Fig 2: Poly-ubiquitination is required for increased autophagy in Bassoon KO neurons.(A–B) Representative images of hippocampal neurons from WT and Bsn KO mice expressing FU-RFP-LC3 only (A) or FU-RFP-LC3 and FU-UbK0 (B) that were fixed and stained with antibodies against Synaptophysin1 (Syp). FU-UbK0 vector co-expresses a soluble eGFP that enables the identification and quantification of axons from infected neurons only. (C–E) Quantification of the number of RFP-LC3 puncta per 10 µm axon (C) (WT = 0.50 ± 0.063, n = 46 axons, 3 independent experiments; Bsn KO = 1.10 ± 0.108, n = 47 axons, 3 independent experiments; WT + UbK0 = 0.70 ± 0.091, n = 40 axons, 3 independent experiments; Bsn KO + UbK0 = 0.55 ± 0.055, n = 40 axons, 3 independent experiments; p****<0.0001, p**=0.0060, p****<0.0001), the number of Syp1 puncta per 10 µm axon (D) (WT = 1.68 ± 0.109, n = 46 axons, 3 independent experiments; Bsn KO = 1.33 ± 0.100, n = 47 axons, 3 independent experiments; WT + UbK0 = 1.52 ± 0.153, n = 40 axons, 3 independent experiments; Bsn KO + UbK0 = 1.48 ± 0.102, n = 40 axons, 3 independent experiments) and the colocalization of RFP-LC3 and Syp1 (E) (WT = 9.23 ± 1.809, n = 46 axons, 3 independent experiments; Bsn KO = 38.19 ± 4.211, n = 47 axons, 3 independent experiments; WT + UbK0 = 16.04 ± 3.269, n = 40 axons, 3 independent experiments; Bsn KO + UbK0 = 16.82 ± 3.014, n = 40 axons, 3 independent experiments; p****<0.0001, p****<0.0001, p****<0.0001). Scale bars: 5 µm. Error bars represent SEM. Data points represent axons. ANOVA Tukey’s multiple comparisons test was used to evaluate statistical significance.

Fig 3: Beclin1 and LC3 are elevated in human abdominal aortic aneurysm (AAA) samples. (A) qPCR analysis of Beclin1 in human control versus AAA tissue (n = 10 samples/group). *P = 0.0015 using student's t‐test. (B) qPCR analysis of LC3 in human versus AAA tissue (n = 10 samples/group). *P = 0.0023 using a student's t‐test. (C) Immunohistochemical staining of Beclin1, LC3, ATG5, and ATG9 in human control versus AAA samples (N = 6 samples/group). IgG staining as controls for each stain and Verheoff Van Geisen stain to depict aneurysm morphology. Each stain was graded by two individual reviewers and then quantified using a student's t‐test. (D and E) Quantification of Western blot of human Beclin and LC3 (n = 4/group) using a student's t‐test.

Fig 4: mRNA expression of HIF-1α (a), LC3 (b), ATG 5 (c), mTOR (d), Beclin 1, (e) and p62 (f) in the lung tissue of rats. Group A: Control; Group B: OVA-sensitized rats; Group C: OVA-sensitized rats stimulated with cold conditions; Group D: intervention with XQLT in OVA-sensitized stimulated with cold conditions. ∗P < 0.05 and ∗∗P < 0.01, compared with Group A; #P < 0.05, ##P < 0.01, compared with either Group B or C XQLT: Xiaoqinglong decoction. OVA: ovalbumin.

Fig 5: Curcumin‐induced cellular ferroptosis was alleviated by CQ. (a) Cell viability was measured after 30 μmol/L curcumin and 20 μmol/L CQ treatment for 24 hours. (b) Representative cell morphological changes in each group of cells (scale bar = 200 μm). (c) Effects of curcumin and CQ on autolysosome and ultrastructural features of mitochondrion in A549 and H1299 cells (scale bar = 10 μm). (d) The effect of CQ on protein levels of P62, Beclin1, LC3, ACSL4, SLC7A11, and GPX4 in curcumin‐treated A549 and H1299 cells. (e) Semi‐quantitative analysis of immunoblotting results. (f)–(h) Effects of curcumin and CQ on MDA, SOD, GSH, and iron content in A549 and H1299 cells. All results are expressed as the mean ± SD. *P < 0.05, **P < 0.002, ***P < 0.0002, ****P < 0.0001 versus curcumin group