Knockdown efficiency and specificity of ID3-siRNA and ID3-shRNA. (A) ID3 knockdown greater than 50% for 48 hrs was confirmed after ID3-siRNA transfection to D283 cells. (B) ID3 showed a selective reduction of the transcript level after ID3-siRNA treatment. (C) Western blot confirmed specific knockdown of ID3 with siRNA. (D) D283 cell lines transfected with ID3-shRNA (D283-ID3-shRNA) showed a significant reduction of ID3 mRNA after puromycin selection. (E) After transfection with ID3-shRNA, ID1, ID2, and ID3 transcripts levels were decreased and ID4 level was significantly increased. Upon ID3 rescue, ID2 and ID3 transcript levels were restored and ID4 level was also normalized. (F) In protein expression, there was a minimal decrease of ID2 expression by ID3-shRNA. The basal ID4 protein expression was negligible in D283 cells. ID4 also showed significant changes in opposite directions of ID3, as indicated in transcript levels.