Figure 2. Angiocrine effects of endothelial Jag1 on Notch2 activation and propagation of Myc⁺ mouse LCs.

(A) Expression level of Notch pathway effecter Hey1, Hes1, and Hey2 in LCs.

(B) Approaches to define Notch pathway activation in mouse LCs. Notch1, Notch2 and Hey1 in LCs was silenced by shRNA (shNotch1, shNotch2, shHey1), and Notch pathway was blocked by compound E. Cell expansion, Notch activation, colony formation, and hepatic invasiveness were then compared.

(C, D) Quantification (C) and representative image (D) of expansion of LCs co-cultured with ECs (LC+EC) or cultured in serum-free medium (LC). Srb, scrambled shRNA; OE Hey1, overexpression of Hey1; n= 4; Scale bar= 25 μm.

(E, F) Notch1 and Notch2 intracellular domains (ICD) were detected in LCs by immunoblot (E) and Notch2 ICD in LCs was examined by immunostaining (F). White arrowheads indicate nuclear Notch2 ICD. Scale bar= 10 μm.

(G) ChIP analysis of RBPJ activity in LCs after Notch inhibition. shN1 and shN2 denote shRNA against Notch1 and Notch2, respectively. n= 4.

(H–K) Aggressive features of LCs were tested after disruption of Notch2-Hey1 pathway. Extra-nodal invasiveness of LCs was examined by intrasplenic injection into NSG mice (H). Hepatic tumor load was examined by H&E (I) and fluorescent microscopy (K). Quantification of hepatic tumor load is shown in (J); n= 4; Scale bar= 100 μm in (I) and 1 mm in (K).

(L) Expression of Notch ligands in feeder ECs after co-culture with LCs (EC + LC); n= 4.

(M, N) LCs were co-cultured with ECs transduced with Scrambled shRNA (EC^Srb) and Jag1 shRNA (EC^shJag1). Notch activation in LCs was tested by Hey1 upregulation (M) and fluorescent intensity of RBPJ-driven GFP reporter in LCs (N); n= 5.

(O, P) Representative image (O) and expansion (P) of LCs cultured alone or co-cultured with EC^ShJag1 or EC^Srb. *, p<0.025; n= 5. Scale bar= 50 μm.

See also Figure S2.