FIGURES AND TABLES
FROM:
Cigarette smoke induces demethylation of prometastatic oncogene synuclein-
in lung cancer cells by downregulation of DNMT3B
H Liu, Y Zhou, S E Boggs, S A Belinsky and J Liu
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Figure 1.
SNCG expression and methylation status in A549 and H292 lung cancer cell lines. (a) SNCG mRNA expression in two lung cancer cell lines was examined by RT–PCR analysis. Two micrograms of total RNA was used in the reaction of reverse transcription in a volume of 20 
l. Two microliters of the RT product was used in PCR with specific primers to SNCG or GAPDH. The RT–PCR products were separated on a 2% agarose gel and stained with ethidium bromide. SNCG protein expression in these two cell lines was examined by Western blot by using 50 g of protein of total cell lysate. MSP was used to assess the methylation status of SNCG CpG island in A549 and H292 cells. M, methylated PCR product; U, unmethylated PCR product. (b) The entire map of CpG island from each cell line was obtained by genomic sequencing as described in the 'Materials and methods' section. Positions are indicated relative to the translation start codon, and each circle in the figure represents a single CpG site. For each cell line, six independent clones were sequenced and each row represents one clone. 
, unmethylated; 
, methylated. (c) A549 cells were treated with 5
M of 5-aza-C for 4 or 6 days. Genomic DNA, total RNA and cell lysates were separated at the end of treatment. The methylation status of SNCG CpG island was determined by MSP; SNCG mRNA and protein expressions were determined by RT–PCR and Western blot analysis.
Figure 2.
Dose- and time-dependent induction of DNA demethylation and reexpression of SNCG in A549 cells by CSE. A549 cells were exposed to the indicated concentrations of CSE for 3 days or to a concentration of 2.5% CSE for different days. The medium was changed every day with fresh medium and CSE. By the end of treatment, genomic DNA and total RNA were isolated from untreated and treated cells. (a) Dose-dependent effects of CSE on SNCG mRNA expression and CpG island demethylation in A549 cells. (b) Time-dependent effects of CSE on CpG island demethylation in A549 cells examined by MSP (left panel). The methylation status of A549 cells treated with CSE for 3 days was further determined by genomic sequencing. (c) Kinetics of induction of SNCG mRNA expression by CSE in A549 cells. (d) Dose-dependent effects of NNK on SNCG mRNA expression in A549 cells.
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Figure 3.
Effects of CSE on the methylation status of TSGs and prometastatic oncogenes. A549 cells were untreated or treated with 5% CSE for 3 or 6 days. Genomic DNA was isolated and modified by sodium bisulfite. MNMSP was conducted as described in the 'Materials and Methods' section. M, methylated PCR product; U, unmethylated PCR products.
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Figure 4.
Effects of CSE on lung cancer cells proliferation and invasion. (a) A549 cells were seeded in 24-well culture plates at a density of 1 
104 cells/well and cultured in 0.5 ml medium containing 10% FBS without or with 5% CSE. At indicated time, cells were trypsinized and viable cells were counted. Values are the mean of triplicate wells. (b) A549 cells were treated with 0, 1.2 and 5% CSE for 3 days. The invasive capacities of cells were determined using two-compartment Boyden chambers and the basement membrane matrigel. The 8 m pore polycarbonate filters were coated with matrigel and used to analyse 5 104 cells in each chamber. After 40 h, filters were fixed, washed and stained. Cells were examined under a light microscope. Under 200 magnification, 10 randomly selected fields were examined and the average number of cells invaded per field was calculated. Photographs of representative views from control and CSE-treated cells are shown in the top panel and the numbers of invaded cells are presented in the bottom graph. The data shown are representative of three separate experiments. **
P<0.01 as compared to untreated control. (c) siRNAs directed to two different regions of SNCG mRNA were cotransfected into A549 cells. A nonspecific siRNA with scrambled sequence was also transfected into A549 cells as transfection control. Twenty-four hour posttransfection, cells were treated with 5% CSE for 3 days. siRNA-transfected cells and nontransfected cells were trypsinized and were tested in Boyden chamber assay for their invasive capacities. *
P<0.05 as compared to without siRNA. (d) H292 cells were transfected with SNCG siRNA or the scrambled control siRNA for 3 days. A portion of transfected cells was used in Boyden chamber assay and rest of cells were lysed for Western blot analysis of SNCG protein expression. *
P<0.05 as compared to without siRNA.
Figure 5.
Correlation of SNCG expression with inhibited DNMT3B expression by CSE in A549 cells. (a) Analysis of DNMT1 expression by western blot. A549 cells were treated with 5% CSE for 1–3 days. Total cell lysate was isolated at the indicated times and analysed for DNMT1 protein expression by Western blotting. (b) Analyses of DNMT3A and DNMT3B mRNA expression by real-time RT–PCR. A549 cells were treated with 5% CSE for a period of 0–72 h. At the indicated time points, total RNA was isolated and quantitative real-time RT–PCR with predeveloped probes to human DNMT3A, DNMT3B and GADPH was conducted to assess the mRNA levels. After normalization with GAPDH, the abundance of DNMT3A or DNMT3B mRNA in untreated cells was defined as 1, and the amounts of each mRNA from CSE-treated cells were plotted relative to that value. The figure shown is representative of three independent experiments in which each sample was assayed in triplicates. The results are mean 
s.d. (c) CSE withdrawal. A549 cells were untreated or treated with 5% CSE for 3 days. Then the fresh medium without CSE was added to cells, and the cells were harvested for genomic DNA and total RNA isolations at the indicated time points. The methylation status of SNCG was determined by MSP, the SNCG mRNA expression was examined by RT–PCR and the mRNA levels of DNMT3B were assessed by quantitative real-time PCR. DNMT3B mRNA in untreated cells was defined as 1, and the amounts of DNMT3B mRNA from CSE-treated cells at different time points after CSE withdrawal were plotted relative to that value. (d) Blocking DNMT3B expression with siRNA induces SNCG mRNA expression. A549 cells were transfected with DNMT3B siRNA or a scrambled RNA (as control) for 3 days. The mRNA levels of SNCG and DNMT3B were assessed by RT–PCR (inset) and real-time quantitative RT–PCR.
Figure 6.
Silencing SNCG transcription by exogenous expression of DNMT3B in H292 cells. H292 cells were transiently transfected with pcDNA-DNMT3B plasmid or the control empty vector (mock). Three days posttransfection, nuclear extracts, total RNA and genomic DNA were isolated from transfected cells. (a) RT–PCR analysis of SNCG and DNMT3B mRNA expressions. (b) Twenty micrograms of nuclear extracts obtained from cDNA-DNMT3B-transfected or mock-transfected cells were incubated with the methyl donor [3H]AdoMet and unmethylated poly(dI-dC)-poly(dI-dC). After incubating at 37°C for 3 h, the reaction mixtures were passed through GF/C filters and precipitated with TCA. Total counts of mean disintegration per minute of [3H]CH3 incorporation retained on the filters represented the level of de novo DNMT activity. The data shown are the mean of duplicates. (c) MSP analysis of SNCG CpG island in mock and pcDNA-DNMT3B-transfected cells. (d) The methylated PCR product from pcDNA-DNMT3B-transfected cells and the unmethylated PCR product from mock-transfected cells were cloned into pCR2.1-TOPO vector. After transformation, plasmid DNAs of 6–7 clones from each DNA sample were sequenced. CpG positions are indicated relative to the translation start codon, and each circle in the figure represents a single CpG site. , demethylated; , methylated. Each row represents a single clone.
Figure 7.
Silencing SNCG expression by DNMT3B not by other DNMTs. H292 cells were transiently transfected with plasmid pcDNA-DNMT1, pcDNA-DNMT3A, pcDNA-DNMT3B or pcDNA3.1 empty vector (mock) individually. Three days posttransfection, cells were harvested and total lysates were prepared for Western blot analysis using anti-SNCG antibody. Overexpression of individual DNMTs in respectively transfected cells was confirmed by real-time RT–PCR analyses. The data shown represent three separate transfection experiments.
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