Fig 1: Mitochondrial translocation of p53 mediated BRD7-enhanced HSC ferroptosis. (A) HSC-LX2 cells with BRD7 knockout and knockin were exposed to erastin (10 µM) for 24 h. Then, the mitochondria, cytoplasm and nucleus were extracted by commercialized kits, respectively. The levels of p53 in cytoplasm, mitochondria, and nucleus were determined by Western blot (n = 3 in every group). (B) HSC-LX2 cells with BRD7 knockin were exposed to erastin (10 µM) for 24 h. The co-localization of p53 (red fluorescence) and mitochondria (mitotracker green) was determined by confocal imaging. Scale bars are 100 µm. Representative photographs were shown (n = 3 in every group). (C) HSC-LX2 cells with BRD7 knockout and knockin were exposed to erastin (10 µM) for 24 h. The expression of total p53 and phosphorylated p53 was detected by Western blot (n = 3 in every group). Serine 392 was substituted by a non-phosphorylatable alanine (S392A mutant) with CRISPR/Cas9 system in HSC-LX2 cells with BRD7 knockin. Then, HSC-LX2 cells were exposed to erastin (10 µM) for 24 h. (D) The levels of p53 in mitochondria were examined by Western blot (n = 3 in every group). (E) Cell viability were determined by CCK8 kit (n = 3 in every group, **P < 0.01, N·S., not significant). (F) The levels of iron, lipid ROS, GSH, and MDA were determined by commercial kits (n = 3 in every group, **P < 0.01, ***P < 0.001). (G) HSC-LX2 cells were transfected with p53 shRNA and BRD7 plasmid, and were exposed to erastin (10 µM) for 24 h. The levels of iron, lipid ROS, GSH, and MDA were determined by commercial kits (n = 3 in every group, **P < 0.01). (H) HSC-LX2 cells were exposed to erastin (10 µM), RSL3 (2.5 µM), and BSO (200 µM) for 24 h. The combination of BRD7 and p53 was examined by immunocoprecipitation (n = 3 in every group). (I) The full-length and different domains of p53 with myc tag were constructed, and then were cotransfected with pcDNA3.1-BRD7 into HSC-LX2 cells. Cell lysates were immunoprecipitated with anti-myc antibody and blotted with anti-BRD7 antibody (n = 3 in every group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: Immunofluorescence staining showing the changes in expression levels of apoptosis-related proteins in retina tissues of diabetic mice treated with FA or not.(A–I) Representative images of immunofluorescence staining. Quantitative results of P53 (J), BAX (K), Bcl2 (L) and cleaved caspase-3 (M) in retina of diabetic mice based on integrated optical density value. (N) Representative images of western blot results. The protein expression levels of P53 (O), BAX (P), Bcl2 (Q) and cleaved caspase-3 (R) in each group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Three mice were used and three sections of each mouse were counted. FA, Ferulic acid. Scale bar = 20 µm.
Fig 3: SIRT1-mediated deacetylation maintains fenestrae in HSECs via inhibiting progerin-associated premature senescence. Freshly primary HSECs, isolated from normal rats and cultured in vitro, were transfected with the SIRT1 adenovirus vector to overexpress SIRT1 (called AV-SIRT1) or nontarget adenovirus vector (called AV-CTR), and then treated with H2O2 (10 µM) for two days. (A) The SA-ß-Gal activity in HSECs on Day 2 in the four groups (AV-CTR, AV-CTR + H2O2, H2O2 + AV-SIRT1, AV-SIRT1) was observed by SA-ß-Gal staining (Scale bar: 25 µm). The black triangles indicated the SA-ß-Gal-positive cells. The SA-ß-Gal-positive cells were quantified in the graph, right. * P < .05 vs the AV-CTR group; # P < .05 vs the AV-CTR + H2O2 group. (B) Primary HSECs were extracted nuclear and cytoplasmic protein and were detected their protein levels. Representative immunoblots of SIRT1, Ac p53 K381, total p53, progerin, Lamin A/C and Lamin B1 in nuclei and cytoplasm of HSECs in four groups (AV-CTR, AV-CTR + H2O2, AV-SIRT1 + H2O2, AV-SIRT1). (C) Interaction of progerin with Ac p53 K381 was detected by the co-IP assay. Progerin of HSECs was individually immunoprecipitated, as well as Ac p53 K381 and progerin subjected to immunoblotting analysis as indicated. (D) The immunocytochemical co-localization of Ac p53 K381 (green) with progerin (red) and F-actin (purple) in primary HSECs on Day 2 in four groups (AV-CTR, AV-CTR + H2O2, AV-SIRT1 + H2O2, AV-SIRT1), visualized by confocal microscopy (Scale bar: 5 µm). Nuclear was showed by DAPI (blue). (E) Magnification of SEM of HSECs in the four groups (AV-CTR, AV-CTR + H2O2, AV-SIRT1 + H2O2, AV-SIRT1), revealing fenestrae structures in HSECs (Scale bar: 2 µm). The black triangles indicated fenestrae in HSECs. The total fenestral diameter was quantified in the graph, right. * P < .05 vs the AV-CTR group; # P < .05 vs the AV-CTR + H2O2 group
Fig 4: Expression of ASK1 and downstream targets following NQDI-1 treatment in vivo. After 250 nmol NQDI-1 treatment for 48 h, the brain cortex was perfused and collected. (A and B) Western blotting detection of ASK1 expression in the sham, HI, DMSO- and NQDI-1-treated brain cortex (n=4; **P<0.01 compared with the sham controls; ##P<0.01 compared with the HI group). (C) Immunofluorescence detection of ASK1 in the sham, HI, DMSO- and NQDI-1-treated brain cortex (n=3; magnification, ×200). DAPI was used to indicate the cell nucleus. (D and E) Western blotting detection of p-JNK, p-c-Jun, p53 and Caspase 3 expression in sham, HI, DMSO- and NQDI-1-treated brain cortex (n=4; **P<0.01 compared with the sham controls; ##P<0.01 compared with the HI group). (F) Immunofluorescence detection of p-JNK in sham, HI, DMSO-and NQDI-1-treated brain cortex (n=3; magnification, ×200). DAPI was used to indicate the cell nucleus. p-, phosphorylated; ASK, apoptosis signal-regulating kinase 1; HI, hypoxia-ischemia; DMSO, dimethyl sulfoxide; DAPI, 4',6-diamidino-2-phenylindole; JNK, c-Jun N-terminal kinase.
Fig 5: Expression of ASK1-associated downstream targets in the brain of the rat HI model. (A and B) Western blotting detection of p-JNK and JNK expression in the sham and HI model at 6, 12, 24 and 48 h after brain insult. GAPDH was used as a loading control (n=4; **P<0.01). (C) Immunofluorescence detection of p-JNK in the brain of the sham and HI model at 6, 12, 24 and 48 h after brain insult. DAPI was used to indicate the cell nucleus (magnification, ×200). (D and E) Western blotting detection of p-c-Jun, c-Jun, p53 and Caspase 3 in the sham and HI model at 6, 12, 24 and 48 h after brain insult. GAPDH was used as a loading control (n=4; **P<0.01). ASK, apoptosis signal-regulating kinase 1; HI, hypoxia-ischemia; DAPI, 4',6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein.
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