Fig 1: Stroke induces the expression of Hmgb2 in microglia. (A) representative images show the expression of Hmgb2 (red) in micriglia (green) in the cortex at 3 days after operation with sham or stroke, the co-expressed Hmgb2 and Iba1 cells were tagged. (B) the lystaes of microglia and non-microglia fractions from mice at 0, 12, 24, 48, 72 hours after operation with sham or stroke were prepared and blotted with anti-Hmgb2 or anti-a-tubulin, as indicated. (C) Relative expression (R.E) levels (defined by normalizing the band intensities of anti-Hmgb2 to the respective anti-a-tubulin) in the individual mice (circles) and their averages per group (triangles) are plotted. Data are mean ± SEM (0.17 ± 0.024 in sham verse 1.02 ± 0.13 in stroke group, n = 5 mice per group, F(5, 24) = 74.61, ns = no significantly differences, ***p < 0.0001, BF ANOVA). (D) representative images show the brain section stained with DAPI (blue) and anti-Iba1 (green) from mice at 3 days after operation with sham or stroke. (E) both the numbers (mean ± SEM, n = 5 mice per group, *p = 0.013 , t-tests) and the soma size (mean ± SEM, n = 15 cells/5 mice per group, ***p < 0.001, t-tests) of the Iba1-labeted cells in the indivudal mice (circles) and their averages per group (triangles) at 3 days after operation with sham or stroke are plotted. (F) The ratios of the major inflammatory factors (IL-1, Cxcl-16, Ctss, IL-6 and TNF-a ) in stroke versus sham in the individual mice (circles) and their averages per group (triangles) are plotted. Data are mean ± SEM (n = 5 mice per group, **p = 0.0091, 0.0014, 0.0059, ***p < 0.0001, t-tests).
Fig 2: miR-40 inhibition suppresses the initiation of atherosclerosis in mice(A) Detection of miR-410 expression level in aorta tissues. (B) Arterial impairment and inflammation. (C) The area of aorta impairment. (D) Serum TNF-a, IL-1, and IL-6 levels in mice. (E) VCAM-1, ICAM-1, and MCP-1 expression in aorta tissues; *p < 0.05. Measured data are described as mean ± standard deviation, analyzed using independent samples t test between two groups; n = 10.
Fig 3: HDAC1 prevents atherosclerosis in mice via promoting IKBa expression(A) HDAC1 and IKBa levels in mice aorta tissues. (B) The area of aortic impairment (×400). (C) VCAM-1, ICAM-1, and MCP-1 levels in aorta tissues. (D) Serum TNF-a, IL-1, and IL-6 expression in mice; *p < 0.05. Measured data are described as mean ± standard deviation. Unpaired t test was used to analyze data between two groups; n = 10.
Fig 4: Blocking miR-410 prevents the development of atherosclerosis(A) miR-410 expression in HUVECs treated with ox-LDL. (B) miR-410 expression in HUVECs after inhibiting miR-410. (C) HUVEC viability. (D) Colony formation to evaluate HUVEC viability. (E) HUVEC apoptosis. (F) The expression of TNF-a, IL-1, and IL-6 in HUVEC culture supernatant. (G) VCAM-1, ICAM-1, and MCP-1 levels in HUVECs; *p < 0.05. Measured data are described as mean ± standard deviation. Unpaired t test was used to analyze data between two groups and Bonferroni or Tukey’s test to analyze data among groups at different time points after repeated-measures ANOVA. All of the experiments were performed with technological triplicate.
Fig 5: Hmgb2 mediates microglia pro-inflammatory response in stroke. (A) representative images show the expression of Hmgb2-SI (red, SI-tdT) and Hmgb2-I (red, I-tdT) in the cortex at 18 days after the injection of the AAV-PHP.eB-Hmgb2-SI/tdT or the AAV-PHP.eB-DIO-Hmgb2-I/tdT virus particles into the tail vein of the Cx3cr1-Cre mice. The sections are stained with anti-Iba1 (green). (B) the numbers and the soma size of the Iba1-labeted cells in the indivudal mice (circles) and their averages per group (triangles)at 18 days after the injection of AAV are plotted (ns = no significantly differences). (C) Hmgb2-I inhibits the Hmgb2 expression in microglia. The microglia lysates are prepared from mice expressing Hmgb2-SI or Hmgb2-I at 1, 2, or 3 days after operation with sham or stroke and blotted with anti-Hmgb2 or anti-α-tubulin, as indicated. (D) Relative expression (R.E) levels (defined by normalizing the band intensities of anti-Hmgb2 blots to the respective α-tubulin) in the individual mice (circles) and their averages per group (triangles) are plotted. Data are mean ± SEM (n = 5 mice per group, ns = no significantly differences, *p = 0.040; **p = 0.003; ***p < 0.0001 between Hmgb2-SI and Hmgb2-I, t-tests). (E) AAV-PHP.eB -DIO-Hmgb2-SI/tdT virus (green symbols) or AAV-PHP.eB -DIO-Hmgb2-I/tdT (blue symbols) virus particles or saline (pink symbols) were injected into the tail vein of the Cx3cr1-Cre mice. 18 days after the injection, mice were operated with sham or stroke. 24 or 72 hours after the operation, the ratios of IL-1, Cscl-16, Ctss, IL-6 and TNF-a in the extracellular space of stroke mice versus sham mice (stroke/Sham) are analyzed and plotted by the individual mice (circles) and their averages per group (triangles). Data are mean ± SEM (n = 5 mice per group, **p = 0.0041, ***p < 0.0001 between Hmgb2-SI and Hmgb2-I, t-tests). (F) the ratios of IL-10 in the extracellular space of stroke mice versus sham mice (stroke/Sham) are analyzed and plotted by the individual mice (circles) and their averages per group (triangles). Data are mean ± SEM (n = 5 mice per group, F (3,16) = 9.506, ns = no significantly differences, **p = 0.0038, 0.0011, 0.0054, BF ANOVA).
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