Figure 3. TLR4 Overexpression Decreases Proliferation and Reduces CSC Maintenance.

(A and B) Transient TLR4 overexpression in CSCs was measured at both the mRNA (A) and protein (B) levels using qRT-PCR and western blotting, respectively. Western blots were also stained for the pluripotency factors SOX2, NANOG, and OCT4. CSCs were nucleofected with pcDNA3-TLR4-YFP and used for experiments 3 days later. Actin was used as an internal control for qRT-PCR and a loading control for western blotting.

(C) Proliferation over 7 days was assessed in CSCs derived from the shown specimens using CellTiter Glo after overexpression of TLR4. Growth was normalized to cells containing an empty vector.

(D) CSCs overexpressing TLR4 were treated with LPS at 500 ng/mL, and proliferation was assessed for 7 days relative to cells containing a control vector.

(E) Limiting dilution analysis was used to estimate stem cell frequencies in CSCs after nucleofection with control or TLR4 overexpression vectors. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods.

All experiments were performed at least three times. Data are represented as mean ± SEM. **p < 0.01 and ***p < 0.001 as assessed by one-way ANOVA. See also Figure S3.