Fig. 4

Caveolin-1-positive early endosomes are signaling hotspots for FSS-induced BMP signaling. Confocal images of Endoglin, Caveolin-1, and DAPI in cells exposed to A HSS or B LSS. Insets show magnified regions. Arrows indicate Endoglin-positive Caveolin vesicles. Scale bars 10 μM. C Quantification of the number of Caveolin-1 vesicles (left) and Endoglin-positive Caveolin-1 vesicles (right). Counting was performed using a self-written ImageJ script (see the “Methods” section for details). D Confocal image of Endoglin, Caveolin-1, and EEA1. Scale bar 1 μM. Insets show single channels in gray scale. The white line indicates the area of intensity profile shown in E. E Intensity profile of Endoglin, Caveolin-1, and EEA1 staining shown in D. F Quantification of EEA1 vesicles (left), Cav-1-positive EEA1 vesicles (middle), and Cav-1 and Endoglin double-positive EEA1 vesicles (right). G Confocal image of Caveolin-1 and PLA of Endoglin and overexpressed ALK1-meGFP. Scale bar 1 μM. The white line indicates the area of intensity profile shown in H. H Intensity profile of Caveolin-1 and Endoglin-ALK1-meGFP PLA staining shown in G. I Confocal image of Caveolin-1, deuterated silicon-rhodamine-labeled BMP9 (BMP9-siR), and DAPI. Scale bars 500 nm. The lower panel schematically depicts the labeling process of BMP9 with deuterated silicone-rhodamine (see the “Methods” section). J Inverted confocal images of SMAD1 show the nuclear accumulation of SMAD1 in LSS over HSS. Scale bar is 10 μM. K Confocal image of Caveolin-1, Endoglin, and SMAD1 shows the location of SMAD1 at Endoglin-positive Caveolin-1 vesicles, indicated by arrows. Scale bar 5 μM. L Quantification of SMAD1-positive Endoglin/Cav-1 vesicles. M Immunoblot using antibodies specific against pSMAD1/5 and Caveolin-1 in cells exposed to 24 h of FSS after Caveolin-1 knock-down in the absence/presence of 50 ng/mL ALK1-Fc. N Densiometric quantification of SMAD1/5 phosphorylation relative to GAPDH expression from immunoblots in M. Data is presented as mean ± SD from 3–5 independent experiments. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test, ****p < 0.0001,***p < 0.001