Model for R2TP complex-mediated recruitment of exosome and Monad-directed amphiregulin mRNA decay.

<p>See text for details.</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

Up-regulated gene in PIH1D1-knockdown MDA-MB-231 cells.

<p>Up-regulated gene in PIH1D1-knockdown MDA-MB-231 cells.</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

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

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

Up-regulated gene in RPAP3-knockdown MDA-MB-231 cells.

<p>Up-regulated gene in RPAP3-knockdown MDA-MB-231 cells.</p