PKCα expression and cell proliferation/migration/invasion inhibition by the truncated-MZF-1 (MZF-1ΔDBD) or-Elk-1 (Elk-1ΔDBD) proteins in SK-Hep-1 cells.

<p>The inhibitory effects of transfection with MZF-1ΔDBD and Elk-1ΔDBD constructs from RT-PCR (A) and Western blot analysis (B) assays. SK-Hep-1 cells were transiently transfected with FLAG vector (5 μg), c-Myc vector (5 μg), FLAG-MZF-1ΔDBD (5 μg), or c-Myc-Elk-1ΔDBD (5 μg). (C and D) Effect of MZF-1ΔDBD and Elk-1ΔDBD on cell growth. The SK-Hep-1 cell was transfected with the indicated dose of MZF-1ΔDBD and Elk-1ΔDBD for 1 and 2 d. Untreated cultures were designated as control (Control). Absorbance values obtained from untreated cells on day 0 after subculture were considered 100%. The migration (E and F) and invasion (G and H) assays were performed on cell cultures treated with 5 μg dose of FLAG vector, MZF-1ΔDBD, c-Myc vector, and Elk-1ΔDBD, as described in the Materials and Methods Section. Values are presented as means ± SE of three replicates from two independent experiments. **, P < 0.01 vs. FLAG or c-Myc vector-transfected group.</p

MZF-1 and Elk-1 form a complex at the MZF-1 and Elk-1 sites of the PKCα promoter.

<p>(A) Interaction between endogenous MZF-1 and Elk-1 proteins in SK-Hep-1 cells. Protein extracts underwent immunoprecipitation (IP) with anti-MZF-1, anti-Elk-1, or control rabbit IgG, as indicated. Proteins were resolved via SDS-PAGE and then underwent immunoblotting (IB) using anti-MZF-1 or anti-Elk-1 antibodies. (B) N-terminal domain of MZF-1 interacts with Elk-1 at the C-terminal domain of the protein. SK-Hep-1 cells were transfected with expression vectors for FLAG-MZF-1ΔDBD (5 μg) or c-Myc-Elk-1ΔDBD (5 μg), as indicated. Protein extracts underwent IP with anti-Myc and anti-FLAG, as indicated. Proteins were resolved via SDS-PAGE and underwent IB using an anti-MZF-1 or anti-Elk-1 antibody. (C) ChIP and Re-ChIP assays. In the ChIP assay (left), the chromatin was pulled down with IgG, MZF-1, and Elk-1 antibodies, whereas in the Re-ChIP assay (right), the pulled down chromatin interacted with Elk-1 and then with MZF-1 antibodies, after which the sequence was reversed. The PCR products shown are the 210 bp bands in the PAGE result. The input represents the purified chromatin for parallel PCR reaction as a positive control. (D) ChIP assay performed on SK-Hep-1 cells transfected with 5 μM sense or antisense of MZF-1 or with 5 μM sense or antisense of Elk-1. PCR was performed on purified chromatin as control (Input) and on chromatin fragments from the IP with the specific antibodies. The data represent 1 of 3 independent experiments with similar results.</p

Activation of PKCα expression by combinations of MZF-1 and Elk-1 directly binding to the promoter.

<p>(A) Transcriptional activation of PKCα promoter activity by wild-type MZF-1 and Elk-1 and DNA-binding deficient mutants MZF-1 (MZF-1ΔDBD) and Elk-1 (Elk-1ΔDBD) via luciferase assays. MZF-1 and Elk-1 synergistically enhance PKCα transcriptional activity when co-transfected to Huh-7 and HepG2 cells, whereas co-transfection of the DNA-binding domain deletion mutant of MZF-1 (MZF-1ΔDBD)/Elk-1 (Elk-1ΔDBD) failed to enhance the PKCα transcriptional activity. Luciferase activity was normalized to the level of β-galactosidase. Transcriptional activity is expressed as fold induction compared with the level obtained with each reporter vector in the presence of empty pcDNA 3.1/<i>Myc</i>-His vectors. The results are the mean ± SE of three independent experiments performed in triplicate. (B) MZF-1 and the Elk-1 binding sites regulate the PKCα promoter activity in Huh-7 cells. Cells were co-transfected with different mutant PKCα promoter luciferase constructs and vectors containing MZF-1 and Elk-1 transcription factors. Luciferase activity was normalized to the level of β-galactosidase. The results are the mean ± SE of three independent experiments performed in triplicate. **, P < 0.01 versus the cells transfected with normal PKCα promoter constructs in each group.</p

Suppression of tumorigenesis in MZF-1ΔDBD-transfected cells.

<p>(A) Tumor growth curve in FLAG (5 μg; ■) or MZF-1ΔDBD (5 μg;●) transfected human SK-Hep-1 xenografts (n = 5). A total of 5 × 10⁷ FLAG- or MZF-1ΔDBD-pretreated cells were injected into the flank region of nude mice. Tumor development was observed in 2 months, and the mice were then sacrificed. Cross-sectional tumor diameters were measured externally, and the approximate tumor volume was calculated as described in the Materials and Methods Section. The results represent the mean ± SE of five developed tumors in the experimental mice. **<i>P</i> < 0.01 vs. FLAG pretreated group. (B) Visible tumor (white arrow) formed by the indicated pretreated cells on day 56. The blots of the anti-FLAG indicated the presence of exogenous MZF-1-(1–72) in cells. β-actin was designed as a housekeeping gene.</p

The two-dimensional plots of gastric adenocarcinomas.

<p>Scatter plots of the first and second important discriminant function plotted against one another. The two-dimensional plots showed the separation of signet ring cell adenocarcinoma, mucinous adenocarcinoma, from and tubular carcinoma and papillary adenocarcinoma (●: signet ring cell adenocarcinoma; □: mucinous adenocarcinoma; Δ: tubular adenocarcinoma; ○: papillary adenocarcinoma).</p

The two-dimensional plots of gastric adenocarcinomas and normal gastric mucosa.

<p>Scatter plots of the first and second important discriminant function plotted against one another. The two-dimensional plots showed the separation of gastric adenocarcinoma from normal gastric mucosa (▽: normal gastric mucosa; ●: signet ring cell adenocarcinoma; □: mucinous adenocarcinoma; Δ: tubular tubular adenocarcinoma; ○: papillary adenocarcinoma).</p

The two-dimensional plots of gastric adenocarcinomas.

<p>The first two calculated principal components (PC1 and PC2), which contained most of the information, were plotted against each other for visualization. The PC1 accounting for the largest Raman spectra variance was 89.85%, and PC2 was 4.45%. The two-dimensional plots showed the separation of signet ring cell adenocarcinoma, mucinous adenocarcinoma, from and tubular carcinoma and papillary adenocarcinoma (●: signet ring cell adenocarcinoma; □: mucinous adenocarcinoma; Δ: tubular adenocarcinoma; ○: papillary adenocarcinoma).</p

sj-docx-1-aut-10.1177_13623613221138690 – Supplemental material for Association of labor epidural analgesia exposure with long-term risk of autism spectrum disorder in offspring: A meta-analysis of observational studies

Supplemental material, sj-docx-1-aut-10.1177_13623613221138690 for Association of labor epidural analgesia exposure with long-term risk of autism spectrum disorder in offspring: A meta-analysis of observational studies by Kuo-Chuan Hung, Jen-Yin Chen, Chung-Hsi Hsing, Chih-Wei Hsu, Ping-Hsin Liu, Ying-Jen Chang, Jui-Yi Chen, Sheng-Fu Chiu and Cheuk-Kwan Sun in Autism</p

Performance report of sample classification by using 20-fold cross validation.

<p>Performance report of sample classification by using 20-fold cross validation.</p

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