1. mir-200c Regulates Induction of Apoptosis through CD95 by Targeting FAP-1 
Robert Schickel, Sun-Mi Park, Andrea E. Murmann, Marcus E. Peter 
Molecular Cell 
Volume 38, Issue 6, Pages (June 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
Altering Levels of miR-200 Changes Sensitivity of Cells to CD95-Mediated Apoptosis
(A) Phase contrast pictures showing morphological changes of cells transfected with scrambled pre-miRNA (scr) and pre-miR-200c (three times over 9 days) or cells stably infected with a retrovirus expressing scrambled oligo (vec) and miR-200c. Levels of mir-200c in retrovirus infected cells were quantified by real-time PCR.
(B) Cells shown in (A) stimulated through CD95. HCT116 cells were transfected with LNA200 seven times prior to stimulation. Cells ectopically expressing miR-200c or cells treated with LNA200 were stimulated with different concentrations of anti-APO-1 for 20 hr and apoptosis was quantified by MTS assay.
(C) CAKI-1 cells transfected as in (B) were treated with 1 μg/ml anti-APO-1 and 1 μg/ml cycloheximide for 20 hr. (D) Cells with increased or reduced levels of miR-200 were treated with 2 μg/ml of staurosporine for 20 hr and cell death was quantified by MTS assay. Asterisks indicate p values ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Note: different CD95 stimuli were used in the assays shown in (B) because type I cells are not sensitive to sCD95L, and type II cells are not sensitive to noncrosslinked anti-APO-1 (Algeciras-Schimnich et al., 2003). Values in graphs (A), (B), and (D) represent the mean ± SD from three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2 FAP-1 Is Expressed in Mesenchymal Cells
(A) Schematic diagram of the 3′UTR of FAP-1 with the location of the putative miR-200b,c/429 target site.
(B) Relative expression of FAP-1 protein in epithelial and mesenchymal cells among the NCI60 cells. The horizontal bars in (B) represent the mean.
(C and D) Correlation between expression level of miR-200c (% of max) (C) or let-7d (D) and FAP-1 protein in the NCI60 cells. Expression values were transformed to rank values in both axes, and straight lines indicate linear regression for transformed rank values. Rho = Spearman Rank Correlation Coefficients between miRNAs and FAP-1 expression, and p values are given.
(E) Detection of FAP-1 and E-cadherin by Western blot analysis of CAKI-1 and ACHN cells infected with lentiviruses expressing control shRNA or shRNAs for FAP-1. β-actin was detected to demonstrate equal loading.
(F) Real-time PCR analysis of FAP-1 and E-cadherin in CAKI-1 cells expressing control shRNA or FAP-1 shRNAs relative to expression of GAPDH. Values in the graph in (F) represent the mean ± SD from three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3 Identification of FAP-1 as a Target of miR-200c
(A) ACHN, CAKI-1, and SKOV3ip cells were transfected with scrambled pre-miRNA or pre-miR-200c. HCT116 cells were serially treated seven times with LNA scrambled or LNA200. Expression of FAP-1 and E-cadherin was determined by Western blot analysis.
(B) HCT116 cells were transfected as in (A). FAP-1 mRNA level was determined by semiquantitative RT-PCR. Actin mRNA was determined as a control.
(C) 293T cells were transfected with reporter plasmids containing the 3′UTR of FAP-1 (psiCHECK 3′UTR FAP-1 WT) or its corresponding mutant (psiCHECK 3′UTR FAP-1mut) together with either 1 pmol of pre-miRNA-scrambled or pre-miR-200c. Reporter plasmids with a fragment of the 3′UTR of ZEB2 (harboring sites 4, 5, and 6 of the seven mir-200 sites found in the full length) and its mutant (Park et al., 2008) were used as positive controls. Renilla luciferase activity was normalized to firefly luciferase activity.
(D) IGROV-1 (miR-200 low) and HCT116 (miR-200 high) cells were transfected with reporter plasmid psiCHECK 3′UTR FAP-1.
(E) IGROV-1 cells were transfected with psiCHECK 3′UTR FAP-1 WT or its mutant together with 1 pmol of scrambled pre-miRNA, pre-miR-200a, or pre-miR-200c. HCT116 cells were transfected with the same reporter plasmids together with 50 nM of LNA scrambled or LNA-200 oligos. ∗p < 0.05, ∗∗p < Values in (C) —(E) represent the mean ± SD from three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4
Targeting of FAP-1 by miR-200c Regulates Sensitivity to CD95-Mediated Apoptosis
(A) HCT116 cells infected with control shRNA or FAP-1 shRNA#2 were serially treated seven times with scrambled LNA or LNA-200. Cells were stimulated with different concentration of sCD95L, and apoptosis was measured by MTS assay.
(B) Real-time PCR analysis of FAP-1, E-cadherin, ZEB1, and ZEB2 in HCT116 cells. The insert shows a Western blot analysis of the same experiment.
(C) Flow cytometric analysis of surface CD95 expression of cells with increased or reduced miR-200. Scrambled oligo or miR-200c was introduced in ACHN, CAKI-1 and HeyA8 cells either transiently or stably. For HCT116 cells, cells were serially treated seven times with scrambled LNA or LNA-200. Percentage of CD95 positivity and mean fluorescence intensity (MFI) were determined. SD, standard deviation. Values in (A) and (B) represent the mean ± SD from three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions