Figure 4. TLR4 Overexpression Compromises Stemness by Repressing the Transcription Factor RBBP5.

(A) The expression of the shown transcription factors was analyzed in CSCs overexpressing TLR4 using qRT-PCR and normalized to cells containing a control vector. CSCs were nucleofected with pcDNA3-TLR4-YFP and used for experiments 3 days later. Actin was used as an internal control.

(B) Protein levels of RBBP5 were analyzed in both CSCs and non-CSCs using western blot. Actin was used as a loading control.

(C) A reporter expressing luciferase under the control of the RBBP5 promoter region was used to measure RBBP5 expression after transient TLR4 overexpression in CSCs. CSCs were nucleofected with pcDNA3-TLR4-YFP and used for experiments 3 days later. Luciferase levels were normalized to those of control (NT) cells.

(D) Levels of RBBP5 and GFAP were determined by western blot in CSCs expressing both control and TLR4-overexpression vectors. Actin was used as a loading control.

(E) CSCs from the shown specimens were differentiated by incubating with DMEM with 10% FBS for the indicated times, and protein levels of RBBP5 were determined by western blot. Actin was used as a loading control.

All experiments were performed at least three times. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 as assayed by one-way ANOVA.