy and aggrephagy, which can be rescued by transcriptionally active Nrf1. (A) Confocal images of FK2 labeled MEF ctrl, NGLY1KO, and NGLY1KO p110 cells after treating them with DMSO or 25 nM CFZ for 20 h. Recovery samples were washed out after CFZ treatment and incubated in fresh complete media for 20 h. Scale bar represents 20 μm. (B) Analysis of the percentage of cells with aggresomes under each condition from (5A) are plotted, based on the confocal microscopy analysis. Number of cells analyzed in each sample is noted underneath the images in B. (C) Western blot analysis of LC3B, and Nrf1 in MEF-ctrl, NGLY1KO, and NGLY1KO cells expressing p110 construct treated with DMSO, 200 nM BTZ (20 h), 60 μM CQ (3 h) or both BTZ (20 h) and CQ (3 h). β-Actin was used as a loading control. (D) Percent autophagic flux after normalizing LC3B-II levels to β-Actin levels from 8C. (E) Confirmation Western blot for Nrf1 protein expression in MEF NGLY1KO, NGLY1KO p110 overexpression, and NGLY1KO 9ND overexpression cells treated with CFZ (200 nM/4 h). α/β-Tubulin was used as the loading control. (F) Confirmation Western blot for GABARAPL1 protein expression in MEF NGLY1KO, and NGLY1KO GABARAPL1 over expression cells (GABAOE) cells. α/β-Tubulin was used for loading control. (G) MEF control (WT), NGLY1KO, NGLY1KO p110, and NGLY1KO 9ND cells were treated with 50 nM CFZ for 20 h, then washed and either treated with 60 μM CQ or fresh complete media for a 20 h recovery (R). Western blot analysis of ubiquitin after treatment and recovery, α/β-Tubulin was used for loading control. (H) MEF control (WT), NGLY1KO, NGLY1KO p110, and NGLY1KO p110-9ND cells were treated with 200 nM CFZ for 20 h. qRT-PCR analysis of ATG4A, GABARAPL1, CTSD, VPS37A, PSMD12, and PSMC4, mRNA levels were normalized to 18s rRNA. (n = 3 biological replicates). Error bars denote mean ± SD. Three biological replicates for qRT-PCR, microscopy and Western blotting were used. P values were calculated by either Student’s t test or two-way ANOVA. *<0.05, **<0.005, ***<0.0005 Source data are available for this figure: SourceData F8.