Application of strain to fibrin gels.

<p>a) Fibrin gels were subjected to continuous tensile strain that was applied using a custom-made device. b) The length of gel extension (<i>L</i>) from the initial gel length (<i>L₀</i>) was defined as the applied strain (<i>L/L₀•100</i>%).</p

Myoblasts in strained fibrin gels.

<p>a) Myoblasts patterning in a strained fibrin gel (Bar: 100 µm). b) The alignment of randomly selected cells was determined by using image-analysis software (Bar: 200 µm). c) Nuclear staining of myoblasts in a strained fibrin gel. The arrows indicate proliferating cells (Bar: 100 µm). d) Linearly aligned cell groups formed in a strained hydrogel (Bar: 400 µm). e) Hematoxylin-eosin (HE) staining of linearly aligned myoblasts sets(Bar: 200 µm). f) HE staining of a longitudinal section of rat skeletal muscle tissue (Bar: 50 µm). g) A cross-section of a strained fibrin gel containing myoblasts. The cells were evenly distributed in the hydrogel, and adjacent cells were not in contact with each other (HE staining, bar: 50 µm). h, i) SEM image of myoblast positions in a strained fibrin gel (h) (Bar: 100 µm) and in a control fibrin gel (i) (Bar: 100 µm). j) HE staining of myoblasts in a control fibrin gel (Bar: 200 µm). k) Cell proliferation in the fibrin gels subjected to different strains. Asterisks indicate significant difference (p<0.01). The red arrow in the figure indicates the strain direction.</p

HUVECs in strained fibrin gels.

<p>a) Alignment of HUVECs in a strained fibrin gel. b) Aligned vessel-like structure in a fibrin gel (arrows). c) HUVECs in a control fibrin gel. d) Cells that developed lumens in a fibrin gel system. Asterisks indicate significant difference (p<0.01). The red arrow in the figure indicates the strain direction. The scale bars are 100 µm for b and c.</p

Orientation of fibrin fibrils and formation of bundle-like structures in fibrin gels.

<p>a, b) AFM images of a control fibrin gel (a) and a strained fibrin gel (b, 50% strain). c, d) SEM images of bundle-like structures formed in a strained fibrin gel (c, 25% strain; d, 100% strain; bar: 5 µm). e) SEM image of a control fibrin gel (bar: 1 µm). f) SEM image of the border of the bundle-like structure in a strained fibrin gel (bar: 1 µm). g) Typical strain-stress curve of a fibrin gel used in this study. h) Representative image of a cross-section of a strained fibrin gel as observed under a phase-contrast microscope (TE2000, Nikon, Japan; bar, 100 µm). The border of the bundle-like structure was highlighted using the brush tool of Adobe Photoshop software (Adobe, CA, USA). i) A cross-section of native rat skeletal muscle tissue (Hematoxylin-eosin staining, HE). Each bundle exhibits a polygonal shape and the morphology resembles that in the cross-section of the strained fibrin gel shown in (h) j) The cross-sectional area of individual bundle-like structures was measured using image-analysis software (Lumina Vision, Mitani, Japan) and extrapolated to circular cross-sections to calculate the average diameter of the bundles under strains of specific magnitudes. The red arrow in the figure indicates the strain direction.</p

Knockdown of Monad increased amphiregulin mRNA.

<p>(A, C, D) Relative mRNA levels were analyzed by real-time RT-PCR at 48 h after the treatment of MDA-MB-231 cells with the indicated siRNA and expressed as percentage to that of control siRNA-treated cells from four independent experiments (mean ± S.E.). Data were normalized based on GAPDH mRNA copy numbers. (B, C) Mouse Monad was overexpressed by doxycycline (Dox). Monad protein levels (B) and relative amphiregulin mRNA levels (C) were analyzed by immunoblotting and real-time RT-PCR, respectively, at 48 h after the treatment of Dox-regulated MDA-MB-231 cells with control or Monad siRNA. *<i>P</i><0.01 vs. control. ^†<i>P</i><0.01 vs. Dox (−).</p

Interaction between amphiregulin mRNA and Monad.

<p>(A) The lysate from MDA-MB-231 cells were immunoprecipitated with anti-Monad antibody. Normal rabbit IgG was used as control. Quantitation of associated mRNA was performed using real-time-RT-PCR and normalized to GAPDH. (B) Binding of recombinant Monad to 3′-UTR of amphiregulin. After RNA pull-down assay using 3′-UTR of sense (+) or antisense (-, control) strand of amphiregulin as probes, separation by SDS-PAGE and silver staining were performed. (C) Interaction of Monad with OIP2. MDA-MB-231 lysate was immunoprecipitated (IP) with control IgG or Monad antibody. Following separation by SDS-PAGE, immunoblotting was performed using anti-OIP2 antibody or Monad antibody.</p

Monad knockdown stabilizes amphiregulin mRNA.

<p>MDA-MB-231 cells were transfected with amphiregulin promoter (A) or 3′-UTR (B) -luciferase (Luc) reporters and after 2 days they were analyzed using luciferase reporter assays. The results represent the mean values with S.E. from three independent experiments. (C) MDA-MB-231cells were treated with either control or Monad siRNA and after 2 days with 5 µg/ml of actinomycin D (ActD). Relative amphiregulin mRNA levels at the different time points were analyzed by real-time RT-PCR and expressed as percentages of the level at the 0-h time point from four independent experiments (mean ± S.E.). Data were normalized based on GAPDH mRNA copy numbers. (D) The decay rates of amphiregulin were determined using the Click-iT Nascent RNA Capture Kit (Invitrogen) and expressed as percentages of the level at the 0-h time point from four independent experiments (mean ± S.E.). Data were normalized based on GAPDH mRNA copy numbers.</p

Secretion of amphiregulin is regulated by Monad.

<p>Amphiregulin secreted from MDA-MB-231cells overexpressing GFP or Monad (A) and treated with control or Monad siRNA (B) was measured by an ELISA. The amphiregulin concentration in the medium was normalized to cell number. Error bars represent the S.E. of three independent experiments. *<i>P</i><0.01 vs. control.</p

Monad does not affect the proliferation of MDA-MB-231 cells.

<p>MDA-MB-231 cells treated with control or Monad siRNA (A) or overexpressing GFP or Monad (B) were plated in 24-well cell culture plate at 2×10⁴cells per well and the proliferation was measured using the MTT assay.</p

Monad regulates cell invasion of MDA-MB-231 cells.

<p>Invasiveness of GFP or Monad overxpressing MDA-MB-231 cells (A), tetracycline-regulated Monad overexpressing MDA-MB-231 cells (B), and control or Monad siRNA treated MDA-MB-231 cells (C) was assayed by a Boyden chamber method 2 days after transfection. In (B), Monad was overexpressed by doxycycline (Dox). Results are presented as means of the number of cells/well with S.E. Cells were cultured in triplicate wells/experiment and the experiment was replicated three times. (C) Knockdown of Monad increased invasiveness of MDA-MB-231 cells. Amphiregulin-neutralizing antibody (1 µg/ml) inhibited the increased invasion by Monad siRNA. *<i>P</i><0.01 vs. control. ^†<i>P</i><0.01 vs. control IgG.</p