Skip to main content
Fig. 1 | The Journal of Physiological Sciences

Fig. 1

From: Gene expression of mesoderm-specific transcript is upregulated as preadipocytes differentiate to adipocytes in vitro

Fig. 1Fig. 1

Expression of Mest in 3T3-L1 cells during adipogenic differentiation. a Representative oil red O staining of lipid droplets on day 7 in undifferentiated (undiff.) and differentiated (diff.) 3T3L1 cells. b Quantitative determination of Oil Red O (a measure of lipid accumulation) in undifferentiated (undiff.) and differentiated (diff.) 3T3-L1 cells. Data are expressed as mean ± SD (n = 6). Double asterisks represents significant oil red O retention in differentiated cells at the P < 0.01 level. c The levels of PPARγ2 expression in 3T3-L1 cells during adipogenic differentiation were measured by RT-PCR and expressed as ratios of the values measured in 3T3-L1 cells on day 1 (n = 3). ND not detected. Double asterisks represents significant differences in untreated cells on day 0 at the P < 0.01 level. d The gene expression values in control 3T3-L1 cells and in cells that had been pretreated with 5 μM 5-aza-dC (denoted by open circles and closed circles, respectively) are expressed as ratios of values measured in untreated 3T3-L1 cells at day 0, normalized against 36B4 mRNA levels. Data are expressed as mean ± SD (n = 3). Double asterisks represents significance compared to untreated cells at day 0 (P < 0.01); single and double hash symbols represent significant differences in gene expression levels in cells pretreated with 5-aza-dC compared with untreated cells (P < 0.05 and P < 0.01 respectively). e Representative RT-PCR results of Mest expression in 3T3-L1 cells. f The genomic region around the transcriptional start site of the Mest gene (−175 to +92 bp) shows as bisulfite-modified DNA (cytosines are converted to thymines). Methylated and unmethylated CpGs are shown as boxed YGs (Y pyrimidine). The Fnu4HI cutting site (GC/NGC) at the gray highlighted area is maintained, since the methylated cytosines in CpG islands are not converted to uracils by bisulfite treatment. The PCR products (319 bp) are cleaved into 222- and 97-bp fragments by Fnu4HI. However, Fnu4HI will not digest if the site is converted to any one of the sequences GTGGT, GTGGC or GCGGT by bisulfite-PCR. Underlined are primer sequences for PCR amplification. Exon1 begins at the double underline. g Genomic DNA was prepared from control 3T3-L1 cells or 3T3-L1 cells pretreated with 5 μM 5-aza-dC on days 0 and 7 after the induction of differentiation. The DNA was treated with bisulfite and amplified by PCR using a specific primer set. The PCR products were incubated in the presence or absence of Fnu4HI. The fragments were subjected to electrophoresis on 2 % agarose gel stained with ethidium bromide. Representative results from the COBRA assay are shown. h DNA methylation state of CpG islands around the Mest gene transcription starting site (TSS) (−175 to +92 bp) in control 3T3-L1 cells and 3T3-L1 cells that had been pretreated with 5 μM 5-aza-dC. Data points are taken from days 0 and 7 after the induction of differentiation. The percentage of methylated DNA was calculated from the band intensity ratio between the Fnu4HI-cleaved PCR fragments and the total amount of PCR product. Data are expressed as mean ± SD (n = 3). Double hash symbols indicates significant difference compared to control (P < 0.01)

Back to article page