Global analysis of transcriptional changes.

<p>A) RNA from three sets of triplicate cultures (passage 3 wild type [P3], passage 5 wild type [P5], and passage 3 <i>Tgif1</i> null MEFs [null]) and was analyzed on Affymetrix expression arrays. A Venn diagram is shown with the numbers of probe-sets that changed significantly in each of three pair-wise comparisons (null – P3, null – P5, and P5 – P3). B) An analysis of the overlap between the null – P3 and null – P5 comparisons is shown as a four-way Venn diagram, allowing overlaps between a maximum of two data-sets. The arrows indicate the direction of the change in signal: For example, pale blue arrow indicates increased signal in the null – P3 comparison. Of the 376 probe-sets that increased in the null – P3 comparison, 176 increased and 50 decreased in the null – P5 comparison, whereas 150 did not change significantly in the null – P5. The distribution of changes in the probe-sets present in the overlaps (176, 25, 50, 140) was analyzed using a 2×2 contingency table and a chi squared test. The chi squared value and p-value are shown above. C) An analysis of overlap of the data from the null – P3 and P5 – P3 comparisons is shown, as in panel B. D) Expression of ten genes from the overlaps shown in B and C was analyzed by qRT-PCR in RNAs from triplicate cultures of wild type and <i>Tgif1</i> null MEFs at both passage 3 and 5. Expression is presented as the average (+ s.d.) in arbitrary units with the P2 wild type set equal to 1 for each gene. Significance levels as determined by ANOVA are indicated above (* p<0.05, ** p<0.01, *** p<0.001).</p

Proliferation defects with transient knock-down of <i>Tgif1</i> in NMuLi cells.

<p>A) NMuLi and NMuMG cells were transfected with siRNAs targeting <i>Tgif1</i>, or with a control pool, and Tgif1 protein levels were analyzed 48 hours later, by western blot. Smad2 levels are shown as a loading control. B) Control and <i>Tgif1</i> knock-down NMuLi and NMuMG cells were analyzed for senescence associated β-gal staining 72 hours after knock-down. The percentage of SA β-gal positive cells is presented as mean + s.d. of triplicate transfections, together with p values. C) Control and <i>Tgif1</i> knock-down NMuLi cells were analyzed for EdU incorporation, as a measure of proliferation. Cells were incubated with EdU for 1 hour, 48 hours after transfection. Data is presented as mean + s.d. of triplicate transfections, together with the p value. D) Expression of the ten genes analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figure 5D</a> was tested in control and <i>Tgif1</i> knock-down NMuLi cells by qRT-PCR from triplicate cultures. Data is shown for <i>Tgif1</i> and the six genes for which differences in expression were significant. All p values were determined by the Student's T test (* p<0.05, ** p<0.01, *** p<0.001, for panel D).</p

Overlap of TGFβ-mediated transcriptional changes with those in <i>Tgif1</i> null MEFs.

<p>A) Data from the comparison of <i>Tgif1</i> null to wild type P3 MEFs was compared to that from MEFs treated with TGFβ for 10 hours (from GSE15871). Total numbers of probe-sets with significant changes, and the overlaps are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figure 5</a>. Chi squared analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figure 5</a> and is shown above. B) Data from the comparison of <i>Tgif1</i> null to wild type P3 MEFs was compared to MEFs treated with TNFα for 4 hours (from GSE3700). Data was analyzed and is presented as in A. C) Twelve genes represented in the overlap shown in panel B were analyzed by qRT-PCR. Six genes each were selected from those that went up in both and those that went down in both. Expression was analyzed in triplicate cultures of passage 3 <i>Tgif1</i> null MEFs treated with a TGFβ receptor kinase inhibitor (1 µM SB-431542), or left untreated. Data is shown as mean (+ s.d.) with the value in the untreated cells set to 1 in each case. * p<0.05, ** p<0.01, *** p<0.001, as determined by the Student's T test. Shown below is the fold change (on a linear scale) in the comparison of the <i>Tgif1</i> null to wild type P3 array data for each gene.</p

Increased DNA damage in MEFs lacking Tgif1.

<p>Passage 2 wild type and <i>Tgif1</i> null cells were analyzed by comet assay, under denaturing conditions. The percentage of total DNA in the tail was quantified for at least 50 cells per condition. A) The percentage of DNA in the tail is plotted for each of two independent batches of wild type and <i>Tgif1</i> null MEFs. Data is plotted as median, 25^th and 75^th percentiles (box) and 5^th and 95^th percentiles (whiskers). p-values determined by the Student's T test are shown. B) The data shown in A, binned into 5% blocks, are plotted to show the distribution. C) Cells were treated with 100 µM H₂O₂ for 20 minutes and analyzed by comet assay at time-points thereafter over a 160 minute time-course. Data are presented as in A, with p-values for comparisons between wild type and Tgif1 null shown where significant. D) representative images of mitotic cells with DNA bridges are shown for <i>Tgif1</i> null cells. Cells analyzed for α-tubulin (green), pHH3 (red), and Hoechst (blue) are shown, together with a merged image of all three colors. Images were captured at 40×. Scale bar = 25 µM. E) Wild type and <i>Tgif1</i> null MEFs were grown on a 3T3 protocol in a standard incubator (5% CO₂ in air [20% O₂]), or in a chamber with 5% CO₂ and 3% O₂. Growth is plotted as relative cumulative cell number, with the starting 300,000 cells at P2 set equal to 1.</p

TGFβ induces growth inhibition and senescence.

<p>A) The TGFβ data-set from GSE15871 was compared to probe-set changes between P3 and P5 wild type MEFs. Data was analyzed and is presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035460#pone-0035460-g007" target="_blank">7</a>. B) The culture conditions over passages 2 to 5 are shown schematically: Arrows below indicate times at which TGFβ (1 pM or 3 pM) was added for the 3T3 assay. The time of addition of TGFβ and EdU is shown above, and the time at which the SAβG assay was performed is shown below. Relative cell proliferation was measured at passage 3 and 5 in cells grown under standard conditions, followed by a single 24 hour treatment with TGFβ at the indicated concentration. The percentage of EdU positive cells is shown. C) Cell proliferation was determined in a 3T3 assay and is shown as cumulative increase in cell number. Cells were cultured under standard conditions or with the addition of 1 or 3 pM TGFβ at the times indicated in panel B. D) The percentage (average + s.d. of triplicate cultures) of wild type cells with positive SAβG staining is shown at passage 5, after continued treatment with 1 pM or 3 pM TGFβ, or under standard conditions. Significant differences between control and plus TGFβ are shown: * p<0.05, ** p<0.01, as determined by the Student's T test. E) A tentative model describing the involvement of Tgif1 and TGFβ in the pathways leading to cellular senescence.</p

<i>Tgif1</i> null MEFs have increased DNA damage foci.

<p>A) Representative images of passage 5 cells stained with an antibody against γH2AX (green) and Hoechst (blue) are shown. Images were captured at 20×. Scale bar = 50 µM. B) The distribution of the number of γH2AX damage foci per nucleus is shown for passage 4 and 5 wild type and <i>Tgif1</i> null cells. Significance values were determined by Chi squared test, comparing the distribution in <i>Tgif1</i> null to that expected based on the wild type. C) The distributions of the number of γH2AX damage foci per nucleus in passage 4 cells were compared to those from cells that had been grown in 3% oxygen from passage 2 to 4. Data is presented and analyzed as in B. Note that the P4 MEFs analyzed for damage foci in 20% oxygen in panels B and C are the same. p-values for the comparisons of wild type to <i>Tgif1</i> null and 3% to 20% oxygen are shown below.</p