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. 2016 Jan 28;6:20007. doi: 10.1038/srep20007

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Copyright © 2016, Macmillan Publishers Limited

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(A) Embryonic day 9.5 embryos showing strong growth defect on Sept7 deletion. (B) Cre transduction induced deletion of Sept7 in immortalized fibroblasts leads to obligate multinucleation. SEPT7-immunostaining, phalloidin staining for F-actin, DAPI for DNA are shown. (C,D) Expression cassette and important features in the bi-cistronic SEPT7 expression vector (C) and the bidirectional Cre-mCherry expression vector (D) used in the study. (E) Schematic representation for the basis of the dual colour flow-cytometric assay for SEPT7 rescue model. (F) Flow-cytometric analysis of double transduced MEFs showing mCherry and dox-inducible GFP signals. (G) Immunoblot analysis showing the Cre-mediated depletion and doxycycline-induced rescue of SEPT7 expression in MEFs. (H) Flow-cytometric analysis for the percentage of mCherry Cre positive cells at the indicated time-points post transduction, in the presence or absence of doxycycline. Wild-type cells transduced with mCherry Cre is shown as control. (I) Representative microscopic images showing the effect of Dox-treatment on Cre-induced multinucleation in double-transduced MEFs. (J) pSERS-SEPT7-IRES-GFP or pSERS-GFP expressing cells were transduced with mCherry Cre and were sorted for mCherry/GFP-double positive cells. The cells were analyzed for multinucleation after SYTOX green staining (student’s t-test, n = 4,*** denotes a p value of 0.00025).