RA and EGF+bFGF effects on Erk phosphorylation and on cell cycle progression.

<p>A: Western blot analysis of hMSC that were cultured for 5 days in either DMEM alone (DMEM) or in the presence of 0.5 µM RA (RA) or in the presence of 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+bFGF), with or without the addition of 0.5 µM RA (EGF+bFGF+RA). As control, cells were cultured with 10%FBS (FBS), or with 0.5 µM RA in 10%FBS (FBS+RA). Whole-cell protein extracts from these differently treated cells were fractionated on a denaturating 12% polyacrylamide gel, transferred to nitrocellulose and detected with anti phosphorylated Erk antibody (pErk1/2). The membrane was stripped twice, one for detection with anti total Erk antibody (Erk1) and the second for β-actin antibody detection used as loading control. B: Densitometry analysis of A. The bars represent relative expression normalized to β-actin expression and referred to this ratio in DMEM. C–J: Cell cycle progression by FACS of hMSC that were cultured for 5 days with DMEM (C), 0.5 µM RA in DMEM (D), 20 ng/ml EGF (E), 5 ng/ml bFGF (F), 5 ng/ml bFGF +20 ng/ml EGF (G), or 5 ng/ml bFGF +20 ng/ml EGF +0.5 µM RA (H). In addition, hMSC were cultured for 2 days in DMEM (I) or in the presence of 0.5 µM RA (J) before replacement of the medium with 20 ng/ml EGF +5 ng/ml bFGF in DMEM for further 2 days. At the end of the experiment the cells were harvested by trypsinization, permeabilized and stained with propidium iodide to measure the DNA content by FACS.</p

Antibodies used for Western blot in this study.

<p>Antibodies used for Western blot in this study.</p

Microarray bioinformatical analysis of the RA effects on hMSC cultured for 5 days without serum.

<p>A: Venn diagram showing the number of differentially expressed transcripts in DMEM and RA treated hMSC when compared to 10%FBS treated cells. B: The genes regulated specifically by RA (i.e. blue or yellow in the diagram depicted in A) were clustered by Ingenuity software. This software contains a database with different transcripts arranged in networks according to their known biological interactions. According to the number of transcripts regulated in each of these networks, the program scores them. The best scored network is depicted here. Direct interactions are represented by continuous arrows and direct by dotted ones. Increase in expression is represented by red color and decrease by green. The number under each transcript name is the logarithmic change in comparison to 10%FBS control cells RNA. The type of interaction is indicated by a label and the type of molecule is indicated by the shape of the box, being both detailed at the right of the figure.</p

hMSC osteogenesis is inhibited by RA and enhanced by EGF+bFGF.

<p>hMSC were cultured for 21 days in osteoblasts differentiation medium without serum supplemented with 10% FBS (A), 10% FBS +0.5 µM RA (C), nothing (D), 0.5 µM RA (E), 20 ng/ml EGF +5 ng/ml bFGF (F), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (G), or were cultured in DMEM supplemented with 10% FBS (B) or with 20 ng/ml EGF +5 ng/ml bFGF (H) (negative controls). To visualize differentiation osteoblasts were stained with alizarin red after fixation in 70% ethanol. Size bars = 100 µm.</p

Primers used for QRT-PCR in this study.

<p>Primers used for QRT-PCR in this study.</p

Periostin can rescue the effect of RA on hMSC osteogenesis.

<p>A: hMSC were treated for 5 days with either DMEM, 0.5 µM RA in DMEM (RA), 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+FGF), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (EGF+FGF+RA), 10%FBS (FBS), or 0.5 µM RA in 10%FBS (FBS+RA). The RNAs were purified and the cDNAs were synthesized using reverse transcriptase. Periostin transcript was quantified in each sample. The error bars represent relative expression normalized to RNF10 expression and referred to the relative expression on DMEM as mean ± s.e.m. All the treatments were performed in triplicates in two independent experiments with cDNA from different donors and the significance of the results was assessed using one way ANOVA and Tukey's multiple comparison test. B–G: hMSC were cultured in osteoblasts differentiation medium supplemented with 0.5 µM RA (B–D), or in 10%FBS +0.5 µM RA (negative control) (E–G), for 21 days over uncoated plastic (B and E), 2.5 µg laminin/well coated plastic (C and F), or 2.5 µg periostin/well coated plastic (D and G). Osteoblasts were stained with alizarin red. Size bars = 100 µm.</p

hMSC adipogenesis is inhibited by RA and enhanced by EGF+bFGF.

<p>hMSC were cultured for 21 days in either adipocytes maintenance medium (days 7–8 and 15–16) or adipocytes induction medium (the rest of the days) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012689#s4" target="_blank">Materials and Methods</a> for seeing the media composition), without serum, supplemented with 10% FBS (A), 10% FBS +0.5 µM RA (C), nothing (D), 0.5 µM RA (E), 20 ng/ml EGF +5 ng/ml bFGF (F), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (G). In addition, hMSC were cultured in DMEM supplemented with 10% FBS (B) or with 20 ng/ml EGF +5 ng/ml bFGF (H) (negative controls). To visualize differentiation, adipocytes were stained with Oil red O after fixation with 4% paraformaldehyde. Size bars = 100 µm.</p

RA and EGF+bFGF treatments affect laminin expression which modulates cell migration and adhesion of hMSC.

<p>A: QRT-PCR analysis of laminin alpha 1 expression in hMSC that were treated for 5 days with either DMEM, 0.5 µM RA in DMEM (RA), 20 ng/ml EGF+ 5 ng/ml bFGF (EGF+bFGF), 20 ng/ml EGF +5 ng/ml bFGF +0.5 µM RA (EGF+bFGF+RA), 10%FBS (FBS), or 0.5 µM RA in 10%FBS (FBS+RA). The error bars represent relative expression normalized to RNF10 expression and referred to the relative expression on DMEM as mean ± s.e.m. B: Wound healing experiments were performed on hMSC that were previously treated for 5 days as in A and also treated with 0.5 µM RA followed by EGF+bFGF at the time of the experiment (preRA+EGF+bFGF). The error bars represent % of the initial gap after 7 hours as mean ± s.e.m. C: Similar wound healing experiments as shown in B but hMSC were previously seeded over either laminin coated (20 µg/well) (laminin) or uncoated plates (plastic) and treated as in A. The error bars represent % of the initial gap after 7 hours as mean ± s.e.m. D: Cell spreading assay of hMSC that were treated for 5 days similarly as in A (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012689#s4" target="_blank">Materials and Methods</a>). The error bars represent cell perimeter as mean ± s.e.m. All the treatments in A–D were performed in triplicates in two independent experiments with different hMSC donors and the significance of the results was assessed using one way ANOVA and Tukey's multiple comparison test (for A and B) or with two way ANOVA followed by Bonferroni post test (for C and D). *P<0.05; **P<0.01; ***P<0.001.</p

Identification, annotation of novel cervical cancer modules and comparison with embryonic stem cell modules using both in silico and in vivo studies (ENCODE) and correlation of module activity with apoptosis and cell cycle.

<p>A. Venn diagram of commonly transcription regulators found to be enriched near the TSS (-3000, +200 bp) of upregulated genes in each gynecological cancer. B. Scatter plot showing the correlation between the HeLa cell line and a cervical cancer array (r = 0.84). C. Heatmap of the correlation of the expression of all gynecological cancers and the corresponding normal samples, with the expression of HeLa, K562, A549, HepG2 and normal brain cells as calculated from more than three independent studies for each tissue or cell line. Accession numbers from all the studies are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142229#pone.0142229.s007" target="_blank">S3 Table</a>. D. Bar graph depicting the enrichment of annotated transcription regulators from ENCODE in HeLa cells in the upregulated genes in cervical cancer patients. E. Heatmap of transcription factor binding site overlap of transcription regulators enriched in cervical cancer upregulated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142229#pone.0142229.s008" target="_blank">S4 Table</a>). F. Heatmap showing the correlation of JUN and AP2 modules with apoptosis, and of E2F/NFY with cell cycle regulation. G. Bar graphs of the correlation of E2F/NFY and MAX/CEBP modules with Myc module for upregulated genes. H. Bar graphs of the correlation of E2F/NFY and MAX/CEBP modules with Prc module for downregulated genes.</p

Endocervix columnar, squamocolumnar and ectocervix squamous gene signatures and ES enrichment in cervical, endometrial and vulvar cancer patients.

<p>A. Enrichment of differentially expressed genes from our gene signatures of cervical, endometrial and vulvar cancer with endocervix columnar, squamocolumnar junction (SC) and ectocervix squamous gene signatures. For significant differences with <i>p</i> < 0.05, the asterisk was used for annotation. B. Enrichment of differentially expressed genes from our gene signatures from cervical, endometrial and vulvar cancer with Myc, Prc and Core modules, identified to play a key role in the formation and establishment of pluripotency in embryonic stem cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142229#pone.0142229.ref033" target="_blank">33</a>]. For significant differences with <i>p</i> < 0.05, the asterisk was used for annotation.</p