Fig 2: Effect of NLRC5 over-expression on autophagy in EESCs (n = 7). (A) Representative image of immunofluorescence staining showing NLRC5 and LC3, Beclin1 are present both in the cytoplasm and nucleus, NLRC5 and LC3, Beclin1 were co-located in the nucleus. Photographs were taken at magnifications of 1600×. (B, C) Representative western blotting and qRT-PCR results showing over-expression of NLRC5 by transfection with NLRC5 plasmid significantly promoted NLRC5, LC3 Beclin1 expressions, and inhibited p62 expression when compared with vector group (**P < 0.01 vs. vector group). (D) Representative images showing LC3 staining in EESCs infected with GFP-RFP-LC3 adenovirus, over-expression of NLRC5 by transfection with NLRC5 plasmid significantly promoted yellow puncta when compared with vector group. Photographs were taken at magnifications of 1,600×, quantification of mean yellow puncta of 10–15 cells per condition is shown (**P < 0.01 vs. vector group). (E) Representative transmission electron microscopy (TEM) showing over-expression of NLRC5 by transfection with NLRC5 plasmid significantly promoted autophagosomes formation when compared with vector group, autophagosomes were highlighted by red arrows (left scale bar: 1µm; right scale bar: 2µm; **P < 0.01 vs. vector group). The expression levels of mRNA were normalized with respect to ß-actin and were calculated using the 2-??Ct method. The protein expression levels were quantified by Image J software and normalized to ß-actin protein levels. The results are represented as the mean ± SEM from at least three independent experiments.

Fig 3: Isorhamnetin suppressed ZEA-induced apoptosis. Ovarian GC were cultured with isorhamnetin (20 µmol/L) for 2 h, before treatment with ZEA (60 µmol/L) for 24 h. (A) Structure of isorhamnetin. (B) The viability of ovarian GC treated with ZEA for 24 h. (C) The effect of pretreatment with different concentrations of isorhamnetin on the viability of ZEA (60 µmol/L)-exposed ovarian GC. (D) Western blot analysis showed Bcl-2, Bax and C-Casp3 expression in ovarian GC treated with ZEA or isorhamnetin. (E to G) Relative quantification (Western blot) of the protein expression of Bcl-2, Bax and C-Casp3 normalized to ß-actin expression. (H) Representative images of mitochondrial distribution in ovarian GC treated with ZEA or isorhamnetin. Scale bars: 200 µm. (I) Western blot analysis of PGC-1 in ovarian GC treated with ZEA or isorhamnetin. (J) Relative quantification (Western blot) of the protein expression of PGC-1 normalized to ß-actin expression. (K) Western blot analysis of LC3 and P62 in ovarian GC treated with ZEA or isorhamnetin. (L to M) Relative quantification (Western blot) of the protein expression of LC3 and P62 normalized to ß-actin expression. Data are expressed as means ± SEM. Different letters mean significant differences (P < 0.05). ZEA = zearalenone; GC = granulosa cell; Bcl-2 = b-cell lymphoma-2; Bax = bcl2-associated x; C-Casp3 = cleaved-caspase-3; PGC-1 = ppar? co-activator-1; LC3 = microtubule-associated protein 1 light chain 3; P62 = the sequestosome 1 protein.

Fig 4: A and B, Bazedoxifene attenuated IL-6-stimulated cell hypertrophy in H9c2 myoblasts (A, scale bar, 20 µm). Bazedoxifene down-regulated the transcripts of ANP and BNP induced by IL-6 (B, n = 4). *P < .05 vs ctrl group; # P < .05 vs IL-6 group without Bazedoxifene. C and D, Western blots with quantification showing the expression of p-STAT3 in H9c2 cells stimulated by IL-6 of 25 ng/mL at serial time-points as indicated. E and F, after serum starvation H9c2 cells were treated without or with Bazedoxifene (10, 15, 20 µmol/L) for 2 h and then administrated with IL-6 (25 ng/mL) in presence of Bazedoxifene for another 30 min. Bazedoxifene inhibited IL-6-induced STAT3 activation. G and H, Treated without or with Bazedoxifene (10, 15, 20 µmol/L) for 2 h, H9c2 cells were stimulated by IL-6 (25 ng/mL) for another 30 min. IL-6 trigger decreased ratio of LC3-?/LC3-? and promoted P62 expression. Bazedoxifene increased the ratio and down-regulated expression of P62 in H9c2 cells. Quantitative data of p-STAT3 or P62 were normalized to those of GAPDH. The conversion of LC3 was quantified by the ratio of LC3-?/LC3-?. Data of control group were set arbitrarily as 1. Data represent means ± SEM. *P < .05 vs ctrl group; # P < .05 vs IL-6 group without Bazedoxifene

Fig 5: Depletion of PARP-1 alone significantly increases the frequency of micronuclei and blocks the ST-dependent autophagy. (a and b) After transfection with p62 siRNA for 48 h, 786-O cells were incubated with ST for 3 h, lysed, and the lysates were precipitated using the antibody against ?-H2AX or Rad51. The immunoprecipitates were resolved by electrophoresis and probed by immunoblotting with the indicated antibodies. (c–f) 786-O cells were transfected with siRNA target PARP-1 for 48 h. (c) Following treatment with ST with or without CQ for 12 h, images were obtained using fluorescence microscopy, and the histogram graph data representing the mean±S.D. were shown in (d), **P<0.01 versus control. (e and f) Cell lysates were prepared and immunoblotting was performed with the indicated antibodies for 3 h. The data represent three independent experiments. **P<0.01, and ns was short for insignificance