Fig 2: Human Epidermal growth factor Receptor 2 (HER2) expression level in SKOV-3 and SKOV-3.ip cells cultured in monolayer and 3D in vitro models. (A) The distributions of SKOV-3 cells (left plot) and SKOV-3.ip cells (right plot) according to fluorescence intensity detected after staining with HER2-specific antibodies (red—monolayer culture, blue—spheroids, green—collagen hydrogel); (B) HER2 expression level in monolayer and 3D models denoted as relative fluorescence values. mnlr, monolayer; sph, spheroids; clgn, collagen hydrogel. “*” indicates significant difference in RF level from monolayer culture (ANOVA, Holm–Sidak’s multiple comparisons test, p < 0.05).

Fig 3: Schematic summary of the proposed mechanisms by which HCV mediates down-regulation of ErbB3 Expression.HCV up-regulates production of the ErbB3 ligand NRG1 (see also Fig 2) in its host cell by a mechanism that involves Sp1 (see also Fig 4). NRG1 in turn mediates down-regulation of ErbB3 transcript expression via an Akt-dependent pathway, which is also ligand-independently activated by HCV itself, and also of ErbB3-protein levels (see also Fig 3). Points which are not part of the scheme are that down-regulation of ErbB3 expression leads to an up-regulation of EGFR and ErbB2 expression by yet unknown mechanisms and promotes viral replication and infectivity.

Fig 4: Reduction of ErbB3 expression on the cell surface is accompanied by increased cell surface expression of EGFR and ErbB2.(A) and (B) Huh9-13 replicon cells were analysed by FACS to determine surface expression of ErbB3 and EGFR (A) or of ErbB3 and ErbB2 (B). (C) and (D) Huh7 cells were transfected with ErbB3 specific siRNA or with control siRNA. 72 hours later cells were prepared for FACS analysis to determine the amounts of ErbB3 and EGFR (C) or of ErbB3 and ErbB2 (D) on the cell surface. Data were analysed using the FlowJo software against the corresponding isotype controls and analysis of EGFR (A and C) or ErbB2 (B and D) surface expression was gated to cells with low expression of ErbB3. Results correspond to the average of the receptors at the cell surface (in precent); error bars indicate SEM from at least 3 independent experiments.

Fig 5: Monoclonal α-Cot-NK92 cell lines have different killing potentials. (A, B) α-Cot-NK92 clones were established through single cell sorting using FACS in an expression level-dependent manner for CAR-Myc. Black triangles with a line indicate the expression level of CAR-Myc. The ability of α-Cot-NK92 clones to lyse AU565 cells was assessed using co-culture with α-HER2-Cot (A: 500 ng/mL) or ZEGFR-Cot (B: 50 ng/mL) for 4 h at E:T ratios of 5:1, 2:1, and 1:1. Ve: vehicle. (C, D) Comparison of cytolytic potency between low (L8) and medium (M2) CAR-expressing α-Cot-NK92 clones through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells in a dose-dependent manner of α-HER2-Cot (C: from a concentration of 500 to 62.5 ng/mL, upper panel) or ZEGFR-Cot (D: from a concentration of 12.5 to 1.56 ng/mL, lower panel). (E) Comparison of cytolytic potency between M2 clone and heterogeneous α-Cot-NK92 cells through co-culture with AU565, A549-Red-Fluc, or MDA-MB-231 cells with α-HER2-Cot (125 ng/mL) or ZEGFR-Cot (6.25 ng/mL). (F) Relative expression level (%) and mean fluorescence intensity (MFI) of HER2 and EGFR in breast cancer cells (AU565) and normal lung cells (WI-38). (G) Cytolytic activity of NK92, monoclonal α-Cot-NK92-L8, and α-Cot-NK92-M2 cells with ZEGFR-Cot or HER2-Cot against AU565 and WI-38 cells. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer.