Fig 2: Loss of MLL1 alleviated neuron apoptosis by inhibiting apoptosis signal regulating kinase 1 (ASK-1)/tumor necrosis factor α (TNF-α) complex formation. (A) Western blot showed that OGD significantly upregulated the expressions of MLL1, total ASK-1, pASK-1, and cleaved caspase-3 in primary cortical mixed cells, while these effects could be blocked by siMLL1 and siASK-1. (B–E) Immunoprecipitation showed that no significant difference was found in the formation of ASK-1/STRAP/14-3-3 complex among different groups, while OGD significantly enhanced the formation of ASK-1/TNF-α complex in primary cortical mixed cells, and this effect could be blocked by siMLL1. (F) Flow cytometry analysis showed that OGD promoted the apoptosis of primary cortical mixed cells, while this effect could be blocked by siMLL1. Data are presented as mean ± SD, *P < 0.05, **P < 0.001. OGD was performed and lasted for 24 h.

Fig 3: Gain of MLL1 promoted neuron apoptosis by enhancing apoptosis signal regulating kinase 1 (ASK-1)/tumor necrosis factor α (TNF-α) complex formation. (A) Western blot showed that overexpression of MLL1 and ASK-1 significantly upregulated the expressions of total ASK-1, pASK-1, and cleaved caspase-3 in primary cortical mixed cells, while siASK-1 blocked the effects induced by MLL1 overexpression. (B–E) Immunoprecipitation showed that no significant difference was found in the formation of ASK-1/STRAP/14-3-3 complex among different groups, while MLL1 overexpression significantly enhanced the formation of ASK-1/TNF-α complex in primary cortical mixed cells. (F) Flow cytometry analysis showed that MLL1 overexpression promoted the apoptosis of primary cortical mixed cells. Data are presented as mean ± SD, **P < 0.001. OGD was performed and lasted for 24 h.

Fig 4: Serum MLL1 level was significantly correlated with the severity of IS. (A,B) MLL1 level in MCAO mouse serum, ischemic core, and ischemic penumbra were all significantly higher than that in the control mouse. (C–G) MLL1 level in MCAO mouse serum was positively correlated with that in the ischemic core and penumbra, and it was also positively correlated with infarct volume and neurological score. (H) No difference was found in serum MLL1 level among patients admitted to hospital at different times after IS onset. (I) Serum MLL1 level of IS patients was significantly higher than that of control. (J–L) Serum MLL1 level was positively correlated with NIHSS score, infarct volume, and serum high-sensitivity C-reactive protein (Hs-CRP) level of IS patients. Data are obtained using qPCR and presented as mean ± SD, *P < 0.05, **P < 0.001. Mice were euthanized at 24 h after MACO. There were 10 mice in each group.

Fig 5: Apoptosis signal regulating kinase 1 (ASK-1) expression was regulated by mixed lineage leukemia 1 (MLL1). (A) Spatial expression pattern of MLL1 in MCAO mouse analyzed by quantitative PCR (qPCR). (B) Immunohistochemistry showed that the expression of MLL1 increased significantly in ischemic penumbra from MCAO mice. (C) Chromatin immunoprecipitation showed that the recruitment of MLL1 to ASK-1 promoter region was enhanced, and the H3K4me3 level in this region was increased. (D) Western blot showed that MLL1 expression increased significantly in primary cortical mixed cells from the oxygen–glucose deprivation (OGD) group. (E) Chromatin immunoprecipitation showed that OGD promoted the recruitment of MLL1 to the promoter region of ASK-1 and upregulated H3K4me3 level in this region. (F) qPCR indicated that MLL1 expression showed a rapid increase within the first 2 h after MACO/OGD, then kept a stable level within 2–48 h. (G) Immunofluorescence showed that OGD upregulated the expressions of MLL1 and cleaved caspase-3 in the same cell, and siMLL1 could reverse these effects. Data are presented as mean ± SD. Compared with MLL1 recruitment in control group, *P < 0.05, **P < 0.001; compared with H3K4me3 level in control group, &P < 0.05, &&P < 0.001. (A–C) Mice were euthanized 24 h after MACO. There were 10 mice in each group. (D,E,G) OGD was performed and lasted for 24 h. The scale bar indicates 5 μm.