Fig 2: PM represses TGEV infection by degrading APN via PIK3C3-mediated autophagy. (A and B) ST cells transfected with PIK3C3 siRNA-4 or NC siRNA for 24 h were treated with PM (10 mM) and infected with TGEV (0.1 MOI) for 16 h. The APN, PIK3C3, ATG14, BECN1, P62, TGEV N, and LC3 were measured by western blot (A), and viral titers and TGEV N mRNA levels were determined by TCID50 and RT-qPCR (B). (C and D) WT ST cells and PIK3C3 KO ST cells were treated with PM (10 mM) and infected with TGEV (0.1 MOI) for 16 h. Then the APN, PIK3C3, ATG14, BECN1, P62, TGEV N, and LC3 were detected by western blot (C), and viral titers and TGEV N mRNA levels were determined by TCID50 and RT-qPCR (D), respectively. (E and F) WT ST cells and PIK3C3 KO ST cells transfected with pCMV-Myc and pCMV-Myc-PIK3C3, respectively, for 24 h were treated with PM (10 mM) and infected with TGEV (0.1 MOI) for 16 h. The APN, PIK3C3, P62, TGEV N, and LC3 were detected by western blot (E). TCID50 and RT-qPCR (F) were performed to detect viral titers and TGEV N m RNA level. All western blot results were calculated by Image J. Results are presented as mean ± SD of data from three independent experiments. ***, P ≤ 0.001.

Fig 3: Markers of AMPK activation. Mice (n = 3–5) were orally gavaged every 4 days for 28 days with TCDD prior to tissue collection. Protein levels were assessed in hepatic extracts using capillary immunoassay analysis. (A) Total AMPK and (B) phosphorylated AMPK (P-AMPK) and (C) the P-AMPK/AMP ratio two were assessed. Capillary immunoassay analysis of (D) total ATG14 and (E) phosphorylated ATG14 at serine 29 (P-ATG14 Ser29), a marker of P-AMPK activation. Bar graphs denote the mean ± SEM. Significance (*p ≤ 0.05) was determined using a one-way ANOVA followed by Dunnett’s post-hoc analysis. Plots were created using GraphPad Prism (v8.4.3).

Fig 4: ULK1 interacts directly with the PRMT5 complex independent of its role in autophagy. (A) Flp-In T-REx 293-GFP-ULK1 and Flp-In T-REx 293-GFP cells were stimulated with 0.1 μg/ml doxycycline for 18 h, followed by 1 h of starvation treatment with EBSS. After cytoplasm extraction (S100) GFP-IP was performed and analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against GFP, PRMT5, WD45, and pICln. (B) Flp-In T-REx 293-GFP-ULK1 and Flp-In T-REx 293-GFP cells were stimulated with 0.1 μg/ml doxycycline for 18 h, followed by treatment with full or starvation medium (EBSS) in the absence or presence of bafilomycin A1 (BafA1; 10 nM) for 1 h. Afterward, cells were harvested, lysed, and cleared cellular lysates were subjected to Tris/Glycine-SDS-PAGE and immunoblotting for GFP, ULK1 pS757, ATG14, ATG14 pS29, p62, LC3, GAPDH, and Actin. (C) Flp-In T-REx 293-GFP-ULK1, -GFP-ULK1/ΔCTD and -GFP cells were stimulated with 0.1 μg/ml doxycycline for 18 h. After cytoplasm extraction (S100) GFP-IP was performed and analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against FIP200, GFP, PRMT5, ATG13, WD45, pICln and ATG101. (D) HEK293T cells were stably transfected with pMSCVbsd-GFP-ULK1 kinase domain (k. dom.) and pMSCVbsd-GFP constructs. Additionally, HEK293T cells were transiently transfected with pcDNA5-FRT-TO-GFP-ULK1 GABARAP domain (GABARAP dom.) and -ULK1 C-terminal domain (CTD) constructs. After cytoplasm extraction (S100) GFP-IP was performed and analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against GFP, FIP200, ATG13, ATG101, PRMT5, WD45, and pICln. (E) S100 extract was generated of HEK293T cells and applied to a Superdex 200 increase column. Fractions were analyzed by Tris/Glycine-SDS-PAGE and immunoblotting using antibodies against ULK1, ATG13, ATG101, PRMT5, WD45 and pICln. (F) S100 extract of HEK293T cells was applied to a Superdex 200 increase column and subsequent immunoprecipitation of endogenous was performed with antibody against ULK1. Immunoprecipitation was analyzed by Tris/Glycine-SDS-PAGE using antibodies against ULK1 and pICln.

Fig 5: p62 bodies regulate autophagosome formation by a spatially concentrated PI3K kinase subunit.a TIRF images showing that droplets formed by LLPS of mCherry-p62 (50 μM), His-Ub8 (15 μM), PI3KC3-C1 (GFP-Becllin1) and GFP-ULK1 (1 μg/μl) fused upon contact in vitro. Scale bar, 5 μm. b Line profiling of a representative section of the p62 droplets, indicated by the white lines in panels (1)–(3) from (a). c Schematic diagram of the sedimentation assay to separate the condensed liquid droplets and the supernatant. In vitro phase separation assay of mCherry-p62 with linear His-Ub8. ULK1, PI3KC3-C1 and ATP were added after the phase separation. mCherry-p62, 50 µM; His-Ubx8: 15 µM; ULK1, (1 μg/μl); PI3KC3-C1, (1 μg/μl); ATP, 10 µM. d The sedimentation and supernatant were separated by centrifugation and analyzed by western blot using antibodies against p-Beclin1, Beclin1, p-ATG14, ATG14 and ULK1. S: supernatant, P: pellet. e The ratio of p-Beclin1 or p-ATG14 to total Beclin1 or ATG14 were analyzed from (d), Data are presented as mean ± SD, n = 3 times measurement of band intensity, p values were calculated using the two-tailed, unpaired t-test, ****p < 0.0001. f In vitro phase separation assay of GFP-p62 with linear His-Ub8, and then PI3KC3-C1 and ATP were added up to the phase separation system. Liposome and mCherry-FYVE were finally added into the above phase separation system. GFP-p62, 50 µM; His-Ub8: 15 µM; PI3KC3-C1, (1 μg/μl); ATP, 10 µM; Liposome, 1 µM; mCherry-FYVE, 1 µM. g Liposomes (PE-NDB) and mCherry-FYVE(X2) around the phase separated p62 bodies were observed from (c). Scale bar, 1 µm. h The number of mCherry-FYVE puncta colocalized with p62 droplet was quantified. Data are presented as mean ± SD, n = 3 independent experiments. w/o means without. p values were calculated using the two-tailed, unpaired t-test. ****p < 0.0001.