For the collection of conditioned media, 2 ×105 HCC cells (usually SK-Hep1 cells, whose conditioned medium contained the most activity for ERBB3 activation) were seeded with regular cultured media in 36-mm dishes overnight; after that, the media were removed from the dishes, washed three times with phosphate-buffered saline, and further cultured in serum-free media for

24 hours. Media were spun for the removal of any insoluble components for 15 minutes at 12,000g and then used to treat cells. For blockade assays, conditioned media were incubated with antibodies against NRGs (200 or 400 ng/mL) for 20 minutes at 37°C to neutralize the biological activity of NRGs and then were used to treat HCC cells. To knock down the expression of EGFR, HER2, ERBB3, or NRG1, cells were transfected with siRNAs or Atezolizumab transduced with lentivirus-based shRNAs targeting EGFR, HER2, ERBB3, or NRG1. siRNAs with randomly scrambled sequences were used as the controls. To guarantee the specificity and to avoid off-target effects, we used two clones of siRNAs or shRNAs for each gene and separately examined the silencing efficiency with respect to their target genes and their effects on the related biological results. For example, we used two clones of siRNA targeting HER2. Silencing

BVD-523 purchase of HER2 expression via both siRNA clones efficiently suppressed the phosphorylation of ERBB3 and its downstream Akt (Supporting Information Fig. 1A). Also, two clones of siRNAs targeting ERBB3 were used, and the specificity for the silencing of ERBB3 expression and for the suppression of phosphorylation

of downstream Akt was examined (Supporting Information Fig. 1B). In addition, consistent effects of both siRNA clones targeting ERBB3 and both siRNA clones targeting HER2 on cell proliferation selleck chemicals llc (Supporting Information Fig. 1C) and tumor sphere formation for HepG2, Huh7, and SK-Hep1 cells were observed (data not shown). Basically, 2 × 105 cells were seeded onto six-well plates and transfected with 5 nM siRNA with Lipofectamine as the transfectant reagent according to the manufacturer’s protocols (Lipofectamine RNAiMAX, Invitrogen). Forty-eight hours after transfection, the cells were harvested or subjected to further assays. RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction were performed as previously described. Immunoblotting analysis and immunohistochemistry assays were conducted as previously described13, 14 (see the Supporting Information). The invading activities of HCC cells were analyzed with Boyden chambers (8-μm pore size; Corning, Inc.), cell motility was assayed with wound healing assays, and cell proliferation was determined via colorimetric sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assays (see the Supporting Information).