Fig 1.
The twenty five biological pathways most significantly (P<0.05) regulated by IL-4 treatment in HCAEC.
(A, B) Pathways represented as histograms are ranked by the–log value (P value). Length of histogram corresponds to the number of genes associated with that specific pathway.
Table 1.
Key genes regulated by IL-4 in ‘Immune response_Oncostatin M signaling via JAK-Stat in human cells’, ‘Development_Regulation of epithelial-to-mesenchymal transition (EMT)’ and ‘Cytoskeleton remodeling_TGF, Wnt and cytoskeletal remodeling’ pathways in HCAEC.
Fig 2.
Metacore™ map showing the signaling context of the genes contained in the ‘Cytoskeleton remodeling_TGF, Wnt and cytoskeletal remodeling’ pathway.
Genes regulated by IL-4 are marked by red and blue thermometer icons representing upregulated and downregulated gene expression respectively.
Fig 3.
Expression of Wnt5A in IL-4 treated HCAEC.
Fold changes in the expression of (A) Wnt5A mRNA in 8 h IL-4 treated HCAEC and HPAEC and (B) IL-6 mRNA in 8 h IL-4 treated HCAEC. Data were obtained from three independent qRT-PCR experiments run with duplicate samples and expressed as the mean ± SEM. *P<0.05. (C) Wnt5A protein expression in non-treated and IL-4 treated HCAEC. Wnt5A is immunostained (red), nuclei (DAPI, blue). Photomicrographs were acquired using a Zeiss Axioskope equipped with AxioCam MRm digital camera and AxioVision Rel.4.6 software. Original magnification, 200×. (D) Mean cellular fluorescent intensity of Wnt5A. Data represent mean ± SD from five different regions. *P<0.05. The experiment was repeated five times, with analogous results. (E) Immunoblot of Wnt5A induction in IL-4 treated HCAEC. In-gel stained 50 kDa band, served as loading control and for densitometry normalization. Left band, non-treated cells; right band, IL-4 treated cells. Data are mean ± SEM from three independent experiments. *P<0.05.
Fig 4.
Phosphorylation of LIMK2 and CFL1 in IL-4 treated HCAEC.
(A) pLIMK2 (pT505) and pCFL1 (pCofilin-1, pS3) were immunostained (red) in HCAEC treated with IL-4 either alone or in combination with Y-27632. F-actin (fluorescent phalloidin, green) and nuclei (DAPI, blue) were also stained. Photomicrographs were acquired using a Zeiss Axioskope equipped with AxioCam MRm digital camera and AxioVision Rel.4.6 software. Original magnification, 200×. (B) Mean cellular fluorescent intensities of pLIMK2 and pCFL1. Data represent mean ± SD from five different regions. *P<0.05. The experiment was repeated three times, with analogous results. (C, D) Immunoblots of pLIMK2, LIMK2, pCFL and CFL in HCAEC treated with IL-4 either alone or in combination with Y-27632. Left band, non-treated cells; middle band, IL-4 treated cells; right band, IL-4+Y-27632 treated cells. Data are mean ± SEM from three independent experiments. *P<0.05.
Fig 5.
Stress fiber formation in IL-4 treated HCAEC.
Live actin-RFP staining showing the formation of actin stress fibers in HCAEC treated with IL-4 or Wnt5A in the absence or presence of sFRP1 and WIF1. Expression of de novo synthesized RFP-actin after transfection as outlined in methods was visually observed up to 24 h. Photomicrographs of stress fiber formation were taken randomly at 12 h after stimulation with IL-4 and Wnt5A alone or in the presence of sFRP1 and WIF1 using Zeiss Axio Observer.Z1 equipped with AxioCam MRm digital camera and ZEN 2012 software. Original magnification, 400×. Independent identical experiments in triplicates were repeated at least three times, with analogous results.
Fig 6.
Assembly of VE-cadherin in IL-4 treated HCAEC.
VE-cadherin immunostained (red) in HCAEC either non-treated (A) or treated with IL- 4 for 8 h (B). Nuclei (DAPI, blue). Photomicrographs were acquired using a Zeiss Axioskope equipped with AxioCam MRm digital camera and AxioVision Rel.4.6 software. Original magnification, 400×.
Fig 7.
ECIS barrier function assays showing the effect of Wnt5A in IL-4 induced hyperpermeability of HCAEC.
Uniform confluent monolayers of HCAEC cultured in stabilized and collagen coated ECIS 8W10E+ arrays were treated with IL-4. TEER of HCAEC monolayers was measured in Ohms continuously every 5 min at multiple frequencies ranging from 62.6 Hz to 64 kHz, normalized to its value at time zero and plotted with respect to time. Stimulations conducted in duplicate wells were grouped and averaged to plot a single curve with error bars representing mean ± SD. Figures shown depict the resistance measurements conducted at 4000 Hz and represent four independent experiments. (A) 1, non-treated Wnt5A-siRNA transfected HCAEC; 2, non-treated non-transfected HCAEC; 3, IL-4 treated Wnt5A-siRNA transfected HCAEC; 4, IL-4 treated non-transfected HCAEC. (B) 1, non-treated negative control siRNA transfected HCAEC; 2, IL-4 treated negative control siRNA transfected HCAEC; 3, IL-4 treated non-transfected HCAEC. (C) Black, non-treated; green, IL-6.
Fig 8.
Automated wound healing assay showing decreased motility of IL-4 treated HCAEC.
Uniform confluent monolayers of HCAEC cultured in stabilized and collagen coated ECIS 8W1E cultureware were treated with IL-4 either alone or in combination with Y-27632. Three hours after treatment, wells were subjected to an elevated electric field to create a wound. Measurements were started immediately after wounding. Impedance of HCAEC monolayer was continuously measured in Ohms every 5 min at multiple frequencies ranging from 62.6 Hz to 64 kHz and plotted with respect to time. Representative figures depict the impedance of wells after wounding was applied. Figures shown depict the measurements conducted at 4000 Hz and represent four independent experiments. (A) Black, non-treated; Blue, IL-4. (B) Black, control with Y27632; Green, IL-4 + Y27632.
Fig 9.
Wnt5A expression in IL-4 activated human macrophages.
Fold changes in the expression of Wnt5A mRNA in IL-4 treated human macrophages. Data were obtained from two independent qRT-PCR experiments run with duplicate samples using RNA isolated from macrophages from four different donors and expressed as the mean ± SEM. *P<0.05 using an unpaired Student’s t- test.