For K19 knockdown experiments, HCC cells were transfected with 10 nmol/L K19‐siRNA (#4427037‐s7998 or #4427037‐s7999; Invitrogen) or control‐siRNA (#4390843; Invitrogen) using Lipofectamine LTX reagent (Invitrogen), according to the manufacturer's protocol. K19 expression was significantly downregulated by both K19 siRNAs (Fig. S1 A). As identical results were acquired with both siRNAs, results of K19‐siRNA (#4427037‐s7999) are shown as representative data.
For K19 overexpression experiments, the human K19 open reading frame was amplified by RT‐PCR and ligated to the CMV6‐AC plasmid (PS100020; OriGene, Rockville, MD) digested with Sgf1 and RsrII. HCC cells were then transfected with this K19 expression vector or with mock vector (PS100020, OriGene) using Lipofectamine LTX reagent (Invitrogen), according to the manufacturer's protocol. K19 expression was significantly upregulated by the K19 expression vector (Fig.S1 B). For quantification of CYFRA 21‐1, culture supernatants of HCC cells were collected 72 h after transfection.
For K19 overexpression experiments, the human K19 open reading frame was amplified by RT‐PCR and ligated to the CMV6‐AC plasmid (PS100020; OriGene, Rockville, MD) digested with Sgf1 and RsrII. HCC cells were then transfected with this K19 expression vector or with mock vector (PS100020, OriGene) using Lipofectamine LTX reagent (Invitrogen), according to the manufacturer's protocol. K19 expression was significantly upregulated by the K19 expression vector (Fig.