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

Fig 6. Tomosyn-1 negatively regulates secretion of AVP vesicles.

Fig 6

(a–k) The effects of overexpression or siRNA treatments on tomosyn-1 expression. Dispersed SFEBq/gfCDM cultured cells were transfected with empty vector (vector) or tomosyn-1 vector (Tomosyn-1), scrambled siRNA (siScr) or Tomosyn-1 siRNA (siTomosyn-1 #1 or #2), or not treated (NT). (a, f, g) After 48 h, the expression levels of tomosyn were analysed by western blotting using anti-tomosyn antibody (Tomosyn, marked with arrowheads at the right). The levels of β-actin in the same samples were determined as a control for protein loading (bottom panels of a, f, g). (b–e) Representative immunostaining for tomosyn (green), copeptin (red), and DAPI (blue) at 48 h after overexpression of tomosyn-1 in dispersed SFEBq/gfCDM cultured cells. The merged image is shown in (e). (h–k) Representative immunostaining for tomosyn (green), copeptin (red), and DAPI (blue) at 48 h after knockdown of tomosyn-1 with siRNA in dispersed SFEBq/gfCDM cultured cells. The merged image is shown in (k). (l) AVP concentration in the media of artificial cerebrospinal fluid cultured cells (aCSF) (non-treated, NT; n = 13), KCl treatment (KCl; n = 13), empty vector with KCl (Vector + KCl; n = 12), and tomosyn-1 vector with KCl (Tomosyn-1 + KCl; n = 12). (m) AVP concentration from scrambled siRNA with KCl (siScr + KCl; n = 13), and siTomosyn-1 with KCl (siTomosyn-1 #1 + KCl group; n = 13, siTomosyn-1 #2 + KCl group; n = 12). Final KCl concentration was 100 mM. Values are expressed as the mean ± SEM. **P < 0.01.