Response of pBRE-Luc in different cells and to different BMP concentrations and duration of BMP exposure.

<p>Cultures of human kidney cell line HEK293T (Panels A-a, C-a, B, D, E and F], human pulmonary arterial endothelial cells HPAEC (Panels A-b and C-b) or human umbilical vein endothelial cells EA.hy926 (Panels A-c and C-c) were transiently transfected with reporter construct pBRE-Luc together with constitutive β-galactosidase expression construct pCH110. 24 hr later the cultures were serum starved for 4 hr, followed by treatment with BMP4 or BMP9 for 15 hr at the indicated concentrations (Panels A-D) or 10ng/ml for the indicated times (Panels E and F). Cell lysates were assayed for β-galactosidase and luciferase activities. Within each experiment, the luciferase data were normalized for β-galactosidase activity in each extract. Each variable was investigated in single (A-b) or triplicate cultures (all other panels). Data are shown as mean ± SE. Asterisks indicate <i>p</i><0.05 for the particular group when compared to the respective BMP-free groups.</p

MxA partially rescues the inhibition of BMP4 signaling PAH-disease associated mutants of BMPR2 (F14, KDF and R899X).

<p>HEK293T cultures were transiently transfected with reporter construct pBRE-Luc together with constitutive β-galactosidase expression construct pCH110 with (solid columns) or without MxA (open columns) and the indicated BMPR2 expression constructs (WT, F14, KDF and R899X). Twenty-four hr later the cultures were serum starved for 4 hr followed by treatment with 0 (Panels A, C; “basal”) or 10 ng/ml BMP4 (Panels B, D) or BMP9 (Panel E) for 15 hr. Cell lysates were assayed for β-galactosidase and luciferase activities. Within each experiment, the luciferase data were normalized for β-galactosidase activity in each extract. Each variable was investigated in triplicate. Data are shown as mean ± SE. Asterisks and pound signs indicate <i>p</i><0.05 for the indicated group when compared to the–MxA control groups or +MxA control groups, respectively.</p

Evidence for MxA endosomes by immunoelectron microscopy.

<p>HEK293T cells were transfected with either pcDNA (Panel A) or the pMxA-HA vector (Panel B). One day later the respective cultures were processed for immuno-EM using anti-MxA rabbit pAb and Protein A-15 nm colloidal gold. Arrows point to the intercellular space between adjacent cells. The boxed inset in Panel B is shown at higher magnification in panel C. Scale bars = 1 μm.</p

MxA-positive endosomes lie alongside microtubules and the RTN4-positive endoplasmic reticulum in HPAECs.

<p>Double-label immunofluorescence imaging was carried out on MxA-vector transfected HPAECs for MxA and RTN4 using a 40x water-immersion (Panel A) or 100x oil-immersion objective (Panel B). Panel A: Double-label analysis for MxA and β-tubulin (scale bar = 10 μm). The area in the white rectangle is also shown at higher magnification (scale bar = 5 μm).Panel B: Double-label analysis for MxA and RTN4 (a marker of the standard ER) (scale bar = 10 μm). The area in the white rectangle is also shown at higher magnification (scale bar = 5 μm). In Panel A, Pearson’s R (with Costes’ automatic thresholding) was 0.142 comparing tubulin and MxA images. In Panel B, Pearson’s R (with Costes’ automatic thresholding) was 0.23 comparing RTN4 and MxA images.</p

Effect of modifying the caveolin- or clathrin-mediated endocytic pathways on productive transcription following of BMP4 or BMP9 signaling.

<p>HEK293T cultures were transiently transfected with reporter construct pBRE-Luc and constitutive β-galactosidase expression construct pCH110 (Panels C and F) or additionally with the indicated expression constructs (Panels A, B, D and E). 24 hr later the cultures were serum starved for 4 hr, followed by treatment BMP4 or BMP9 at 10 ng/ml for 15 hr as indicated (Panels A, B, D and E) or with dynasore at the indicated concentrations together with BMP4 or BMP9 for 3 hr (Panels C and F). Cell lysates were assayed for β-galactosidase and luciferase activities. Within each experiment, the luciferase data were normalized for β-galactosidase activity in each extract. Each variable was investigated in triplicate. Data are shown as the mean ratio of the normalized luciferase activities in response to 10 ng/ml BMP4/9 compared to that in the absence of BMPs (“fold-inducibility”); error bars denote ± SE. Asterisks indicate <i>p</i><0.05 for the particular group when compared to the control groups in Panels A, B, D and E or to the dynasore-free groups in Panels C and F. Cav-1, caveolin-1; CLT-HC, clathrin heavy chain.</p

Endogenous MxA in HPAECs localizes to endosomes that lie alongside ER tubules.

<p>HPAEC cultures were exposed to IFN-α for 16 hr at the indicated concentrations. Panel A illustrates Western blots for MxA and β-actin of whole cell extracts (80 μg protein/lane); quantitation of the blot shown in terms of MxA induction normalized to β-actin is shown in the graph on the right. Panel B shows single-label immunofluorescence for MxA. Scale bar = 10 μm. Panel C shows double-label immunofluorescence for MxA and RTN4 of the periphery of cell in an IFN-treated culture (1000 IU/ml). The boxed inset in the merged panel is illustrated at higher magnification in the lower right. Scale bars = 10 μm. In Panel C, Pearson’s R (with Costes’ automatic thresholding) was 0.143 comparing RTN4 and MxA images.</p

MxA endosomes carry early endosome markers and RSmad1/5/8.

<p>Double-label immunofluorescence imaging was carried out on MxA-vector transfected HEK293T cells for the indicated protein markers of early endosomes (EEA1, CLT-LC, Rab5), the transcription factors RSmad1/5/8 and caveolin-1-GFP and LAMP2 as indicated in Materials and Methods. Arrows indicate MxA-positive structures also positive for the respective second antigen. Scale bar = 10 μm. Pearson’s R (with Costes’ automatic thresholding) comparing MxA images with those of EEA1, CLT-LC, Rab5, RSmad1/5/8, LAMP2 and Cav-1-GFP were respectively 0.879, 0.704, 0.908, 0.709, 0.277 and 0.211.</p

IFN-α and MxA enhance basal and productive transcription in response to BMPs.

<p>Panel A: HAPECs in 35 mm plates were transfected with the pBRE-luc reporter and the β-gal expression plasmids using Lipofectamine 3000. The next day the cultures were either left untreated or exposed to IFN-α (3000 IU/ml) for 12 hrs. Cultures were then either continued as such (“Basal”) or exposed to BMP4 (30 ng/ml) for another 15 hr. Graph summarizes pooled data from two independent experiments in terms of the BRE-Id1-luciferase/β-gal activity normalized to the IFN-free groups (“CTR”) as 100%. Asterisks indicate <i>p</i><0.05 comparing the IFN-containing groups with the IFN-free groups. Panel B: Expression of MxA in HEK293T cultures transfected with the MxA-HA expression vector. Whole-cell lysates were prepared 24 or 48 hr after transient transfection. Immunoblots were carried out using equal aliquots protein (80 μg) from such extracts. Panels C-E: HEK293T cultures were transiently transfected with reporter construct pBRE-Luc together with the constitutive β-glycosidase expression construct pCH110 and indicated amounts of the constitutive MxA-HA expression construct. Twenty-four hr later the cultures were serum starved for 4 hr, followed by exposure to BMP4 or BMP9 at 10 ng/ml for 15 hr (Panels D and E) or left untreated (Panel C) in triplicate for each variable. Cell lysates were assayed for β-galactosidase and luciferase activities. Panel C shows the increase in basal luciferase activity in response to MxA alone; Panels D and E show data for fold-inducibility (mean ± SE). Asterisks indicate <i>p</i><0.05 for the particular group when compared to the respective MxA-free groups in Panel C, D and E.</p

Perturbation of the clathrin-mediated endocytic pathway and disruption of microtubules dynamics abrogated the enhancing effect of MxA on transcriptional activation by BMP4.

<p>HEK293T cultures were transiently transfected with reporter construct pBRE-Luc together with constitutive β-galactosidase expression construct pCH110 with (solid columns) or without MxA (open columns). 24 hr later the cultures were serum starved for 4 hr and then pretreated with 15 μM dynasore (Panels A and B), 10 μg/ml nocodazole (Panel C) or 20 nM paclitaxel (Panel D) at 37°C for 30 min, followed by treatment with 0 or 10 ng/ml BMP4 or BMP9 together with each chemical at 37°C for another 3 hr (Panels A and B) or 15 hr (Panels C and D). Cell lysates were assayed for β-galactosidase and luciferase activities. Within each experiment, the luciferase data were normalized for β-galactosidase activity in each extract. Each variable was investigated in triplicate. Data are shown as mean ± SE; as normalized luciferase activity in Panels A and B, and as fold-inducibility in Panels C and D. Asterisk and pound signs indicate <i>p</i><0.05 for the indicated group when compared to the–MxA DMSO group or the +MxA DMSO groups, respectively.</p

Perturbation of the clathrin-mediated endocytic pathway and increased caveolin expression abrogated the enhancing effect of MxA on transcriptional activation by BMP4.

<p>Panels A-D: HEK293T cultures were transiently transfected with reporter construct pBRE-Luc together with constitutive β-galactosidase expression construct pCH110 with (solid columns) or without MxA (open columns) and the indicated expression constructs. Twenty-four hr later the cultures were serum starved for 4 hr, followed by treatment with BMP4 at 10 ng/ml for 15 hr or left untreated. Data are shown as fold-inducibility (mean ± SE). Asterisk and pound signs indicate <i>p</i><0.05 for the indicated group when compared to the–MxA control groups or +MxA control groups, respectively. “NS” denotes for <i>p</i>>0.05 for the indicated groups.</p