Figure 7.

lclPTEN-L Cell-to-Cell Transfer at Lower Donor/Recipient Cell Ratios and with Neural Stem Cell Donor Cells

(A) Analysis of lclPTENL cell-to-cell transfer was performed as described in Figure 6C, except that the ratio of donor U87MG/lclPTENL cells to U87MG/mCherry cells was approximately 1:3, not 1:1. (B) Analysis of PTENL cell-to-cell transfer with a donor to recipient cell ratio of 1:3. (C) U87MG/lclPTEN-L cells were cocultured with U87MG/mCherry cells at a 1:1 ratio and treated without or with doxycycline for 72 hr. Cells were then labeled with EdU for 2 hr and fixed. mCherry/EdU double-positive cells and total mCherry⁺ cells were then counted under immunofluorescence microscopy for 10 randomly selected fields of view. (D) Neural stem cells were generated from iPSCs as described in Materials and Methods. These were transduced with lentiviruses for Tet activator expression and expression of lclPTEN-L, and selected with G418 and puromycin. Cells were then treated without or with doxycycline for 48 hr, and total cell lysates were analyzed by western blot with antibody to PTEN. (E) Neural stem cells engineered for inducible expression of lclPTEN-L were cocultured in neural stem cell media with U87MG cells labeled with mCherry. Videomicroscopy was used to determine distance traveled for both cell populations in the cocultures in the absence and presence of doxycycline. (A–D) Bar graphs show the mean ± SEM for distance traveled (in pixels). *p < 0.05 by the Mann-Whitney rank-sum test.