H520-Rnd3 and H358-Rnd3 cells have a lower proliferation rate.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) The generation of an H358 cell line stably expressing GFP-tagged Rnd3. The Rnd3 expression was verified by both Rnd3 and GFP antibodies. (<b>C</b>) <b>&</b> (<b>D</b>) The generation of an H358 cell line stably expressing GFP-tagged Rnd3. The Rnd3 expression was verified by both Rnd3 and GFP antibodies. (<b>E</b>) <b>&</b> (<b>F</b>) H358-Rnd3 has a lower proliferation rate compared to H358 cells as detected by BrdU incorporation. (<b>G</b>) <b>&</b> (<b>H</b>) H520-Rnd3 has a lower proliferation rate compared to H520 cells as detected by BrdU incorporation. (<b>I</b>) <b>&</b> (<b>J</b>) Cell number was quantified at different time point. An average of three samples at each time point was presented in this figure. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D.</p

Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.

<p>(<b>A</b>) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (<b>B</b>) <b>&</b> (<b>C</b>) A western blot to detect phosphorylated MYPT1, NICD in cells. (<b>D</b>) <b>&</b> (<b>E</b>) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (<b>F</b>) <b>&</b> (<b>G</b>) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *<i>p</i><0.05 compared to control (group 0); #<i>p</i><0.05 compared to group 1; $<i>p</i><0.05 compared to group 3. Data represent means ± S.D.</p

Rnd3 is down-regulated in non-small lung cancer cell lines, H520 and H358.

<p>(<b>A</b>) Rnd3 mRNA detected by qRT-PCR is down-regulated in H358 and H520 compared to HBEC. (<b>B</b>) Rnd3 protein expression levels in cells by western blot. (<b>C</b>) Densitometry quantification of western band intensity in B. (<b>D</b>), (<b>F</b>), (<b>H</b>) <b>&</b> (<b>I</b>) A western blot to detect phosphorylated MYPT1, phosphorylated MLC2, ROCK1 and NICD in cells. (<b>E</b>), (<b>G</b>) <b>&</b> (<b>J</b>) Densitometry quantification of western band intensity showed up-regulation of Rho Kinase activity and NICD expression in H358 and H520 cells compared to HBEC. Western blots were quantified from three independent experimental repeats. BrdU-positive cells were quantified from 8 images taken from four slides. Data represent means ± S.D.</p

Rnd3 inhibits proliferation by promoting NICD degradation in H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) Hes1 was up-regulated in H520 and H358 cells compared to HBEC cells. (<b>C–F</b>) Hes1 expression decreased when Rnd3 was stably overexpressed in H358 and H520 cells. (<b>G</b>) <b>&</b> (<b>H</b>) Transient overexpression of Rnd3 in H358 cells down-regulated NICD and Hes1 in a dosage dependent manner. (<b>I</b>) <b>&</b> (<b>J</b>) Inhibition of proteasome activity by MG132 (final concentration of 15 µM) abolished the Rnd3 dosage dependent NICD down-regulation in H358 cells. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D. *<i>p</i><0.05 compared to control (group 0); <b>#</b><i>p</i><0.05 compared to group 1; <b>$</b><i>p</i><0.05 compared to group 3. Data represent means ± S.D.</p

Inhibition of Notch signaling prevented proliferation of H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) The application of compound E blocks the proliferation of H358 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (<b>C</b>) <b>&</b> (<b>D</b>) The application of compound E blocks the proliferation of H520 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. BrdU-positive cells were quantified from 8 images taken from four slides. Data represent means ± S.D.</p

Reintroduction of Rnd3 corrects Rho and Notch signaling in H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) Decreased NICD expression in H520-Rnd3 cells compared to H520 cells. (<b>C</b>) <b>&</b> (<b>D</b>) Decreased Rho Kinase activity in H520-Rnd3 cells compared to H520 cells detected by antibodies specific for pMYPT1 and pMLC2. (<b>E</b>) <b>&</b> (<b>F</b>) Decreased NICD expression in H358-Rnd3 cells compared to H358 cells. (<b>G</b>) <b>&</b> (<b>H</b>) Decreased Rho Kinase activity in H358-Rnd3 cells compared to H358 cells detected by antibodies specific for pMYPT1 and pMLC2. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D.</p

Additional file 2 of Genome-wide association analyses identified novel susceptibility loci for pulmonary embolism among Han Chinese population

Additional file 2: Table S1. Independent genome-wide significant lead SNPs associated with pulmonary embolism (PE) in the discovery stage and replication stage. Table S2. The allele frequency of identified loci in 1000Genomes. Table S3. Replication of associations for the known loci in our cohort. Table S4. Association results for genes that were significant in FUMA gene-based analysis. Table S5. Polygenic risk score variants. Table S6. Polygenic risk score (PRS) quantile and odds ratio (OR)

Additional file 1 of Genome-wide association analyses identified novel susceptibility loci for pulmonary embolism among Han Chinese population

Additional file 1: Fig. S1. Regional association plot at genome-wide association study (GWAS) genome-wide significant loci. Fig. S2. Principal component analysis (PCA) plot of Han Chinese PE cohort. Fig. S3. FUMA Manhattan plot and QQ plot of genome-wide association study (GWAS) meta-analysis. Fig. S4. The transfection efficiency of cellular experiments for FABP2. Fig. S5. Low-density lipoprotein cholesterol (LDL-C) levels of patients with different genotypes of rs1799883. Fig. S6. Forest plot for the association of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) with PE. Fig. S7. Ancestry-specific polygenic risk score (PRS) ROC plot. Fig. S8. Performance of different PRSVTE in the CURES testing set