Fig 1: Mammary tumor growth in EE and SE housing.(A) Growth of tumor volume in SE and EE mice, EE decreased tumor growth rate (n = 10–12 per group). (B) Significant decrease of tumor volume observed at day 6, 8 and 10 shown in a box and whisker plot showing median, quartiles, and extreme values (outliers are shown as circles) (n = 10–12 per group). (C) 21 days after the injection, the tumors were excised from SE and EE mice and weighted (n = 8 in each group). (D) Representative EO771 adenocarcinoma dissected day 21 after orthotopic injection into the right fourth mammary fat pad. (E) Upper panels: Representative hematoxylin and eosin-stained tumor sections excised from EE and SE mice, 21 days after the injection. Lower panels: Tumor sections were labeled by indirect immunofluorescence staining for active caspase 3, for Ki67 or for CD31 (green) and with DAPI as nuclear counterstain (blue). Right panel: The immunolabeled cells for active caspase 3 and Ki67 and the CD31 positive area was measured using ImageJ software (n = 3 per group). The SE levels were set at 100%. Scale bars: 100 µm. Data presented are the mean ± SEM; * = p<0.05, ** = p<0.01, NS = not significant.
Fig 2: Angiogenesis was defective in the placental labyrinth zone of Pdcd5 knockout. (a–c) Immunohistochemical staining of Vegf (a), Vegfr-2 (b) and Pecam-1 (c) in the labyrinth zones of Pdcd5+/+ and Pdcd5–/– placentas at E12.5
Fig 3: A similar magnitude of reduction in muscle degeneration and improvement in angiogenesis in the ischemic limbs of mice following Stempeucel®-1 and Stempeucel®-1A treatment: a Treatment with Stempeucel®-1 and Stempeucel®-1A significantly protected muscle fiber loss of adductor and gastrocnemius muscles compared to vehicle control. b Protection from muscle weight loss was observed upon treatment with Stempeucel®-1 and Stempeucel®-1A compared to the vehicle-treated group. c–f Histological analysis of muscle section of the sham control, vehicle control, and Stempeucel-treated groups. Red arrow indicates vacuolar degeneration, green arrow—mononuclear cells infiltration. Yellow arrow—muscle necrosis and black arrow—muscle degeneration. Intramuscular administrations of Stempeucel®-1 and Stempeucel®-1A survive in ischemic limb tissue, secrete paracrine factors, and recruit or proliferate CD31 positive endothelial cells. Representative images of IHC staining for g–j mCD31; k–n HNA; o–r hVEGF; positive cells in limb muscles of sham control, vehicle control, and Stempeucel-treated animals at day 28. Positive areas are marked with black arrows. Photographs were taken at × 40 magnification, and the scale bar shows the 50 μm
Fig 4: Endothelial-targeted Lrrc8a KO mice exhibit reduced phosphorylated endothelial nitric oxide synthase (p-eNOS) and mild angiotensin-II stimulated hypertension.(A) Strategy for endothelium targeted Lrrc8a ablation to generate eLrrc8a KO. (B) Isolation of murine primary endothelial cells from WT and eLrrc8a KO using tdTomato reporter mice. (C–D) Current-voltage relationships of volume-regulatory anion channel (VRAC) current in isotonic (Iso, 300 mOsM) and hypotonic (Hypo, 210 mOsm) solution in response to voltage ramps from -100 to +100 mV over 500 ms in WT (C) and KO (D) primary murine endothelial cells. DCPIB (10 µM) inhibition in C (WT). (E) Mean outward (+100 mV) and inward (-100 mV) currents from WT (n = 3 cells) and eLrrc8a KO (n = 3 cells). (F) Ex vivo aorta sprouting assay performed in aortic rings isolated from WT and eLrrc8a KO mice and cultured in FGM media for 3 days at 37°C. Immunofluorescence staining with antibodies to LRRC8A (green), CD31 (red), LRRC8A+CD31+ (Merge) demonstrating endothelial-targeted Lrrc8a deletion. (G) Quantification of LRRC8A immunofluorescence staining in WT (n = 15) and eLrrc8a KO (n = 15) endothelial cell tubes. (H–I) Representative images of aortic sections from WT (n = 3) and eLrrc8a KO (n = 3) mice immunostained with p-eNOS (H, green), and immunofluorescence quantification of regions of WT (n = 5) and eLrrc8a KO (n = 5) endothelium (I). Scale bar in (H) is 50 µm. (J–L) Tail-cuff systolic blood pressures of male (J) and female (K) WT (n = 5 males and 7 females) and eLrrc8a KO (n = 5 males and 12 females) mice, and systolic blood pressures of male WT (n = 4) and eLrrc8a KO (n = 5) mice under basal conditions and after 4 weeks of chronic angiotensin-II infusion (L). Statistical significance between the indicated values is calculated using a two-tailed unpaired Student’s t-test. Error bars represent mean ± s.e.m. *p<0.05; **p<0.01; ***p<0.001.Figure 7—source data 1.Source data for Figure 7E.Figure 7—source data 2.Source data for Figure 7I.Figure 7—source data 3.Source data for Figure 7J.Figure 7—source data 4.Source data for Figure 7K.Figure 7—source data 5.Source data for Figure 7l.
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