Figure 5.

EVs produced by MYCN-expressing neuroblastomas induce mitotic index and histone H3 (T11) phosphorylation in a PKM2-dependent manner. (a) Western blot analysis demonstrating the downregulation of PKM2 expression by two different siRNAs. An actin antibody was used as loading control. (b) siRNA knock-down of PKM2 in TET21-N donor cells inhibits histone H3 phosphorylation in recipient cells (n = 3). (c) Mitotic index. The mitotic index is expressed as the percentage of cells in mitosis (prophase, metaphase, anaphase and telophase) over the total number of cells (n = 3). (d) SH-EP cells were transfected with pEGFP-C1 or pEGFP-C1-PKM2 and stained with an antibody against phospho-histone 3 (T11) after 48 h. The number of transfected and phospho-histone H3 (T11) positive cells was quantified in each condition (n = 3). Scale bar, 100 μm. (e) Western blot analysis demonstrating downregulation of Rab27a expression by siRNA. An actin antibody was used as loading control. (f) Western blot analysis of CD63-GFP overexpressing cells in the presence or absence of the anti-Rab27a siRNA. An actin antibody was used as loading control. (g) siRNA knock-down of Rab27a in TET21-N donor cells inhibits histone H3 phosphorylation in recipient cells (n = 3). Error bars represent mean values ± s.e.m. *p ≤ 0.05, **p ≤ 0.01.