Figure 3. Sphere Formation in TKOs or RB1^−/− MEFs Generates Cells with Characteristics of a Tumor SP.

Immunostaining for Abcg2 and CD133 is shown on the left, and Hoechst dye staining is shown on the right.

(A) TKOs in subconfluent monolayer culture.

(B) Cells derived from TKO spheres after 2 weeks in suspension culture. Similar results were seen with cells derived from RB1^−/− MEF spheres.

(C) Quantification of side population (SP) (Hoechst−, Abcg2+, CD133+) cells.

(D) TKO and RB1^−/− MEF sphere-derived cells were separated into SP (Hoechst−, CD133+, Abcg2+) and MP (Hoechst+, CD133−, Abcg2−) and placed in culture (day 0). Then at the indicated times, the cells were again examined to quantify the appearance of MP cells within the SP population, and SP cells within the MP population.

(E) Hoechst− SP cells from RB1^−/− MEF spheres.

(F) Higher-power view of a cell from (E).

(G) A Hoechst− cell in (E) gives rise to a Hoeschst+ cell on day 2 in culture.

(H) Population of cells arising from a single Hoechst− from (E) after 5 days in culture.

(I) Hoechst+ MP Cells.

(J) Higher-power view of a single MP cell.

(K) A Hoechst+ cell gives rise to two additional Hoeschst+ cells on day 2 in culture.

(L) Population of cells arising from a single Hoechst+ after 5 days in culture.

(M) Quantification of SP (white bars) and MP (blue bars) cells arising from single SP or MP cells. Results are from five different single SP or MP cells.

Error bars indicate standard deviations.