Additional file 1: of Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication

Confocal sections of lipid droplet accumulation in a macromeront. 25 confocal sections of the composite Z-stack shown in Figure 3E are depicted. Nuclei were stained by DAPI (blue) whilst lipid droplets were shown in green by Bodipy 493/503 staining. Scale bars represent: 10 Οm

Impact of TASK-1 inhibitor A293 on vasoconstriction of WT and TASK-1 KO mice.

<p>A293 induced constriction of WT intra- <b>(A)</b> and pre-acinar <b>(B)</b> arteries at normoxia and reduced hypoxia-induced vasoconstriction. In KO intra- <b>(C)</b> and pre-acinar <b>(D)</b> arteries at normoxia, the constrictory effect of A293 persisted. Data are presented as mean ± SEM. <i>n</i> is number of vessels/number of animals. Tests of significance were done at the time points indicated by arrows. *, #, §, P≤ 0.05 and **, ##, §§,  $ P≤ 0.01 (Mann–Whitney U-test).</p

Impact of TASK-1 inhibitor anandamide on vasoconstriction of WT mice.

<p><b>(A)</b> Effect of anandamide on intra-acinar arteries. Anandamide induced constriction at normoxia and reduced hypoxia-induced vasoconstriction significantly at one time. <b>(B)</b> Effect of anandamide on pre-acinar arteries. Anandamide did not induce constriction at normoxia. However, it reduced hypoxia-induced vasoconstriction significantly after 20 min. Data are presented as mean ± SEM. <i>n</i> is number of vessels/number of animals. Tests of significance were done at the time points indicated by arrows. *, #, §, $ P≤ 0.05 and **, ## P≤ 0.01 (Mann–Whitney U-test).</p

One-Dimensional (1-D) and Two-Dimensional (2-D) gel electrophoresis followed by anti-TASK-1 western blotting.

<p><b>(A)</b> 1-D Gel electrophoresis followed by western blotting with anti-TASK-1 antibody lot AN 02. Immunoreactivity was prominent in the cerebellum and heart and was weak in lung extracts of TASK-1 wild-type (WT), heterozygous (HZ), and knockout (KO) mice. <b>(B)</b> 1-D Gel electrophoresis followed by western blotting with anti-TASK-1 antibody lot AN 08. Labelling with anti-TASK-1 antibody was detectable in lung and heart extracts of WT and KO animals, but was absent in the cerebellum of both mouse strains. Staining was absent in control without primary antibody. <b>(C)</b> 2-D Gel electrophoresis of the cerebellum of WT and TASK-1 KO mice followed by anti-TASK-1 western blotting. Comparable labelling patterns were present in samples of both mouse strains with anti-TASK-1 antibody lot AN 02. Immunoreactive spots were identified as glial fibrillary acidic protein isoform (GFAP2), and mitochondrial cytochrome b-c1 complex subunit 1 (cyt b-c1). Molecular weights (kDa) are presented on the left side of blots. The solid lines indicate assembled blots.</p

TASK-1 potassium channel is not critically involved in mediating hypoxic pulmonary vasoconstriction of murine intra-pulmonary arteries

<div><p>The two-pore domain potassium channel KCNK3 (TASK-1) is expressed in rat and human pulmonary artery smooth muscle cells. There, it is associated with hypoxia-induced signalling, and its dysfunction is linked to pathogenesis of human pulmonary hypertension. We here aimed to determine its role in hypoxic pulmonary vasoconstriction (HPV) in the mouse, and hence the suitability of this model for further mechanistic investigations, using appropriate inhibitors and TASK-1 knockout (KO) mice. RT-PCR revealed expression of TASK-1 mRNA in murine lungs and pre-acinar pulmonary arteries. Protein localization by immunohistochemistry and western blot was unreliable since all antibodies produced labelling also in TASK-1 KO organs/tissues. HPV was investigated by videomorphometric analysis of intra- (inner diameter: 25–40 μm) and pre-acinar pulmonary arteries (inner diameter: 41–60 μm). HPV persisted in TASK-1 KO intra-acinar arteries. Pre-acinar arteries developed initial HPV, but the response faded earlier (after 30 min) in KO vessels. This HPV pattern was grossly mimicked by the TASK-1 inhibitor anandamide in wild-type vessels. Hypoxia-provoked rise in pulmonary arterial pressure (PAP) in isolated ventilated lungs was affected neither by TASK-1 gene deficiency nor by the TASK-1 inhibitor A293. TASK-1 is dispensable for initiating HPV of murine intra-pulmonary arteries, but participates in sustained HPV specifically in pre-acinar arteries. This does not translate into abnormal rise in PAP. While there is compelling evidence that TASK-1 is involved in the pathogenesis of pulmonary arterial hypertension in humans, the mouse does not appear to serve as a suitable model to study the underlying molecular mechanisms.</p></div

Laser-assisted Microdissection (LMD) and expression of TASK-1 in WT murine intra-pulmonary arteries.

<p><b>(A)</b> LMD. The white line indicates the luminal diameter of a pre-acinar artery. It represents the maximum distance between intimal surfaces at right angle (90°) to the longitudinal axis. The area of vessel to be picked was first marked (blue lines; left panel). The marked piece of tissue (missing in right panel) was catapulted into the lid of a reaction tube and processed for RT-PCR. <b>(B)</b> RT-PCR of laser-assisted microdissected samples, agarose gel. ØRT is sample of cardiomyocytes processed without Reverse Transcriptase (RT). β-Actin was used as internal control. Heart served as positive control. H₂O is control without cDNA template.</p

Hypoxia-induced changes in Pulmonary Arterial Pressure (ΔPAP) of isolated, perfused, and ventilated lungs of WT and TASK-1 KO mice.

<p>ΔPAP was referenced to the onset of 180 min of hypoxic ventilation period (time set at 0). Normoxic PAP and its increase during hypoxic ventilation were neither different between KO and in WT mice <b>(A)</b> nor when comparing WT mice in the presence or absence of the TASK-1 inhibitor A293 <b>(B)</b>. Data are presented as mean ± SEM. <i>n</i> refers to number of animals used. HPV: hypoxic pulmonary vasoconstriction.</p

Videomorphometric analysis of the hypoxic response of intra- (A) and pre-acinar (B) arteries of WT and TASK-1 KO mice.

<p>The luminal area of vessels before exposure to hypoxia was set as 100%, and changes in the luminal areas are presented as relative values. Hypoxia-induced constriction of intra-acinar arteries was comparable in both mouse strains, whereas in KO pre-acinar arteries, it started to fade earlier as compared to WT. Data are presented as mean ± SEM. <i>n</i> is number of vessels/number of animals. Tests of significance were done at the time points indicated by arrows. *, #, §, $ P≤ 0.05 and **, ## P≤ 0.01 (Mann–Whitney U-test).</p

TASK-1 Knockout (KO) mice and expression of TASK-1 and TASK-3 mRNA in murine organs.

<p><b>(A)</b> Genotype identification of TASK-1 wild-type (WT), heterozygous (HZ), and KO mice by PCR, agarose gel. <b>(B)</b> Expression of TASK-1 and TASK-3 mRNA in WT murine organs, RT-PCR, agarose gel. ØRT is lung sample processed without RT. β-Actin was used as internal control. Heart served as positive control. H₂O is control without cDNA template.</p

NET formation in LPS-induced lung injury mouse model.

<p>(A) Immunofluorescence staining of lung sections from mice after 24 h intratracheal LPS administration was performed, as compared to the control section, for DNA/histone (red), neutrophil elastase (green) and DAPI (blue). The higher magnification views of the insets (1, 2 and 3), which were randomly chosen, showed co-localization of neutrophil elastase (green) and DNA/histone (red) in NET structures. (B) Immunofluorescence staining of sections from PBS- or LPS-treated mice was performed for DNA/histone (red), CD46 (green) as a cell membrane marker and DAPI (blue). The randomly chosen insets (4, 5 and 6) showed NET formation in LPS-treated lungs in higher magnification views as appeared by extracellular chromatin, disintegration of the cell membranes as well as weak signal for DAPI (indication of chromatin decondensation). DAPI alone and DNA/histone alone were also shown for the insets 4, 5 and 6. In A and B, yellow arrows indicate some of the tissue destruction areas adjacent to NET. Shown are representative pictures of >10 fields of tissue staining. (C) Immunofluorescence staining of lung sections from mice after 24 h intratracheal LPS administration was performed for myeloperoxidase (MPO, red), citrullinated histone H3 (Cit His3, green) and DAPI (blue). The higher magnification views (right column) of the selected areas showed co-localization of myeloperoxidase with citrullinated histone H3 which indicate NET formation.</p