Fig 2: Inhibition of SUMO pathway partially restored DAPK1 protein levels at the presence of TSC2. (A) HEK293T was transfected with HA-DAPK1, Flag-TSC2, and different E2 siRNAs. (B,C) HEK293T cells were transfected with HA-DAPK1, LacZ, TSC2 and four different SENPs as indicated. Cell lysates were extracted 48 h post-transfection and immunoblotted with respective antibodies. The intensity of the bands was quantified using LICOR Odyssey software and represented by bar graphs. The experiments were repeated three times (n = 3), and representative images are presented. The representative western blot images are from different gels and each lane was loaded with the same amount of proteins. Data of triplicate assays are presented as mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, no significance. “+” indicated that the plasmid was transfected, “-” indicated that the plasmid was not transfected.

Fig 3: DAPK1 expression and its association with inhibition of CD4+ T cell proliferation by hMSCs.(a). Representative images of hMSCs cultured in vitro and stained with anti-DAPK1 antibody (red) and DAPI (blue). Scale bar, 50 µm. (b). Immunoblots of hMSC extracts isolated from six healthy donors and blotted with anti-DAPK1 and anti-ß-actin antibodies. (c). CCK-8 assay to assess cell viability of six hMSC lines. (d). The CD4+ T cell proliferation rates (means of three independent experiments) following co-culture of PBMCs incubated with CFSE and stimulated through CD3/28 with the above six hMSC lines (hMSCs1 to hMSCs6) were 49.94%, 47.06%, 60.64%, 40.14%, 69.16%, and 56.48%, respectively. Non-activated PBMCs were used as controls (mean proliferation rate 0.99%). When stimulated through CD3/28 (PBMC+CD3/28), the proliferation rate was extremely high (mean proliferation rate, 82.39%). (e). Correlation of relative DAPK1 expression levels and CD4+ T cell proliferation. R=-0.897, p = 0.015. The levels of DAPK1 were normalized to those of ß-actin, and the DAPK1/ß-actin ratio of hMSC5 was set as 1. Data are presented as means ± SEMsDAPK1, death-associated protein kinase 1; hMSC, human mesenchymal stem cell; DAPI, 4',6-diamidino-2-phenylindole; PBMC, peripheral blood mononuclear cell; CFSE, carboxyfluorescein diacetate succinimidyl ester

Fig 4: Expression of CDC20 and DAPK1 and their correlation with CYP1B1 in clinical samples. a Representative immunostaining of CDC20, DAPK1 and CYP1B1 from matched samples including normal tissue, Low CYP1B1 RCC and High RCC tissues. Intracellular CDC20 accumulation was positive in some cancer cells, whereas majority of the normal kidney cells were negative. Strong cytoplasmic staining of DAPK1 was more common in the normal kidney tissues than in RCC samples. b CDC20 expression was significantly higher in RCC than normal kidney tissues. There was a positive correlation between the expression of CDC20 and CYP1B1 in RCC, though it did not reach statistical significance. DAPK1 expression was significantly down-regulated in RCC as compared to normal tissues. The expression level of DAPK1 was also significantly correlated with Low and High CYP1B1 expression in RCC. *, P < 0.05. **, P < 0.01.***, P < 0.001

Fig 5: Genetic deletion of DAPK1-KD protects against stroke damage. (A) Representative MR images show the area sizes of cerebral infarct. Bar graph shows the quantification of infarct volumes (n = 5 for DAPK1-KD-/- mice; n = 7 for DAPK1-KDloxp/loxp mice).*P < 0.05 vs. DAPK1-KDloxp/loxp mice. (B) Immunoblots of the extracts from the ischemic DAPK1-KD-/- and DAPK1-KDloxp/loxp mice cortex probed with pS262, Tau, PSD95, GluA1, and Synapsin 1 antibodies. Bar graph shows the quantification of the protein levels (n = 5 mice). *P < 0.05 vs. DAPK1-KD-/- mice, #P < 0.05 vs. DAPK1-KDloxp/loxp mice. (C) Representative images show the FJ-labeling in the cortex and striatum. Scale bar in serial pictures is 2.5 mm. Curve lines summarize the FJ-labeled area (n = 4). *P < 0.05 vs. DAPK1-KDloxp/loxp mice. (D) Golgi staining indicated the dendrites in the cortex and numbers of spines (bar graph) in DAPK1-KD-/- and DAPK1-KDloxp/loxp mice 24 h after MCAO (n = 5 mice). Scale bar: 100 µm in the upper panel, 10 µm in the lower panel. *P < 0.05 vs. DAPK1-KDloxp/loxp mice. (E) Overall neurological scores (N.S.) and (F) the performance on rotarod test were analyzed throughout the indicated period after MCAO-treated DAPK1-KD-/- and DAPK1-KDloxp/loxp mice. *P < 0.05 vs. DAPK1-KDloxp/loxp mice. Data are presented as mean ± SEM.