Fig 1: HPSE/SDC-1/TNF-a axis plays a crucial role in necroptosis of HUVECs in vitro.a, b SDC-1, TNF-a mRNA (a) and protein (b) expressions in HUVECs were detected by qRT-PCR and western blotting, respectively. c Soluble SDC-1 and TNF-a concentrations in the supernatant were detected by ELISA assay. d RIP1, RIP3, MLKL, and p-MLKL protein levels in HUVECs were determined by western blotting. e SDC-1 and TNF-a distributions in HUVECs were observed by fluorescence microscope (scale bars: 50 µm). f The number of DAPI staining HUVECs significantly increased after HPSE or SDC-1 expression was inhibited. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: HPSE induces necroptosis of HUVECs in vitro.a The survival rate of HUVECs was determined by CCK-8 assay. b Flow cytometry dot plot of HUVECs in two groups. c Apoptotic index of HUVECs in shCtrl group was significantly higher than that in shHPSE group. d DNA electrophoretogram of HUVECs of both groups showed only a long DNA fragment (length 27 kb). e, f RIP1, RIP3, and MLKL (p-MLKL) mRNA (e) and protein (f) levels in HUVECs of two groups were detected by qRT-PCR and Western blotting, respectively. g, h Morphology of HUVECs were observed by fluorescence microscopy (g, scale bars: 50 µm) and transmission electron microscopy (h, scale bars: 5 µm), respectively. Representative images are shown. Membrane rupture (red triangle), release of the intracellular contents, cell swelling (black arrow), and nuclear fragmentation (red arrow) could be found in shCtrl group. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: Expression of key proteins of necroptosis in HUVECs.a–c TNFR, TRADD (a), caspase-8 and FADD (b) mRNA and their protein expressions (c) in co-cultured HUVECs were validated by qRT-PCR and western blotting analysis, respectively. d–f NF-?B (d) and p38 MAPK mRNA (e) and their protein expressions (f) in co-culured HUVECs were detected by qRT-PCR and western blotting analysis, respectively. g The proposed model of HPSE/SDC-1/TNF-a axis and p38 MAPK pathway in HPSE induced necroptosis. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: HPSE expression is up-regulated in HCC.a, b HPSE mRNA (a) and protein (b) expression levels in peritumor tissues (P) and tumor tissues (T) of 6 HCC patients were detected by qRT-PCR and western blotting, respectively. c HPSE expressions in peritumor tissues and tumor tissues of 88 HCC patients were measured by IHC assay (scale bars, 200 µm). d The average IHC score of HCC tissues of 88 patients was significantly higher than that of peritumor tissues. e, f HPSE mRNA (e) and protein (f) expression levels in LO2 cell line and three kinds of HCC cell lines were measured by qRT-PCR and western blotting, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5: HPSE on necroptosis of vascular endothelial cells in vivo.a The two groups of nude mice showed ascites. b Histofluorescence showed GFP-positive HCC cells in the liver tissue of mice in NS group, and no positive cells in Nec-1 group (scale bars, 50 µm). c Representative images of HE staining of the liver tissues are shown (scale bars: 50 µm). d Necrosis score of MEVCs in NS group was significantly higher than that of Nec-1 group. e CD31 IHC staining showed that the integrity of liver microvessel in NS group was seriously damaged (scale bars: 200 µm). f Double immunofluorescent analysis showed the damage of microvessel integrity (CD31 staining, green) and increased necroptosis (MLKL staining, red) in NS group (scale bars: 50 µm). g Ten size-fixed fields (0.1 × 0.1 mm2) were randomly selected to quantify the number of DAPI-stained cells. *P < 0.05, **P < 0.01, and ***P < 0.001.
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