Fig 2: Cardiomyocyte-specific RORα KO mouse hearts have compromised mitochondrial function and abundancein vivo.A, TUNEL (red), DAPI (blue), and wheat germ agglutinin (green) staining of WT and CMKO hearts with summary TUNEL+ cells (%); oxidative stress as assayed by (B) MitoSOX and (C) DCF. D, swelling of isolated mouse heart mitochondria in calcium uptake assay. E, in vivo autophagic flux as measured by immunoblotting for LC3B 2 h after injection of BFA or vehicle. F, quantitative PCR for mitochondrial DNA (Cox1B, D-loop, and ND2) relative to nuclear DNA (Actb) by mouse age. G, fractional shortening as measured by echocardiography (n = 3–6 per group). H, fibrosis as assayed by Masson Trichrome staining. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by one-way ANOVA with Tukey's post hoc test (A–E) or unpaired t test (F–H). DCF, dichlorofluorescein; LC3B, microtubule-associated protein 1A/1B-light chain 3; ns, not significant; OD, optical density.

Fig 3: Level of autophagy-related protein expression in different cells was correlated with their cell motility.Protein expression was evaluated by western blotting. Cells (104 cells/Transwell) were seeded on matrigel-coated Transwell inserts, and incubated for 16 h. The migrated cells were stained with crystal violet and images were acquired at 40× magnification. (a) Protein expression of AhR, BNIP3, LC3, and ATG12-5 in A549, H1299, and CL1-5 cells. Full-length blots are presented in Supplementary Fig. S13. (b) Cell migration potential analysed by BafA1 treatment (5 nM) compared to the control among A549, H1299, and CL1-5 cells. (c) Protein expression of AhR, E-cadherin, and vimentin by BafA1 (5 nM) treatment of A549, H1299, and CL1-5 cells. Full-length blots are presented in Supplementary Fig. S14. (d) Immunofluorescence microscopy of LC3B (green) in A549 and CL1-5 cells following transfection with shAhR and pcDNA3.1-AhR vector. Nuclei were visualized by staining with Hoechst 33258 (blue). The number of puncta were quantified by Image ProTM, data were presented as percentages normalized to the control (100%). (e) Multifocal, well-demarcated, metastatic foci (arrow) were detected in the lung, there were no tumour detected in AhR and AhR + BafA1 groups. The metastatic tumour cells grew in a nest or sheet pattern and showed areas of glandular differentiation and papillary architecture (HE staining). The quantified data were analysed and expressed as the mean ± SD from three independent experiments. **P < 0.01; ***P < 0.001 compared to the control group.

Fig 4: Cortical ES treatment activated the PI3K/Akt pathway and suppressed autophagy to protect the brain from ischemia/reperfusion injury after tMCAO. Western blotting for PI3K, Akt, extracellular signal-regulated kinase (ERK), P62, and LC3B. (A) Representative pictures of Western blot. The upper panel represents the 2,3,5-triphenyltetrazolium chloride (TTC)-stained view of demarking core and penumbral region. (B,C) Phosphorylation of PI3K and Akt were significantly upregulated in ES groups. (D) There was no significant change in the phosphorylation of ERK. (E,F) In the ES group, there were increased P62 and reduced LC3B, indicating suppression of autophagy by ES (n = 5 for each group). *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Fig 5: IGFBP3 enhanced IR-induced apoptosis. a Cell cycle analysis of OEC-M1 PB and OEC-M1 PB-IGFBP3 cells at 6, 24, 48 and 72 h after exposure to 10 Gy IR. Lfet: Representative DNA histograms of flow cytometric cell cycle analysis based on propidium idodide (PI) uptake. Red line: non-irradiated cells; blue line: irradiated cells. Right: Percentage of cells at each phase of the cell cycle. b Apoptosis assay of OEC-M1 PB and OEC-M1 PB-IGFBP3 cells treated with 10 Gy IR using Annexin V and PI. Upper: A representative diagram of flow cytometric analysis with different quadrants indicating different stages of apoptosis (lower left quadrant: healthy; lower right: early apoptosis; upper right: late apoptosis). Lower: Percentage of apoptotic cells following IR. c Immunoblot of cytochrome c, cleaved caspase-3, and LC3B in OEC-M1 PB and PB-IGFBP3 cells at 48 and 72 h after exposure to 10 Gy IR. The cells treated with 10 μM cisplatin served as a positive control. Protein levels were normalized to expression of α-tubulin, an internal control. The ratios of LC3B II/I were determined by dividing the LC3B II (14 KDa) level by LC3B I (16 kDa) from each cell. d Effects of different doses of 3-MA (100 and 500 μM) and CQ (10 and 20 μM) on survival of ectopic IGFBP3 or vector expressing cells with or without irradiation. Cell viability was decided using the MTS assay at 48 and 72 h after irradiation. The relative cell viability was normalized to corresponding untreated control cells. Results from one of at least two independent experiments are shown. Values are expressed in mean ± SE. *p < 0.05; **p < 0.01; ***p < 0.001