Figure 2. IRS2 supports self-renewal of CSC in a PI3K-dependent manner.

(A and B) IRS1^−/−, IRS2^−/− SUM-159 cells (A) and Irs1^−/−, Irs2^−/− PyMT cells (B) expressing empty vector (EV), IRS1, or IRS2 were assayed for mammospheres in B27+ supplement.

(C) Irs1^−/−, Irs2^−/− PyMT cells expressing EV or Irs2 were assayed for mammospheres with B27+ or B27− supplement.

(D and E) IRS2^−/− SUM-159 cells (D) or Irs2^−/− PyMT cells (E) were assayed for mammospheres in B27+ supplement.

(F and G) ALDH activity was examined in SUM-159 cells (F) and PyMT cells (G). The ALDH inhibitor DEAB was included as a negative control.

(H) Irs2^fl/fl PyMT and Irs2^−/− PyMT cells were injected into the mammary fat pads of NOD/SCID mice in 10-fold serial dilutions.

(I and J) IRS1^−/−, IRS2^−/− SUM-159 cells (I) and Irs1^−/−, Irs2^−/− PyMT cells (J) expressing EV, Irs2-WT (WT), or Irs2-Y5F (Y5F) were assayed for mammospheres in B27+ supplement.

(K and L) IRS1^−/−, IRS2^−/− SUM-159 cells (K) and Irs1^−/−, Irs2^−/− PyMT cells (L) expressing EV, Irs2-WT, or Irs2-Y5F were assayed for ALDH activity.

(M) Irs1^−/−, Irs2^−/− PyMT cells expressing EV, Irs2-WT, or Irs2-Y5F were injected into the mammary fat pads of NOD/SCID mice in 10-fold serial dilutions. For (H) and (M), data are presented as a log-log plot, and the frequency of stem cells is calculated by extreme limiting-dilution analysis. The mammosphere data shown represent the mean ± SD of a representative experiment performed three times independently. The ALDH data shown represent the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

See also Figures S2 and S3.