Fig 2: HspB1 deficiency results in increased neutrophil but slightly decreased macrophage infiltration of wounds.A, Neutrophil infiltration in d1 wound granulation tissue assessed by IHC for neutrophil elastase (NE) and macrophage infiltration at d3 detected by F4/80 staining. B, Plot shows elastase positive neutrophils (NE+) per high power field (hpf); 7 fields/wound; 2 wounds per mouse; n = 4 mice; **P<0.01. B, Plot as for (B) showing F4/80-positive cells/hpf; *P<0.05.

Fig 3: VPA/HPTA-activated macrophages are highly phagocytic in breast tumors at the early stage of treatment in vivo (A) IHC was performed using the cleaved caspase-3 to identify apoptotic bodies within macrophages, representative images and quantitation are shown. Phagocytosis of breast tumor cells was quantified as the proportion of F4/80+ macrophages (red) that contain intracellular EpCAM+ (green), a marker of breast tumor cells by immunofluorescence, representative images (B) and quantitation (C) are shown. Data were normalized to untreated group. Each data point in the graphs was from three independent experiments (mean ± SD). P-values were calculated by Student’s t-test (*P < 0.05, **P < 0.01).

Fig 4: Characterization of the developmental program of meningeal lymphatics. (A) Expression of GFP and LYVE-1 adjacent to the venous sinuses of whole-mount immunostained meninges of Prox1GFP lymphatic reporter mice at P0 and P21 (P0 n = 9; P21 n = 6). Bars, 500 µm. (B) Expression of GFP and PDPN adjacent to the venous sinuses of whole-mount immunostained meninges of Prox1GFP lymphatic reporter mice at P0 and P21 (P0 n = 6; P21 n = 6). Bars, 500 µm. (C) Expression of YFP and LYVE-1 in the dura mater of Flt4YFP lymphatic reporter mice detected by whole-mount immunostaining at P0 and P21 (P0 n = 6; P21 n = 3). Bars, 500 µm. (D) LYVE-1 and PDPN expression shown in the meninges of P0 and P21 wild type mice (P0 n = 8; P21 n = 8). Bars, 500 µm. (E) PECAM and LYVE-1 expression adjacent to the superior sagittal sinus at P0, P3, P5, P8, P13 and P21 in wild type mice (P0 n = 6; P3 n = 5; P5 n = 2; P8 n = 2; P13 n = 3; P21 n = 7). Bars, 500 µm. (F) GFP signal detected by fluorescent stereo microscopy (n = 18) in meninges of Prox1GFP lymphatic reporter mice at P0. Bars, 500 µm. (G) GFP and LYVE-1 expression detected by confocal microscopy after whole-mount immunostaining of meninges of Prox1GFP lymphatic reporter mice at P0. Bars, 200 µm. (H,I) LYVE-1 and F4/80 expression shown in the meninges of wild type mice at P0 and P21 imaged by fluorescent stereo microscopy (P0 n = 9; P21 n = 6; bars, 500 µm) (H) or confocal imaging (bars, 200 µm) (I). (J) GFP and F4/80 expression in the meninges of P0 and P21 Prox1GFP reporter mice (P0 n = 3; P21 n = 3). Bars, 500 µm. Representative images are shown in all panels.

Fig 5: High level of Cisd2 protects the liver from age-related pathological changes, namely hepatic steatosis, inflammation, and fibrosis during natural aging. (a) Immunoblotting of Cisd2 protein in the livers of wild-type (WT) mice and Cisd2TG mice at 3-mo or 26-mo old (n = 3–4). (b) Body weight and (c) ratios of liver/body weight. (d) Serum AST levels (n = 6). (e) Serum ALT levels (n = 6). (f) Serum ALP levels (n = 6). (g) The liver sections of 3-mo WT, 3-mo Cisd2TG, 26-mo WT, and 26-mo Cisd2TG mice stained with H&E, IHC of F4/80 (Kupffer cell marker) or Ki67, and Masson's trichrome. Overt fat deposition, hepatocyte ballooning, inflammation (arrow), and fibrosis were detected in the naturally aged livers. (h) Quantitative analysis of liver fibrosis was performed by measuring the positive area of Masson's trichrome staining. For each mouse, three micrographs (100×) were used for the quantification (n = 3). (i) Quantitative analysis of nuclear Ki67-positive hepatocytes. For each mouse, three micrographs (400×) were used for the quantification. (j) Total triglyceride (TriG) levels in the liver tissues. See Figure S2 for microphotographs at low magnification. Scale bar, 50 µm. Data are presented as mean ± SD. *p < 0.05; **p < 0.005. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase