EV targets RhoA signaling pathway in human podocytes.

<p>(A) Biochemical assay to measure the activation level of the GTPase. The Rho-binding domain (RBD) of the RhoA effector rhotekin was used to affinity-precipitate the active fraction of endogenous RhoA (GTP-RhoA) from cell lysates (representative example from 3 independent experiments). Tubulin was used as loading control. (B) Quantification of total RhoA protein (n = 3 experiments). For quantification, total RhoA protein was normalized with respect to tubulin from whole cell lysates. (C) To quantify the amount of active RhoA protein, GTP-bound RhoA was normalized with respect to total RhoA (n = 3 experiments). (D) Western-blot analysis of MLC protein (representative example from 4 independent experiments). GAPDH = loading control, MLC = total MLC protein levels, pMLC = active, phosphorylated MLC protein. (E) Quantification of total MLC protein (n = 4 experiments). For quantification, total MLC was normalized to GAPDH from whole cell lysates. (F) Quantification of phosphorylated MLC protein (n = 4 experiments). Phosphorylated MLC was normalized to total MLC from whole cell lysates. (G) Western blot analysis of MLC protein after treatment with the ROCK inhibitor Y-27632 (10 µM for 1 h; n = 2 independent experiments). (H) Actin cytoskeleton (phalloidin-TRITC, grey) after treatment with Y-27632. DAPI was used for nuclear staining (blue). Scale bar = 100 µm. MeOH = solvent for EV. Data are means ± SD.</p

EV inhibits migration in human podocytes.

<p>(A) Representative phase contrast images after 0 h and 12 h of migration. Scale bar = 500 µm. (B) Quantification of migration efficiency by measurement of cell-free area after 12 h of migration (n = 3 experiments, ≥5 images per condition). (C) Phase contrast time-lapse studies of living cells. Migration was tracked following the nuclei in the phase contrast movie. Lower panel: Tracks were depicted on white background for better contrast. Scale bar = 500 µm. MeOH = solvent for EV. Data are means ± SD.</p

EV inhibits mTORC1 and mTORC2.

<p>(A) Western blot analysis to measure the activation level of Akt (representative example from 3 independent experiments). GAPDH = loading control, Akt = total Akt protein levels, pAkt = active, phosphorylated Akt protein. (B) For quantification, total Akt protein was normalized with respect to GAPDH. (C) To quantify the amount of active Akt protein, phosphorylated Akt was normalized with respect to total Akt. (D) Western blot analysis of p70S6K protein (representative example from 3 independent experiments). GAPDH = loading control, p70S6K = total p70S6K protein levels, p-p70S6K = active, phosphorylated p70S6K protein. (E) For quantification, total p70S6K was normalized to GAPDH. (F) Phosphorylated p70S6K was normalized to total p70S6K.</p

Aberrant distribution and size of focal adhesions is recovered by EV in human podocytes.

<p>(A) Actin (phalloidin-TRITC) and paxillin (antibody staining) images are presented in gray scale for maximum contrast. The merge image depicts paxillin in green and actin in red. DAPI was used to visualize nuclei (blue). White arrows depict focal adhesion localization. Scale bar = 25 µm. (B) Quantification of focal adhesion size (n = 3 experiments, ≥10 images per condition). (C) Quantification of the distance of focal adhesions from the cell periphery (n = 3 experiments, ≥10 images per condition. MeOH = solvent for EV. FAs = focal adhesions. Data are means ± SD.</p

EV prevents disruption of the actin cytoskeleton in human podocytes.

<p>(A) Actin (phalloidin-TRITC, grey) and nuclear staining (DAPI, blue). Scale bar = 100 µm. (B) Quantification of cell size (n = 5 experiments, ≥25 images per condition). (C) Number of central actin stress fibers within a distinct area (50 µm²) (n = 3 experiments, 20 cells per condition). (D) Quantification of cell numbers (n = 5 experiments, ≥25 images per condition). (E) Hoechst nuclear staining for the detection of apoptosis (n = 3 experiments, ≥50 images per condition). Apoptotic cells were defined as percentage of fragmented nuclei. MeOH = solvent for EV. Data are means ± SD.</p

Cytoskeletal organization in differentiated human podocytes.

<p>(A) Confocal imaging of the actin cytoskeleton and focal adhesions. Actin was visualized by phalloidin-TRITC and focal adhesions by paxillin antibody staining. Actin and paxillin images are presented in gray scale to preserve maximum contrast. Merge image shows actin in red and paxillin in green. Yellow arrow in the paxillin image: Weak paxillin staining along stress fibers. White arrow in the enlarged image: Strong accumulation of paxillin in focal adhesions at the ends of stress fibers. Scale bar = 20 µm. (B) Confocal imaging of the actin cytoskeleton (phalloidin-TRITC, grey) revealed distinct cellular structures reminiscent of dynamic actin rich protrusions (yellow arrows). Scale bar = 25 µm. (C) Phase contrast movies of control podocytes confirmed several dynamic protrusions generated in more central regions in addition to the peripheral extensions. Upper image depicts the first frame of a representative phase contrast movie (Movie S1). Lover panel shows the image sequence of the enlarged region (white box) with two dynamic protrusions (yellow stars).</p