Cre-induced deletion of SEPT7 in fibroblasts leads to proliferation block and obligate multinucleation.

<p><b>A</b>, Adenoviral Cre-expression in <i>Sept7</i>^flox/flox primary mouse embryonic fibroblasts (MEFs) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004558#pgen.1004558-AgetaIshihara1" target="_blank">[26]</a> leads to deletion of SEPT7 and, as judged by immunofluorescence, to a strong reduction of expression of the other core-septins SEPT6/2. <b>B</b>, Retroviral Cre-transduction of <i>Sept7</i>^flox/flox immortalized mouse tail fibroblasts causes complete deletion of the <i>Sept7</i> gene in two different experiments (#1, #2- independently immortalized lines) (control: non-integrating control virus). Genotyping by PCR as indicated in Supporting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004558#pgen.1004558.s001" target="_blank">Figure S1</a>. <b>C</b>, Western blot detection of multiplicity of infection (MOI)-dependent reduced SEPT7 levels in <i>Sept7</i>^flox/flox immortalized mouse tail fibroblasts. Expression of SEPT6/2/9 and anillin is significantly reduced. <b>D,E,</b> Adenoviral Cre-expression in primary <i>Sept7</i>^flox/flox MEFs decreases immunofluorescence detection of the proliferation marker Ki67 (D) and increases histone H3 phosphorylation (E). The percentage of positive cells is indicated. <b>F,</b> SEPT7-depleted <i>Sept7</i>^flox/flox immortalized mouse tail fibroblasts fail to proliferate (3 days post transduction). <b>G,</b> Retroviral Cre-transduced SEPT7-depleted cells are overgrown by non-depleted cells, as indicated by the relative increase in the floxed allele after 12 days in culture. <b>H,</b> Immunofluorescence analysis showing obligatory multi-nucleation of <i>Sept7</i>-KO tail fibroblasts after 11 days post transduction. DAPI is used for nuclear staining and the WGA as counter stain. Arrowheads indicate multi-nucleated, SEPT7-negative cells.