Journal: Molecular Biology of the Cell

Article Title: Shroom2 regulates contractility to control endothelial morphogenesis

doi: 10.1091/mbc.E10-06-0505

Figure Lengend Snippet: Shrm2 knockdown stimulates endothelial angiogenesis. (A and B) Murine yolk sacs at E9.5 stained with PECAM and Shrm2 antibodies show Shrm2 localization at cellular junctions in both large vessels (A) and the capillary plexus (B). (C and D) C166 endothelial cells were treated with a nontargeting siRNA (siControl) (C) or a Shrm2-specific siRNA (siShrm2) (D) and were stained with Shrm2 (inset) and ZO1. (E) Shrm2 knockdown from two different siRNAs was confirmed via Western blot. α-Tubulin was used as a loading control. (F–H) siControl (F), siShrm2–7 (G), or siShrm2–8 (H)–treated C166 cells were plated on matrigel to examine angiogenic potential. Arrow indicates an area that has failed to undergo angiogenesis. (I–K) siControl (I) and siShrm2 (J) C166 cells were grown as spheroids for use in a collagen sprouting angiogenesis assay. Quantification of collagen sprouting angiogenesis is shown in (K). The numbers of branch tips are represented as the mean ± SD (n = 7 spheroids). (L) Western blot of Shrm2 knockdown in HUVECs. (M and N) Matrigel angiogenesis assay for siControl (M) and siShrm2 (N)–treated HUVECs. Scale bars = 25 μm in A–D; 1 mm in F–H; 125 μm in I, J, M, and N.

Article Snippet: Additional antibodies were as follows: PECAM, E-cadherin (BD Biosciences, Franklin Lakes, NJ); ZO1 (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin (Sigma-Aldrich, St. Louis, MO); Rock1, pMLC2–Ser-19, ppMLC2–Thr-18/Ser-19, p-cofilin, and pFAK–Tyr-397 (Cell Signaling, Danvers, MA); Rock2 (Bethyl Labs, Montgomery, TX); p-MYPT (Millipore, Temecula, CA); and GFP, TO-PRO-3, goat anti–mouse, goat anti–rat, or goat anti–rabbit secondary antibodies conjugated to Alexa-488 or Alexa-568 (Invitrogen, Carlsbad, CA).

Techniques: Staining, Western Blot, Angiogenesis Assay

Journal: Molecular Biology of the Cell

Article Title: Shroom2 regulates contractility to control endothelial morphogenesis

doi: 10.1091/mbc.E10-06-0505

Figure Lengend Snippet: Stable knockdown of Shrm2 in murine ES cells enhances vasculogenesis. (A–C) ES cells stably transfected with the parental vector pSuper-GFPneo r (pSuper) (A) or pSuper-shShrm2 (shShrm2) (B) were stained to detect GFP and Shrm2. Stable knockdown was confirmed by Western blotting (C). E-cadherin was used as a loading control. (D and E) After differentiation, pSuper (D) and shShrm2 (E) cells were stained with PECAM to visualize the resulting endothelial network. Boxed regions in D and E are enlarged in Di and Ei. While distinct boundaries exist between control endothelial and surrounding cells (Di), numerous filopodia-like extensions are found throughout the Shrm2-deficient endothelium (Ei) (compare arrows). Scale bars = 25 μm in A and B; 100 μm in D and E.

Article Snippet: Additional antibodies were as follows: PECAM, E-cadherin (BD Biosciences, Franklin Lakes, NJ); ZO1 (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin (Sigma-Aldrich, St. Louis, MO); Rock1, pMLC2–Ser-19, ppMLC2–Thr-18/Ser-19, p-cofilin, and pFAK–Tyr-397 (Cell Signaling, Danvers, MA); Rock2 (Bethyl Labs, Montgomery, TX); p-MYPT (Millipore, Temecula, CA); and GFP, TO-PRO-3, goat anti–mouse, goat anti–rat, or goat anti–rabbit secondary antibodies conjugated to Alexa-488 or Alexa-568 (Invitrogen, Carlsbad, CA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Staining, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Shroom2 regulates contractility to control endothelial morphogenesis

doi: 10.1091/mbc.e10-06-0505

Figure Lengend Snippet: Figure 1: Shrm2 knockdown stimulates endothelial angiogenesis. (A and B) Murine yolk sacs at E9.5 stained with PECAM and Shrm2 antibodies show Shrm2 localization at cellular junctions in both large vessels (A) and the capillary plexus (B). (C and D) C166 endothelial cells were treated with a nontargeting siRNA (siControl) (C) or a Shrm2-specific siRNA (siShrm2) (D) and were stained with Shrm2 (inset) and ZO1. (E) Shrm2 knockdown from two different siRNAs was confirmed via Western blot. α-Tubulin was used as a loading control. (F–H) siControl (F), siShrm2–7 (G), or siShrm2–8 (H)–treated C166 cells were plated on matrigel to examine angiogenic potential. Arrow indicates an area that has failed to undergo angiogenesis. (I–K) siControl (I) and siShrm2 (J) C166 cells were grown as spheroids for use in a collagen sprouting angiogenesis assay. Quantification of collagen sprouting angiogenesis is shown in (K). The numbers of branch tips are represented as the mean ± SD (n = 7 spheroids). (L) Western blot of Shrm2 knockdown in HUVECs. (M and N) Matrigel angiogenesis assay for siControl (M) and siShrm2 (N)–treated HUVECs. Scale bars = 25 μm in A–D; 1 mm in F–H; 125 μm in I, J, M, and N.

Article Snippet: Additional antibodies were as follows: PECAM, E-cadherin (BD Biosciences, Franklin Lakes, NJ); ZO1 (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin (Sigma-Aldrich, St. Louis, MO); Rock1, pMLC2– Ser-19, ppMLC2–Thr-18/Ser-19, p-cofilin, and pFAK–Tyr-397 (Cell Signaling, Danvers, MA); Rock2 (Bethyl Labs, Montgomery, TX); pMYPT (Millipore, Temecula, CA); and GFP, TO-PRO-3, goat anti– mouse, goat anti–rat, or goat anti–rabbit secondary antibodies conjugated to Alexa-488 or Alexa-568 (Invitrogen, Carlsbad, CA).

Techniques: Knockdown, Staining, Western Blot, Control, Angiogenesis Assay

Journal: Molecular Biology of the Cell

Article Title: Shroom2 regulates contractility to control endothelial morphogenesis

doi: 10.1091/mbc.e10-06-0505

Figure Lengend Snippet: Figure 3: Shrm2 interacts with Rock and promotes endothelial contractility. (A) A GST-tagged mShrm2 SD2 or a GST-tagged Shroom binding domain (SBD) of hRock1 were incubated with total cell lysate from T23 cells engineered to express myc-Shrm2 under the tetracycline response element promoter. Following GST pull down, the Shrm2 SD2 interacts with endogenous Rock1, and the Rock1 SBD interacts with full-length myc-Shrm2. (B and C) siControl (B) or siShrm2 (C) C166 cells were stained with Rock1 (Bi and Ci) and ZO1 (Bii and Cii) antibodies. Arrows indicate loss of Rock localization to tight junctions after Shrm2 knockdown. (D) Contractility of siControl or siShrm2 cells with or without the Rock inhibitor Y27632 was assessed through the ability of a monolayer to contract a collagen gel. Quantification is graphed as the percentage of area of the original after 4 h, represented by the mean ± SD (n = 3). (E) Phosphorylation of Rock effectors was visualized by Western blotting. α-Tubulin was used as a loading control. Representative blots from three independent experiments are shown. p-MLC2, phospho-myosin light chain 2 (Ser-19); pp-MLC2, diphospho- myosin light chain 2 (Thr-18/Ser-19); p-MYPT, phospho-myosin phosphatase binding subunit 1 (Thr-696); pFAK, phospho- focal adhesion kinase (Tyr-397). (F–I) Control (F and G) and Shrm2 knockdown (H and I) C166 cells were stained for ppMLC (Fi–Ii) and either ZO1 (F and H) or actin (G and I). Loss of Shrm2 leads to loss of pp-MLC2 at both stress fibers and cell–cell junctions (compare arrowheads). (J–M) Stress fiber organization was examined by immunostaining for actin in C166 cells treated with siControl (J), siShrm2 (K), siControl and Y27632 (L), and siShrm2 and Y27632 (M). Inset is a merge for apical Shrm2 (green) and basal stress fibers (red). Scale bars = 25 μm.

Article Snippet: Additional antibodies were as follows: PECAM, E-cadherin (BD Biosciences, Franklin Lakes, NJ); ZO1 (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin (Sigma-Aldrich, St. Louis, MO); Rock1, pMLC2– Ser-19, ppMLC2–Thr-18/Ser-19, p-cofilin, and pFAK–Tyr-397 (Cell Signaling, Danvers, MA); Rock2 (Bethyl Labs, Montgomery, TX); pMYPT (Millipore, Temecula, CA); and GFP, TO-PRO-3, goat anti– mouse, goat anti–rat, or goat anti–rabbit secondary antibodies conjugated to Alexa-488 or Alexa-568 (Invitrogen, Carlsbad, CA).

Techniques: Binding Assay, Incubation, Staining, Knockdown, Phospho-proteomics, Western Blot, Control, Immunostaining