Figure 3 | Scientific Reports

Figure 3

From: A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

Figure 3

Molecular structure and biological functions of SIN3A c.2830 C>T; p.Gln944* (A) Molecular structures of SIN3A-WT and the SIN3A c.2830 C>T; p.Gln944* nonsense mutant are shown in the upper panel and lower panel, respectively. (B) The sequenced reads of the coding region in SIN3A using SOLiD5500 were shown. (C) The point mutation of SIN3A c.2830 C>T; p.Gln944* as detected in breast cancer tissue was confirmed by Sanger sequencing. (D) Alignments of amino acid sequences adjacent to Gln944 in SIN3A in various species. The PAH4 domain on SIN3A is highly conserved in various species from yeasts to humans. (E) ESR1 mRNA expression was increased in MCF7 cells transfected with the SIN3A c.2830 C>T; p.Gln944*. MCF7 cells were transfected with the indicated vectors together with pMACS4.1 and were incubated with 100 nM E2 for 24 hrs. The ESR1 mRNA was measured by qPCR after the separation with an autoMACS pro. The data represent the means ± SE obtained in 4 independent experiments. Statistical significance was calculated by ANOVA with the Tukey–Kramer multiple comparison test (*P < 0.05). (F) The intracellular localizations of SIN3A were observed in MCF7 cells transfected with the SIN3A mutant. MCF7 cells were transfected with the indicated vectors together with GFP-tagged ERα expression vector. The cells were incubated in culture medium containing TMR ligand for Halo-tag stain and Hoechst33342 for nucleus stain at 24 hrs after transfection. SIN3A (red), ERα (green) and nucleus (blue) were observed under a confocal microscope LSM710. The pictures show representative results obtained from 6 independent experiments. Scale bar shows 20 µm. (G) Changes in intracellular localizations of SIN3A-WT or SIN3A c.2830 C>T; p.Gln944* were calculated from the total fluorescence intensities in the nucleus and the cytoplasm using an IN Cell analyzer 2000 and IN Carta software. The data represent the means ± SE obtained from 190 cells transfected with empty vector, 183 cells transfected with SIN3A-WT, and 341 cells transfected with SIN3A c.2830 C>T; p.Gln944*. Statistical significances were determined by ANOVA with the Tukey–Kramer multiple comparison test (*P < 0.05).

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