Fig 1: Short interfering RNA (siRNA) knockdown of CD133 alters Daoy MB cell function.(A) Western blot analysis of CD133 expression 48 h after transfection with siCD133-1, siCD133-2, scramble siRNA, or no transfection. β-actin served as a loading control (left). Quantification of relative percent CD133 expression, measured by the densitometry of western blot bands and normalized to β-actin (Scramble set to 100%) (right). (B) Fold change of the number of adhered cells per image after transfecting with siCD133-1, siCD133-2 or scramble siRNA, then culturing in ACM or DMEM (control) (DMEM scramble set to 1). (C) Average number of invading cells per image, counted on the lower surface of Boyden chambers with ACM or DMEM in the lower well, after transfecting with siCD133-1, siCD133-2 or scramble siRNA. (D) Total number of neurospheres counted with diameter length between 75–100 μm or >100 μm. Cells were transfected with siCD133-1, siCD133-2 or scramble siRNA and then cultured in the respective media for 48 h. Quantified values expressed as mean +/- SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 2: ACM effects on CD133 surface expression and neurosphere formation.(A) Percentage of CD133 positive cells cultured in ACM or DMEM (control), measured by flow cytometry at 24, 48, and 72 h. (B) Percentage of CD15 positive cells cultured in ACM or DMEM (control), measured by flow cytometry at 24, 48, and 72 h. (C) Representative images of Daoy cells cultured in neurosphere conditions using the respective media for 48 h. Scale bar is 100 μm. (D) Total number of neurospheres counted in all images between 75–100 μm or >100 μm at 48 h, cultured in the respective media. (E) Western blot analysis of Nestin, (F) Oct-4A and (G) Sox2 expression in respective neurosphere culturing conditions at 48 h. β-actin served as a loading control (left). Quantification of relative fold change of Nestin, Oct-4A and Sox2 expression, measured by the densitometry of western blot bands and normalized to β-actin (DMEM condition set to 1) (right). Quantified values expressed as mean +/- SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Supplier Page from OriGene Technologies for CD133 (PROM1) Human siRNA Oligo Duplex (Locus ID 8842)