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. 2016 Oct 12;11(10):e0164544. doi: 10.1371/journal.pone.0164544

Fig 5. Arginine vasopressin (AVP)-secreting neurons from mouse embryonic stem cells express tomosyn.

Fig 5

(a) Flow chart showing the method for culturing modified embryonic stem (ES) cells differentiating in serum-free medium (SFEBq)/growth factor-free chemically defined medium (gfCDM). DFNB = DMEM/F12 supplemented with 7 g/L glucose, N2 and B27. (b–e) Immunostaining with copeptin (red), NeuN (green), and DAPI (blue) in dispersed SFEBq/gfCDM cultured cells. A merged image is shown in the right panel. White scale bars indicate 20 μm. (f–k) Immunolocalization of proteins in dispersed SFEBq/gfCDM cultured cells, immunostaining with anti-tomosyn (green), anti-copeptin (red), or anti-SNAP25 (red) antibodies as analysed with confocal microscopy. Merged images are shown in the right panels. White scale bars indicate 25 μm. (l) AVP levels with or without 100 mM KCl stimulation in SFEBq/gfCDM cultured cells (see Methods section). AVP concentrations in the media of artificial cerebrospinal fluid cultured cells (aCSF) (non-treated, NT; n = 8), or 100 mM KCl treatment (KCl; n = 8) are shown. Values are expressed as the mean ± SEM. **P < 0.01 versus NT (non-treated artificial spinal fluid). (m) Tomosyn-1 mRNA expressions in dispersed SFEBq/gfCDM cultured cells. The amount of mRNA was determined using quantitative RT-PCR. The values are normalized to β-actin mRNA and are expressed as the mean ± SEM.