Western blot.

<p>Representative images of western blots performed in all tested LCLs, subsequently quantified as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195388#pone.0195388.g004" target="_blank">Fig 4</a>.</p

HLC outcome obtained by microarray expression profile of A-T samples.

<p>A total of 675 differentially expressed transcripts allowed us to classify AT129RM and AT50RM as similar to each other, while the other A-T samples behaved differently. The same behaviour pattern was inferred by western blot analysis.</p

Western blot analysis of all tested LCLs.

<p>The protein abundance of selected targets in sample AT129RM is in agreement with the 2DE outcome (paired t-test p<0.05) except for 14.3.3 ζ/δ (see text). Only the AT50RM sample behaved in a similar manner to the AT129RM sample, despite the ATK13RM and ATK36RM cell lines. The W-N graphic reports the whole lane normalization data of the WB experiments.</p

2DE representative images.

<p>A-T and WT samples treated or not treated with Dexa. Each gel image was elaborated with Melanie software. Three technical replicates were used.</p

Veen diagram.

<p>The splicing and expression outputs were compared and plotted to show differences about spliced and altered expression genes between WT and AT. Only small amounts of gene symbols were shared in all tested comparisons.</p

Proteins regulated by Dexa inferred by the 2DE experiments.

<p>All comparisons scores. The bold text column shows the protein ratio (“<b>U</b>” upregulated, “<b>D</b>” downregulated and “<b>=“</b> as unvaried) of the indicated comparison, while the plain text reports the gene expression ratio (U upregulated, D downregulated and “<b>=“</b> as unvaried), the variance analysis p value and the FDR p value.</p

FKBP5, TMEM2 and NFIL3 gene expression by qPCR.

<p>The well-known genes altered by Dexa administration have been tested by qPCR in order to validate the microarray procedure.</p

Additional file 1: Figure S1. of ATM splicing variants as biomarkers for low dose dexamethasone treatment of A-T

Melting analysis of IEDAT qPCR amplicons. During SYBR green qPCR of the IEDAT samples, although only one PCR product was observable by melting step at the end of the experimental, some Tm discrepancies were noted among the samples. A different kind of ATMdexa1 target was probably amplified without affecting the PCR performance. (TIFF 41 kb

Molecular and Functional Characterization of Three Different Postzygotic Mutations in <i>PIK3CA</i>-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors

<div><p>Background</p><p><i>PIK3CA</i>-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic <i>PIK3CA</i> mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed.</p><p>Methods</p><p>We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 <i>in vitro</i>.</p><p>Results and Conclusion</p><p>Our data indicate that patients’ cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.</p></div

Clinical and mutational spectrum of the three index cases.

<p><b>a</b> Patient 1, clinically diagnosed with MCAP, showing diffuse capillary malformation at the age of 2 months and cutaneous syndactyly between the 2^nd and 3^rd toes. The <i>PIK3CA</i> c.241 G>A [p.E81K] mutation detected by Sanger sequencing in affected cells and tissues of patient 1 showed varying levels of the mutant allele depending on the tissue tested. The mutation was absent in the patient's blood and in her parents. <b>b</b> Macrodactyly of the right 4^th finger in patient 2, diagnosed with FAO, at the age of 17 years. Sequence of <i>PIK3CA</i> exon 20 in blood and cultured fibroblasts obtained from patient 2 showing that the mutation is undetectable in these samples. <b>c</b> Patient 3, at the age of 15 months before surgical intervention; note the disproportion of the left 2^nd and 3^rd fingers and the subcutaneous mass at the left deltoid region. Sanger sequencing validation of the c.3140 A>T [p.H1047L] mutation detected with targeted deep sequencing in the biopsy from the 2^nd finger of patient 3. <b>d</b> List of samples and mutations detected with targeted deep sequencing. Coverage indicates the mean average of reads on target in the regions of interest (ROI) while frequency denotes the percentage of reads with the mutation.</p