Fig 2: Exposure of HCE-T cells to CSE resulted in aberrant autophagic ferritin processing and accumulation of a cleaved form of ferritin. (A,B) HCE-T cells were exposed to CSE for the indicated times. Protein lysates were then analyzed by immunoblotting using antibodies directed against ferritin and ß-actin (loading control). (B) Quantitation of the fold increase of each form of ferritin (heavy chain, black arrowheads; light chain, white arrowheads; cleaved form, arrowheads filled with diagonal lines) after exposure to CSE. (C–E) HCE-T cells were incubated without (control) or with 5% CSE in the absence or presence of either 25 µM chloroquine (CQ) or 10 nM bafilomycin A1 (BafA1) for 24 h. Protein lysates were then analyzed by immunoblotting using the indicated antibodies. (D,E) show the quantification of ferritin (the cleaved form) and p62, respectively, both normalized to ß-actin. (F) Representative images of intracellular ferritin distribution in HCE-T cells with or without 5% CSE exposure for 12 h. The cells were stained with anti-ferritin antibody (green), LysoTracker Red (red), anti-p62 antibody (cyan), and Hoechst 33342 (blue). The boxed regions are enlarged at the upper right corners. The arrows represent p62 and LysoTracker Red double positive organelles, which comprise autolysosomes. The arrowheads show the autolysosomes that contain ferritin. Bar = 20 µm. The data are represented as the mean ± SEM (n = 6). *P < 0.05, Student’s t-test vs. Control (Cont); #P < 0.05, ##P < 0.005, Student’s t-test vs. CSE.

Fig 3: Exposure of HCE-T cells to HTPs induced cell death and accelerated ferritin cleavage. (A) Representative images of PI (magenta) and Hoechst 33342 (blue) staining after exposure to HTPs in the presence or absence of 1 mM NAC. (B) The bars show the PI-positive cell ratios. (C) Cell viability was measured with a CCK-8 assay. (D) Immunoblotting of HCE-T cell extracts exposed to HTPs. (E) Quantitation of the fold increase of each form of ferritin (heavy chain, black arrowheads; light chain, white arrowheads; cleaved form, arrowheads filled with diagonal lines) after exposure to HTPs. (F) Representative images of PI (magenta) and Hoechst 33342 (blue) staining after exposure to nicotine-free HTPs (NF-HTPs). (G) The bars show the PI-positive cell ratios. (H) Cell viability was measured with a CCK-8 assay. (I–K) Immunoblotting of extracts derived from HCE-T cells incubated without (control) or with either CSE (5%), HTPs (1 or 5%), or NF-HTPs (1 or 5%) for 24 h was conducted using the indicated antibodies. (J,K) show the cleaved form of ferritin and p62 (both normalized to ß-actin), respectively. Bars = 100 µm (A,F). The data are represented as the mean ± SEM (n = 6). *P < 0.05, **P < 0.005, ***P < 0.001, Student’s t-test vs. Control (Cont); †P < 0.05, ††P < 0.005, †††P < 0.001, Dunnett’s test vs. Control (Cont); ###P < 0.001, Student’s t-test vs. 10% HTPs or NF-HTPs.

Fig 4: Mitophagy is limited in cardiomyocyte-specific RORa KO mouse hearts.A, quantitative RT-PCR (qRT-PCR) for Cre, RORa, and ROR? genes in the heart, liver, and kidney from CMWT and cardiomyocyte-specific RORa KO mice (n = 3 per group). B, qRT-PCR for RORa in the heart tissue from CMWT and CMKO mice exposed to normoxia (21% O2) or to hypoxia (10% O2) for 8 h. C, immunoblotting from WT and CMKO heart lysates with summary densitometry (ImageJ). D, representative LC3B immunofluorescence images of mouse heart sections (=3 per mouse) and ImageJ summary quantification. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired t test (A) or one-way ANOVA with Tukey's post hoc test (B–D). Atg7, autophagy related 7; Atg10, autophagy related 10; Bnip3, BCL2 interacting protein 3; Cre, Cre recombinase; DAPI, 4',6-diamidino-2-phenylindole; Hif1a, hypoxia inducible factor1 alpha; Hsp60, heat shock protein 60; LC3B, microtubule-associated protein 1A/1B light chain 3; PINK1, PTEN-induced kinase 1; p62, SQSTM1.