Effect of OCTN1 knockdown on proliferation of P19-NPCs.

<p>P19 cells were cultured in the presence of retinoic acid for 3-NPCs, and siRNA for OCTN1 or negative control siRNA was transiently transfected. (A) Total RNA was extracted for quantitative RT-PCR analysis of OCTN1 at 1 and 3 DIV after the transfection of control siRNA (white columns) and siOCTN1 (black columns). Each value is normalized by the expression level of GAPDH mRNA and represents the mean ± S.E.M. (n = 4–5). (B) P19-NPCs cultured for 3 DIV after the transfection were fixed with 4% PA, followed by immunocytochemical detection of OCTN1 (green), NPC marker nestin (red) and nuclear marker DAPI (blue). The merged image is also shown. (C) P19-NPCs were transiently transfected with siOCTN1 (black symbols) or negative control siRNA (white symbols), then cultured for 3 DIV, and the cells were incubated with [³H]ERGO at 37°C, followed by determination of [³H]ERGO uptake. Each value represents the mean ± S.E.M. (n = 3). (D) Phase-contrast micrographs were obtained for P19-NPCs cultured for 1 to 3 DIV after the transfection. (E) The area of neurospheres was quantified with ImageJ in P19-NPCs transfected with negative control siRNA (white columns) or siOCTN1 (black columns) and cultured for 1 to 3 DIV. *P<0.05, significant difference from the corresponding control value. N.D., not detectable.