Fig 2: Effects of FMOD on autophagy and apoptosis in chicken granulosa cells. (A) Relative FMOD mRNA expression levels in normal follicles and atresia follicles. (B) Relative mRNA expression levels of FMOD in ctrl and FMOD silenced cells. (C) Relative mRNA expression levels of PRLR, FSH-β and LH-β in ctrl and FMOD silenced cells. The histogram below shows the gray value analysis of protein. (D) Western blot analysis FMOD protein levels in ctrl and FMOD silenced cells. (E) Relative mRNA expression levels of Belin1, ATG5 and ATG7 in ctrl and FMOD silenced cells. (F) Western blot analysis the expression protein levels of LC3B and Beclin1 in ctrl and FMOD silenced cells. The histogram on the right shows the gray value analysis of protein. (G) Relative mRNA expression levels of caspase-3, caspase-8 and caspase-9 in ctrl and FMOD silenced cells. (H) Apoptosis rates in ctrl and FMOD-silenced cells. (I) The ultrastructure of apoptotic bodies was observed by electron microscopy. (J) Western blot analysis the expression protein levels of caspase3 and capase8 in ctrl and FMOD silenced cells. The histogram below shows the gray value analysis of protein. Date represent mean ± S.E. n = 3 independent cell cultures in each group. *P < 0.05, **P < 0.01.

Fig 3: Irisin prevents cell apoptosis in the OIR retinae. (A) Representative images of TUNEL staining in the retinae of normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin mice. (B) The quantitative analysis of TUNEL-positive cells related to A. (C) Western blot analysis and quantification of active caspase 3 expression in normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin retinae at P17. (D) qPCR analysis of TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) mRNA in normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin retinae at P17. Data were normalized to the normoxia + saline and OIR + saline groups. β-Actin was used as an internal control. (E) Western blot analysis and quantification of NF-κB and pNF-κB protein in the retinae from normoxia + saline, normoxia + irisin, OIR + saline, and OIR + irisin mice upon P17 (n = 3–4). *P < 0.05. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Fig 4: Apoptotic effects of TSN on MKN-45 cells. (A) MKN-45 cells were seeded into 6-well plates and treated with varying concentrations of TSN for 48 h, incubated with acridine orange, and visualised using laser confocal microscope. (B and C) Early apoptotic effects of TSN were evaluated by flow cytometry analysis after staining with annexin V-FITC and PI. (D) The mRNA expression of Caspase-3, 8, 9, BAX, BCL2, FAS, Cyt c and APAF1 was detected using reverse transcription quantitative PCR in TSN-treated MKN-45 cells. (E and F) Protein expression levels of Caspase-3, 8, 9, Bax, BCL2, Fas, Cyt c and APAF-1 were detected using western blotting in TSN-treated MKN-45 cells. ß-actin was used as an internal control. All results are expressed as the mean ± SEM of three independent experiments; n=3; *P<0.05, **P<0.01, ***P<0.001 vs. 0 nM. Cyt c, cytochrome c; TSN, toosendanin; APAF1, apoptotic protease activating factor 1; 5-FU, fluorouracil.