H-RasV12 induces cell cycle arrest in immortalized <i>FHL2^−/−</i> MEFs.

<p>(A) Immortalized wt, <i>FHL2^−/−</i> and <i>FHL2^−/−-</i>restored MEFs were infected with pBabe-RasV12 or control pBabe retrovirus and selected for drug resistance. (B) All <i>FHL2^−/−</i> cells were positive in β-galactosidase assay due to the insertion of <i>Lac Z</i> cDNA at the <i>FHL2</i> locus. (C) Ki-67 immunofluorescence analysis in vector- and H-RasV12-transduced <i>FHL2^−/−</i> MEFs. Nuclei were counterstained with DAPI. (D) Expression of Ras, p53, p21^CIP1, p16^INK4a and cyclin D1 in H-RasV12- (R) or vector- (V) transduced wt and <i>FHL2^−/−</i> cell lines. Equal amounts of protein extracts were analyzed by immunoblotting.</p

Absence of foci formation in immortalized <i>FHL2^−/−</i> MEFs upon expression of H-RasV12.

<p>(A) Focus formation assay of MEFs transiently transfected with H-RasV12 or empty vector. Plates were stained with Giemsa at 3 weeks after transfection. Data are representative of at least 3 independent experiments using 3 spontaneously immortalized clones. To restore FHL2 expression, primary <i>FHL2^−/−</i> MEFs were infected with a retrovirus expressing FHL2 (pBabeFHL2puro) and cultured according to the 3T3 protocol. Two <i>FHL2</i>-restored <i>FHL2^−/−</i> cell pools were obtained and used in the study. pBabe vector was used as a control (Babe). (B) Chart depicting at least three independent experiments. Mean colony numbers±standard deviation are shown. Bottom: Immunoblot analysis of FHL2 expression in <i>FHL2</i>-restored cell line. (C) Analysis of H-RasV12 expression in MEFs transfected with either empty (V) or H-RasV12 (R) expression vector. Samples were processed for immunoblotting 48 h after transfection. Actin was used as loading control.</p

Immortalized <i>FHL2^−/−</i> MEFs stably expressing cyclin D1 are susceptible to transformation by oncogenic Ras.

<p>(A) Increased cyclin D1 expression in immortalized <i>FHL2^−/−</i> MEFs after introduction of cyclin D1-HA transgene was confirmed by immunoblotting with anti-cyclin D1 and anti-HA antibodies. (B) Representative phase contrast images of immortalized wt and <i>cyclin D1-HA/FHL2^−/−</i> cell lines transduced with H-RasV12 or empty vector. (C) Soft agar assay. Cells were initially seeded at 5×10⁴ cells/plate and cultured for 10 days. Images are representative of three wt and two <i>cyclin D1-HA/FHL2^−/−</i> cell lines transduced with H-RasV12. (D) Western blot analysis of p53 induction by doxorubicin in the indicated cell lines as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003761#pone-0003761-g003" target="_blank">Fig. 3A</a>.</p

FHL2 deficiency accelerates enterocyte movement.

<p>(A) Representative images of BrdU-positive cells along the crypt-villus axis at 2 h and 48 h post-BrdU labelling in <i>Apc^Δ14/+FHL2^+/+</i> and <i>Apc^Δ14/+FHL2^−/−</i> mice. (B) Distribution of BrdU-positive cells at 48 h. The crypt base was set as position 0. Three mice of each genotype were analyzed. (C) Representative images of BrdU-positive cells along the crypt-villus axis at 48 h post-BrdU labelling in wt and <i>FHL2^−/−</i> mice. Original magnifications: X200 (A and C).</p

Reversal of the cell cycle arrest in H-RasV12-transduced <i>FHL2^−/−</i> MEFs.

<p>(A) The indicated cell lines transduced by H-RasV12 or control vector are shown at cell cycle arrest stage (passage 0) and after reversal of the cell cycle arrest (passage 10). (B) Analysis of the p53 protein in cells at passage 10 after H-RasV12 transduction. Cells were treated with doxorubicine as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003761#pone-0003761-g003" target="_blank">Fig. 3A</a>. (C) Western blot analysis of cell cycle regulators in wt and <i>FHL2^−/−</i> cell lines at passage 10 after transduction with H-RasV12 (R) or empty vector (V). (D) Growth curves of the indicated cell lines after reversal. Equal numbers of cells were initially plated in triplicate wells, and cells were counted at indicated times. Mean values±standard deviation from three independent experiments are shown. Data presented are representative of 3 independent H-RasV12- and vector-expressing MEF lines.</p

Up-regulation of FHL2 in intestinal tumors from <i>Apc^Δ14/+</i> mice and human colon tumors.

<p>(A) FHL2 immunostaining of normal intestine and adenoma from <i>Apc^Δ14/+</i> mouse. Original magnifications, X200. (B) Quantitative RT-PCR analysis of FHL2 expression in adenoma and adjacent normal tissue from five <i>Apc^Δ14/+</i> mice. FHL2 expression was normalized to 18S RNA. The ratio of the FHL2/18S signal in NT was arbitrarily set at 1. The average values and standard deviations for three independent experiments from five <i>Apc^Δ14/+</i> mice are shown. Average fold induction in adenomas is 1.73 (<i>p</i> = 0.011, Student's <i>t</i> test). (C) FHL2 expression in normal human colon (a), in human low-grade (b) and high-grade colon dysplasia (c) and in human colon carcinoma (d) by immunohistochemistry. Control staining on high-grade dysplasia and human colon carcinoma was performed as FHL2 staining without the primary anti-FHL2 antibody. Original magnifications, X200. Part of the image of human colon carcinoma was further enlarged to better visualize individual cells (e).</p

Figure 4

<p>Expression of p53 and p21^CIP1 target genes in <i>FHL2</i>-null cells (A) Left: GSEA plot using a gene set of up-regulated p53-dependent genes from the Molecular Signatures Database (MSigDB) (NES = 2.35, FDR<0.01, <i>p</i><0.0001). Enrichment score (ES) reflects the correlation of the gene set with FHL2^−/− (KO) and WT genotypes. Middle: Core group of genes of the up-regulated p53-dependent gene set that contributed to the ES. The fold change (KO/WT) value for genes that have more than one probe set corresponds to the mean value. Right: Expression profile of the deregulated genes at fold change ≥1.4 in 3 different clones for each genotype. Data are plotted as a heatmap where red and blue correspond to high and low expression in log₂-transformed scale. (B) GSEA Analysis using down-regulated p21^CIP1-dependent gene set of MSigDB (NES = −2.25, FDR<0.01, <i>p</i><0.0001). Expression profile of the top down-regulated genes at fold change ≤0.8 that are differently expressed in the FHL2^−/− compared to wt clones. Abbreviations: NES: normalized enrichment score, FDR: false discovery rate. (C) Quantitative RT-PCR analysis of Cdkn1a (p21^CIP1), Pltp, Ube2c, Ccdc99, Tubb3 transcripts in three independent wt and <i>FHL2^−/−</i> immortalized clones. Data were normalized to 18S RNA. Results are expressed as relative expression of each gene/18S RNA assuming wt cells as 1.0. Average values and standard deviations for three independent experiments are shown.</p

Loss of FHL2 reduces intestinal polyp multiplicity.

<p>(A) Representative images of intestines from 11-month-old <i>Apc^Δ14/+FHL2^+/+</i> and <i>Apc^Δ14/+FHL2^−/−</i> mouse. Note the marked decrease in the number of polyps in <i>Apc^Δ14/+FHL2^−/−</i> mice. (B) Total number of intestinal polyps was counted in 11 <i>Apc^Δ14/+FHL2^+/+</i>, 16 <i>Apc^Δ14/+FHL2^−/+</i> and 18 <i>Apc^Δ14/+FHL2^−/−</i> mice at 11-month-old. Compared to <i>Apc^Δ14/+FHL2^+/+</i> littermates, significant reduction in polyp number was observed in <i>Apc^Δ14/+FHL2^−/+</i> (<i>p</i><0.0046) and <i>Apc^Δ14/+FHL2^−/−</i> mice (<i>p</i><0.0001). (C) Polyp distributions are expressed as the percentages of mice having 0–9, 10–19, 20–29, 30–39 or more than 40 polyps in the intestine. (D) FHL2 deficiency had no effect on tumor growth. The percentages of polyps in each mouse with sizes less than 2 mm, between 2 to 5 mm and more than 5 mm are presented.</p

Analysis of the p53/ARF pathway in immortalized <i>FHL2^−/−</i> MEFs.

<p>(A) Functional analysis of the p53 protein. After induction by doxorubicin (doxo), p53 was immunoprecipitated from immortalized MEFs and analyzed by immunoblotting. Wild type clones are indicated by capital letters and <i>FHL2^−/−</i> clones by lower-case letters. p53 induction was determined as the ratio of the relative p53/actin intensities after doxo treatment to p53/actin intensities without doxo in each cell line. (B) Western blot analysis of p19^ARF expression in seven immortalized clones of each FHL2 genotype. (C) Human papillomavirus E6 oncoprotein restores Ras-mediated transformation in immortalized <i>FHL2^−/−</i> MEFs. Cells were infected with E6-expressing retrovirus (HPVE6) or control virus (Babe), and then transiently transfected with H-RasV12 plasmid or control vector, cultured for 3 weeks without splitting and stained. Data are representative of three independent experiments in two independent E6-transduced <i>FHL2^−/−</i> MEF clones. (D) Dominant negative p53 (p53DD) was expressed alone or coexpressed with H-RasV12 by retroviral vectors in immortalized <i>FHL2^−/−</i> MEFs.</p

Activation of the Wnt signalling in the intestine of <i>Apc^Δ14/+FHL2^−/−</i>mice.

<p>(A) Immunostaining for β-catenin in adenomas (a, b), for cyclin D1 in adenomas (c, d) and normal intestine (e, f) and for c-myc in normal intestine (g, h) in <i>Apc^Δ14/+FHL2^+/+</i> (a, c, e, g) and <i>Apc^Δ14/+FHL2^−/−</i> mice (b, d, f, h). Sections were photographed at the same magnification. Original magnifications: X200. (B) Activation of the Wnt target genes cyclin D1 and c-myc in adenomas is unaffected by FHL2 deletion. Real-time RT-PCR analysis of cyclin D1 and c-myc expression was performed with intestinal tumor (T) and adjacent normal tissue (NT). Cyclin D1 and c-myc expression was normalized to 18S RNA and normalized values in normal intestine of <i>Apc^Δ14/+FHL2^+/+</i> was arbitrarily set at 1. The average values and standard deviations from two independent experiments from four mice of each genotype are shown. The p value is calculated according to Student's test.</p