Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: DEGs in non-metastatic vs. metastatic samples from GEO database. (A,B) Analysis of DEGs between 17 noninvasive BAC and 23 AC-mixed subtype invasive lung adenocarcinomas (GSE27716). A total of 2,172 DEGs, namely, 2,077 downregulated and 95 upregulated genes, were obtained (|logFC|>0.4; adj.P.value<0.01); (C,D) Analysis of DEGs in A549 cells before and after TGFβ1 induced EMT (GSE49644). A total of 2,513 DEGs, namely, 1,506 downregulated genes and 1,007 upregulated genes, were obtained (|logFC|>0.8; adj.P.value<0.001); (E,F) Analysis of DEGs in H358 cells with or without TGFβ1 treatment (GSE49644). A total of 2,295 DEGs, namely, 1,412 downregulated genes and 883 upregulated genes, were obtained (|logFC|>0.8; adj.P.value<0.001). The analysis results of DEGs are presented as heatmaps and volcano plots; (G,H) Venn diagrams showing upregulated (H) and downregulated (G) genes whose dysregulated expression pattern was shared by two GEO microarray database; (I) Analysis of DEGs between HBE-Ctrl and HBE-5Aza cells. HBE cells were treated with 5-Aza (10 μmol/L) for 96 h, and the medium was exchanged every day; (J) Venn diagrams show overlapping genes shared by 506 upregulated genes induced by 5Aza and 18 metastatic-associated downregulated genes. DEG; differentially expressed genes; GEO; Gene Expression Omnibus; BAC; bronchioloalveolar carcinomas; AC, adenocarcinomas; TGFβ1, transforming growth factor β1; EMT, epithelial-to-mesenchymal transition; HBE, human bronchial epithelial.

Article Snippet: Recombinant human TGFβ1 protein (R&D systems, Minneapolis, USA, #240-B-002) was prepared as a stock solution of 20 μg/mL in sterile 4 mmol/L HCl containing 1 mg/mL bovine serum albumin.

Techniques: Expressing, Microarray, Gene Expression

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: Stomatin is essential for transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT) in A549 cells. (A) Representative images of TGFβ1-induced EMT in A549 cells. Scale bars represent 100 μm; (B) A549 cells were treated with 5 ng/mL TGFβ1 for indicated times, and the expressions of stomatin, E-cadherin, Vimentin, N-cadherin and Slug were assessed by western blotting. β-actin was used as a loading control; (C,D) Immunofluorescences were performed to demonstrate the inhibition of stomatin by TGFβ1. E-cadherin (C) and Vimentin (D) staining indicated the induction of EMT in A549 by TGFβ1. Scale bars represent 30 μm. (E) Semiquantitative scoring was performed on stomatin and TGFβ1 staining in lung adenocarcinoma samples. Stomatin was negatively correlated with TGFβ1 in stage III (r 2 =−0.501, P=0.048) tumors; (F) A549-NC and A549-STOM cells were exposed to TGFβ1 (5 ng/mL) for 48 h. The expression levels of N-cadherin, E-cadherin, and stomatin were analyzed by western blotting. β-actin was used as a loading control.

Article Snippet: Recombinant human TGFβ1 protein (R&D systems, Minneapolis, USA, #240-B-002) was prepared as a stock solution of 20 μg/mL in sterile 4 mmol/L HCl containing 1 mg/mL bovine serum albumin.

Techniques: Western Blot, Control, Inhibition, Staining, Expressing

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: Stomatin is inhibited during transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT) in Panc-1 and MDA-MB-231 cells. (A) Panc-1 cells were treated with 10 ng/mL TGFβ1 for the indicated times, and expressions of stomatin, E-cadherin, and N-cadherin were assessed by western blotting. β-actin was used as a loading control; (B,C) Immunofluorescence microscopy was performed to demonstrate the inhibition of stomatin expression by TGFβ1. E-cadherin (B) and Vimentin (C) staining indicated the induction of EMT in Panc-1 by TGFβ1. Scale bars represents 30 μm; (D) MDA-MB-231 cells were treated with 10 ng/mL TGFβ1 for 2 d, and the expression of stomatin was assessed by real-time polymerase chain reaction (PCR). *, P<0.05. **, P<0.001. Three independent experiments were performed; (E) Semiquantitative scoring was performed, and stomatin scores had no correlation with TGFβ1 scores in stage I and II (r=0.1, P=0.477, n=53) lung adenocarcinomas.

Article Snippet: Recombinant human TGFβ1 protein (R&D systems, Minneapolis, USA, #240-B-002) was prepared as a stock solution of 20 μg/mL in sterile 4 mmol/L HCl containing 1 mg/mL bovine serum albumin.

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Inhibition, Expressing, Staining, Real-time Polymerase Chain Reaction

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: Mechanisms of stomatin inhibition during transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT). (A) A549 cells were treated with or without 5 ng/mL TGFβ1. After 12 h, cells were treated with 5 μg/mL cycloheximide (CHX) for the indicated times with or without TGFβ1. Endogenous stomatin was detected by western blotting. β-actin was used as a loading control; (B) A549 cells were stimulated with 5 ng/mL TGFβ1 for 12 h before 10 μg/mL actinomycin D (AcD) treatment; the cells were incubated with AcD for the indicated times. The expression of stomatin was determined by quantitative real-time polymerase chain reaction (PCR). Three independent experiments were performed; (C) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d with or without 5-Aza. Expressions of E-cadherin, and stomatin were analyzed by real-time PCR. *, P<0.05, **, P<0.001. Three independent experiments were performed.

Article Snippet: Recombinant human TGFβ1 protein (R&D systems, Minneapolis, USA, #240-B-002) was prepared as a stock solution of 20 μg/mL in sterile 4 mmol/L HCl containing 1 mg/mL bovine serum albumin.

Techniques: Inhibition, Western Blot, Control, Incubation, Expressing, Real-time Polymerase Chain Reaction

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: During transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT), stomatin was downregulated through methylation-dependent transcription mechanism. (A) Real-time polymerase chain reaction (PCR) was performed to validate the transcriptional change of stomatin identified in the microarray. **, P<0.001; (B) Two CpG islands spanning from −768 to −223 bp and from −143 to +122 bp upstream of STOM gene were identified by EMBOSS cpgplot program (upper). Schematic representation of −768/−223 and −143/+122 (marked with the grey square) CpG islands in the upstream of stomatin gene (lower). Red box marked the exon. The translation start site was position +1; (C) Measuring stomatin promoter methylation levels by Sequenom MassARRAY assay; (D) Sequenom MassARRAY analysis of methylation percentages of CpG sites at the stomatin promoter. The top panel showed the methylation percentages of 30 sites in human bronchial epithelial (HBE) and A549 cells. Dots with different color depths represent different methylation status. Dark dots represent high degrees of methylation. Light dots represent low degrees of methylation. The methylation percentages of 30 CpG sites were compared by two-tailed paired Student’s t test. In line graph, each dot represents an individual CpG dinucleotide. Green dots represent CpG dinucleotides in HBE cells. Red dots represent CpG sites in A549 cells. The corresponding detection sites were connected in a straight line. Real-time PCR was performed to evaluate the expression of stomatin in HBE and A549 cells (P<0.01); (E) The same sequenom MassArray and statistical analyses were also implemented to assess the methylation status of the indicated promoter region of stomatin in A549 and A549-5Aza cells (P<0.01); (F) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d or 5 d with or without 5-Aza. Expression of DNMT3A and stomatin was analyzed by western blotting. β-actin was used as a loading control; (G) Sequenom MassArray analysis was implemented to assess the stomatin promoter methylation levels in A549, A549-TGFβ1, and A549-TGFβ1/5-Aza cells (P=0.005, P=0.004, respectively).

Article Snippet: Recombinant human TGFβ1 protein (R&D systems, Minneapolis, USA, #240-B-002) was prepared as a stock solution of 20 μg/mL in sterile 4 mmol/L HCl containing 1 mg/mL bovine serum albumin.

Techniques: Methylation, Real-time Polymerase Chain Reaction, Microarray, Sequenom Massarray Assay, Two Tailed Test, Expressing, Western Blot, Control

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: Stomatin is essential for transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT) in A549 cells. (A) Representative images of TGFβ1-induced EMT in A549 cells. Scale bars represent 100 μm; (B) A549 cells were treated with 5 ng/mL TGFβ1 for indicated times, and the expressions of stomatin, E-cadherin, Vimentin, N-cadherin and Slug were assessed by western blotting. β-actin was used as a loading control; (C,D) Immunofluorescences were performed to demonstrate the inhibition of stomatin by TGFβ1. E-cadherin (C) and Vimentin (D) staining indicated the induction of EMT in A549 by TGFβ1. Scale bars represent 30 μm. (E) Semiquantitative scoring was performed on stomatin and TGFβ1 staining in lung adenocarcinoma samples. Stomatin was negatively correlated with TGFβ1 in stage III (r 2 =−0.501, P=0.048) tumors; (F) A549-NC and A549-STOM cells were exposed to TGFβ1 (5 ng/mL) for 48 h. The expression levels of N-cadherin, E-cadherin, and stomatin were analyzed by western blotting. β-actin was used as a loading control.

Article Snippet: The lung cancer tissue microarray slides included 69 cases of LUAD with detailed information of each patient were purchased from Shanghai Outdo Biotech Co., Ltd. Immunohistochemistry of these slides was performed using antibodies, such as TGFβ1 (abcam, #ab215715) and stomatin (Proteintech, Rosemont, USA, #12046-1-AP).

Techniques: Western Blot, Control, Inhibition, Staining, Expressing

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: Stomatin is inhibited during transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT) in Panc-1 and MDA-MB-231 cells. (A) Panc-1 cells were treated with 10 ng/mL TGFβ1 for the indicated times, and expressions of stomatin, E-cadherin, and N-cadherin were assessed by western blotting. β-actin was used as a loading control; (B,C) Immunofluorescence microscopy was performed to demonstrate the inhibition of stomatin expression by TGFβ1. E-cadherin (B) and Vimentin (C) staining indicated the induction of EMT in Panc-1 by TGFβ1. Scale bars represents 30 μm; (D) MDA-MB-231 cells were treated with 10 ng/mL TGFβ1 for 2 d, and the expression of stomatin was assessed by real-time polymerase chain reaction (PCR). *, P<0.05. **, P<0.001. Three independent experiments were performed; (E) Semiquantitative scoring was performed, and stomatin scores had no correlation with TGFβ1 scores in stage I and II (r=0.1, P=0.477, n=53) lung adenocarcinomas.

Article Snippet: The lung cancer tissue microarray slides included 69 cases of LUAD with detailed information of each patient were purchased from Shanghai Outdo Biotech Co., Ltd. Immunohistochemistry of these slides was performed using antibodies, such as TGFβ1 (abcam, #ab215715) and stomatin (Proteintech, Rosemont, USA, #12046-1-AP).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Inhibition, Expressing, Staining, Real-time Polymerase Chain Reaction

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: Mechanisms of stomatin inhibition during transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT). (A) A549 cells were treated with or without 5 ng/mL TGFβ1. After 12 h, cells were treated with 5 μg/mL cycloheximide (CHX) for the indicated times with or without TGFβ1. Endogenous stomatin was detected by western blotting. β-actin was used as a loading control; (B) A549 cells were stimulated with 5 ng/mL TGFβ1 for 12 h before 10 μg/mL actinomycin D (AcD) treatment; the cells were incubated with AcD for the indicated times. The expression of stomatin was determined by quantitative real-time polymerase chain reaction (PCR). Three independent experiments were performed; (C) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d with or without 5-Aza. Expressions of E-cadherin, and stomatin were analyzed by real-time PCR. *, P<0.05, **, P<0.001. Three independent experiments were performed.

Article Snippet: The lung cancer tissue microarray slides included 69 cases of LUAD with detailed information of each patient were purchased from Shanghai Outdo Biotech Co., Ltd. Immunohistochemistry of these slides was performed using antibodies, such as TGFβ1 (abcam, #ab215715) and stomatin (Proteintech, Rosemont, USA, #12046-1-AP).

Techniques: Inhibition, Western Blot, Control, Incubation, Expressing, Real-time Polymerase Chain Reaction

Journal: Chinese Journal of Cancer Research

Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

doi: 10.21147/j.issn.1000-9604.2019.06.09

Figure Lengend Snippet: During transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT), stomatin was downregulated through methylation-dependent transcription mechanism. (A) Real-time polymerase chain reaction (PCR) was performed to validate the transcriptional change of stomatin identified in the microarray. **, P<0.001; (B) Two CpG islands spanning from −768 to −223 bp and from −143 to +122 bp upstream of STOM gene were identified by EMBOSS cpgplot program (upper). Schematic representation of −768/−223 and −143/+122 (marked with the grey square) CpG islands in the upstream of stomatin gene (lower). Red box marked the exon. The translation start site was position +1; (C) Measuring stomatin promoter methylation levels by Sequenom MassARRAY assay; (D) Sequenom MassARRAY analysis of methylation percentages of CpG sites at the stomatin promoter. The top panel showed the methylation percentages of 30 sites in human bronchial epithelial (HBE) and A549 cells. Dots with different color depths represent different methylation status. Dark dots represent high degrees of methylation. Light dots represent low degrees of methylation. The methylation percentages of 30 CpG sites were compared by two-tailed paired Student’s t test. In line graph, each dot represents an individual CpG dinucleotide. Green dots represent CpG dinucleotides in HBE cells. Red dots represent CpG sites in A549 cells. The corresponding detection sites were connected in a straight line. Real-time PCR was performed to evaluate the expression of stomatin in HBE and A549 cells (P<0.01); (E) The same sequenom MassArray and statistical analyses were also implemented to assess the methylation status of the indicated promoter region of stomatin in A549 and A549-5Aza cells (P<0.01); (F) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d or 5 d with or without 5-Aza. Expression of DNMT3A and stomatin was analyzed by western blotting. β-actin was used as a loading control; (G) Sequenom MassArray analysis was implemented to assess the stomatin promoter methylation levels in A549, A549-TGFβ1, and A549-TGFβ1/5-Aza cells (P=0.005, P=0.004, respectively).

Article Snippet: The lung cancer tissue microarray slides included 69 cases of LUAD with detailed information of each patient were purchased from Shanghai Outdo Biotech Co., Ltd. Immunohistochemistry of these slides was performed using antibodies, such as TGFβ1 (abcam, #ab215715) and stomatin (Proteintech, Rosemont, USA, #12046-1-AP).

Techniques: Methylation, Real-time Polymerase Chain Reaction, Microarray, Sequenom Massarray Assay, Two Tailed Test, Expressing, Western Blot, Control

Journal: Cancer Science

Article Title: Methylation‐associated silencing of miR‐9‐1 promotes nasopharyngeal carcinoma progression and glycolysis via HK2

doi: 10.1111/cas.15103

Figure Lengend Snippet: MiR‐9‐1 is downregulated by promoter methylation in nasopharyngeal carcinoma (NPC). A, CpG islands in miR‐9‐1. The transcription start site (TSS) is indicated by a curved arrow. The regions analyzed by methylation‐specific PCR (MSP) and MassARRAY are shown. B, Relative miR‐9‐1 expression in normal nasopharyngeal NP69 cells and NPC cell lines. Glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH ) was used as the endogenous control. C, Relative miR‐9‐1 expression in 11 normal nasopharyngeal tissues (N) and 16 NPC tissues (T). U6 was used as the endogenous control. * P < .05. D, Quantitative MassARRAY methylation analysis of miR‐9‐1 in normal tissues (NT), NPC tissue (P), NP69 cell lines, NPC cell lines. E, F, MSP analysis of miR‐9‐1 promoter methylation in NPC cell lines and tissue specimens. M, methylated; U, unmethylated; L, marker; 1‐14, NPC tissue specimen numbers. G, Demethylation treatment with 5‐Aza‐2’‐deoxycytidine (DAC) restored the expression of miR‐9‐1 in NPC cell lines

Article Snippet: The Sequenom MassARRAY platform (Agena Bioscience) was applied to examine the promoter methylation level of miR‐9‐1 quantitatively.

Techniques: Methylation, Expressing, Marker

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Cluster dendrogram based on all autosomal Infinium probes distinguishes placentas of different gestational age . Dendrogram showing the relationship between placental samples from three gestational ages based on DNA methylation levels (β-values) of all analysable Infinium probes. All samples clustered within their gestational age group, with no overlap between gestations, suggesting there are consistent genome-scale DNA methylation patterns associated with each gestational age. First trimester samples clustered away from second and third trimester samples, indicating that overall Infinium methylation patterns are more similar in second and third trimester compared to first trimester.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: DNA Methylation Assay, Methylation

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Number of probes showing differential methylation between first, second and third trimester placental tissue.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Unsupervised clustering based on probes with Δβ > 0.2 between First and Third trimester . HeatMap showing unsupervised clustering of all placenta samples (x-axis) based on 954 probes with a Δβ > 0.2 between First and Third trimester (y-axis). The majority of differentially methylated probes show higher methylation in third trimester (883 probes) compared to only 71 probes with lower methylation in third trimester. Second trimester placentas cluster as a separate group, and show a methylation profile that is an intermediate of first and third trimesters. Green corresponds to low methylation and Red to high methylation.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Variance levels of probes in the third compared to the first trimester . Scatter plot of probe variance ( s 2 ) at first trimester (x-axis) and third trimester (y-axis). Dots represent individual probes and the vertical red dotted line marks s 2 = 0.02 for first trimester, while the horizontal red dotted line marks s 2 = 0.02 for third trimester. Probes on the outside of the red line are deemed 'variable'. This analysis revealed that there are 73 probes (A) which are highly variable in both first and third trimester. Only 33 probes (B) were variable in first, but not third trimester, while 279 probes (C) were variable in third, but not first trimester. This analysis suggests that most of the variable probes become so throughout pregnancy, supporting the hypothesis that accumulating environmental factors contribute to inter-individual variation in DNA methylation, in term placenta.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: DNA Methylation Assay

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Correlation between methylation and gene expression change between first and third trimester . Methylation difference (Δβ) between first and third trimester (x-axis) was plotted against gene expression log fold change (y-axis) between first and third trimester. A positive change in log fold expression indicates higher expression in first trimester, while a positive change in methylation indicates higher methylation in the third trimester. Therefore, the top left panel includes genes which showed lower methylation and higher expression in first compared to third trimester. The three highlighted genes (CCR7, GNLY and CCL21) ranked highly in IPA analysis. Grey dots represent Infinium probes. Black dots represent specific genes of interest.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation, Gene Expression, Expressing

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Average methylation of all samples for first, second and third trimester . Methylation Index (MI) was calculated for each sample by calculating the mean of all analysable Infinium β-values (26, 162 probes) for that sample. The MIs were then grouped by gestation and shown as box and whisker plots. First and second trimester placentas show a similar overall level of methylation (p = 0.46) with median MIs of 0.238 and 0.241, respectively. Third trimester samples show significantly elevated average MI values (median = 0.256) relative to both first and second trimester, indicating that there is a significant increase in methylation level from second to third trimester.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation, Whisker Assay