Additional file 1: of The protein kinase Cmk2 negatively regulates the calcium/calcineurin signalling pathway and expression of calcium pump genes PMR1 and PMC1 in budding yeast

Figure S1. Amino acid sequence comparison between ScCMK2, SpCMK2 and ScRCK2. (PDF 88 kb

Characterization of HUVECs by morphology and immunofluorescence.

<p>(A) Confluent cells exhibited typical cobblestone morphology; insert image at 200X magnification. (B and C) Immunofluorescence of samples labeled for VE-cadherin (B) or Von Willebrand factor (C). The immunoreactive positive cells were stained red and imaged at 400X magnification.</p

The correlation of COX-2 and PTPRJ expression in aortic endothelial cells from whole-genome array expression analysis GSE39264.

<p>(A) Differential expression of PTPRJ from analysis of a MAEC whole-genome array. *P<0.05 versus untreated group. (B) Differential expression of COX-2 from analysis of a MAEC whole-genome array. *P<0.05 versus untreated group. (C) Correlation of COX-2 and PTPRJ expression from analysis of a MAEC whole-genome array. (Pearson correlation coefficient, −0.87; n = 22; P<0.01, two-tailed). Genes were selected from the microarray data using a threshold of 10% FDR (false discovery rate). P values for fold change between untreated and treated MAECs were determined by <i>t</i>-test; P values for fold change were adjusted using Benjamini–Hochberg.</p

Injury-induced down-expression of PTPRJ is correlated with increased COX-2.

<p>(A–D) Representative sections of hematoxylin and eosin-stained arterial samples from control (A and B) and BI (C and D) rabbits. BI significantly induced neointimal hyperplasia. Images acquired at 200X magnification. (E) Morphometric data *P<0.05 versus uninjured group; n = 3 in each group. (F–G) PTPRJ and COX-2 expression quantified by qRT-PCR. Values were normalized to ß-actin expression. *P<0.05 versus uninjured group. (H) Correlation of COX-2 and PTPRJ expression (r = −0.74, P≤0.01).</p

PTPRJ expression is rescsued by the COX-2 selective inhibitor Celecoxib.

<p>(A) Western blot analysis of PTPRJ and COX-2 in protein extracts from HUVEC cells stably over-expressing COX-2 and treated for 24 h with 20 µM or 10 µM Celecoxib. Expression is normalized to GAPDH. Relative levels of PTPRJ were quantified using Image J. (B) ELISA quantification of PGE₂ in HUVEC supernatant; data is expressed as concentration of PGE₂. ** P<0.01 compared to the 0.1% DMSO-treated control group. (C) Relative COX-2 and PTPRJ expression in HUVEC cells treated with varying concentrations of Celecoxib. Expression is normalized to GAPDH. Relative levels of COX-2 and PTPRJ were quantified using Image J. *P<0.05.</p

PTPRJ expression is down-regulated by COX-2 over-expression.

<p>(A) Western blot analysis of PTPRJ and COX-2 in protein extracts from HUVEC cells transiently transfected for 48 h with COX-2 plasmids. Expression is normalized to GAPDH. Relative protein levels were quantified using Image J. (B) ELISA quantification of PGE₂ in the supernatant of HUVECs after transfection with COX-2 plasmids. The data is expressed as PGE₂ concentration. ** P<0.01 versus control group. (C) Correlation of COX-2 and PTPRJ expression in HUVECs after transfection with COX-2 plasmids. (Pearson correlation coefficient, −0.90, P<0.01). (D) Relative PTPRJ expression in HUVEC cells transfected with varying concentrations of COX-2 plasmids and quantified by qRT-PCR. Values were normalized to PRL22 expression. *P<0.05 versus control group.</p

Primers used for the real time analysis.

<p>Primers used for the real time analysis.</p

Supplementary Methods, Supplementary Figures 1-10, Supplementary Tables 1-17, Supplementary References from Genome-Wide Association Study Identifies a New Locus at 7q21.13 Associated with Hepatitis B Virus–Related Hepatocellular Carcinoma

Supplementary Figure 1. An overview of the study workflow; Supplementary Figure 2. The principal components analyses (PCA) of samples in the discovery stage and reference samples from the 1000 Genomes Project data. Supplementary Figure 3. Manhattan plots of the genome-wide P values from the association tests on HBV-related HCC. Supplementary Figure 4. Quantile-quantile plots of the observed P values from the association tests on HBV-related HCC. Supplementary Figure 5. Forest plots for rs10272859 across all studies. Supplementary Figure 6. Expression levels of CDK14 mRNA were significantly higher in HCC tissues compared with adjacent non-tumor liver tissues. Supplementary Figure 7. The at-risk G allele of rs10272859 was significantly associated with higher mRNA levels of CDK14 in liver tissues based on the TCGA data. Supplementary Figure 8. The SNPs at 7q21.13 interact physically with the promoter region of CDK14 in GM12878 cells. Supplementary Figure 9. Kaplan-Meier estimates of the overall survival time for the HCC patients stratified by CDK14 mRNA expression levels. Supplementary Figure 10. Kaplan-Meier estimates of disease-free survival time for patients with HBV-related HCC stratified by genotypes of rs10272859. Supplementary Table 1. Summary description of the populations used in this study. Supplementary Table 2. Summary of the quality controls in proband-parent trios. Supplementary Table 3. Summary of SNP imputation in the discovery stage. Supplementary Table 4. The amount of SNPs with various P values in TDT for the 189 trios in the discovery stage. Supplementary Table 5. Summary of the SNPs that have been reported significantly associated with HBV-related HCC in previous GWASs. Supplementary Table 6. Summary of SNPs that have been reported to be significantly associated with HBV-related phenotypes in previous GWASs. Supplementary Table 6. Summary of SNPs that have been reported to be significantly associated with HBV-related phenotypes in previous GWASs (Continued). Supplementary Table 7. Summary of the top 15 SNPs in the discovery stage. Supplementary Table 8. Primers and probes used in this study. Supplementary Table 9. Summary of the replication studies for the 14 SNPs newly identified in the discovery stage. Supplementary Table 10. Stratification analyses of rs10272859 by gender and age. Supplementary Table 11. The associations between genotypes of rs10272859 and mRNA levels of nearby genes. Supplementary Table 12. The predicted functional relevance of rs10272859 and the other 74 SNPs in strong linkage disequilibrium with rs10272859 at the ~110 Kb region of 7q21.13. Supplementary Table 13. Details for the genotypes of rs10272859 and the prognosis of patients with HBV-related HCC. Supplementary Table 14. Multivariate analyses of overall survival time and disease-free survival time of patients with HBV-related HCC. Supplementary Table 15. Genotype distribution of rs10272859 across TNM stages in patients with HBV-related HCC. Supplementary Table 16. Powers for various genetic effects and various minor allele frequencies. Supplementary Table 17. The allele and genotype frequencies of rs10272859 in different populations.</p