Figure 6. TMVs transfer dsDNA to recipient cells, leading to protein expression and behavioral changes.
(A) Using absolute quantification by qRT-PCR, SRY genomic DNA was identified and measured within TMVs treated with benzonase or co-treated with Triton X-100. The data are presented as means, with whiskers indicating maximum and minimum values. n = 9. The p value was obtained by an unpaired 2-tailed t test.
(B) Total RNA was isolated from 3 benzonase-treated independent pools of LOX, TMVs, or LNCaP cells. SRY mRNA was then examined by RT-PCR.
(C) SRY protein was examined by western blot in lysates from whole cells or isolated TMVs from the male cell lines DU145 or LOX.
(D) SRY genomic DNA is reduced when measured by qRT-PCR on equal numbers of benzonase-treated TMVs from LOX or LOXcGAS–KD cells. The data are presented as means, with whiskers indicating maximum and minimum values. n = 6. The p value was obtained by an unpaired 2-tailed t test.
(E) SRY genomic DNA can be detected by qRT-PCR in female mammary cell lines, MDA-MB-231, or MCF-10A, following incubation with male (LOX) TMVs. Maximal threshold cycle (Ct) count for each qRT-PCR run was set to 40, and SRY was undetected in untreated MDA-MB-231 or MCF-10A cells. The data are presented as means ± SDs. n = 5. The p value was obtained by an unpaired 2-tailed t test.
(F) SRY protein can be detected by western blotting in female cell lines, MDA-MB-231, or MCF-10A, when incubated with LOX TMVs.
(G) Incubation with LOX TMVs increases growth rate in MCF-10A cells, while loss of cGAS mitigates this increase. The data are presented as means ± SDs for each time point. n = 8. The p value was obtained by the Wilcoxon rank-sum test.
(H) Treatment with LOX TMVs leads to SRY protein expression and increases anchorage-independent growth as measured by mammosphere sphere-forming efficiency. The data are presented as means, with whiskers indicating maximum and minimum values. n = 12. The p value was obtained by an unpaired 2-tailed t test. Scale bar, 75 μm.
(I) SRY shRNA reduces TMV-mediated changes to anchorage-independent growth as measured by mammosphere sphere-forming efficiency and blocks expression of SRY protein in mammosphere cultures. The data are presented as means ± SDs. n = 12 (sphere-forming efficiency), n = 5 (protein expression). The p value was obtained by ANOVA with Sidak’s correction for multiple comparisons (mammospheres) or 2-tailed t test (protein levels).