Fig. 3: Overexpression of GDF1 induced HCC tumour-lineage plasticity.

a Foetal mouse livers at different developmental stages were collected, and the relative expression of GDF1 and several developmental biomarkers was measured by qPCR. Shaded areas show the standard error of the means, n = 3 independent experiments. b A sphere-formation assay was performed in PLC-8024 cells and Huh7 cells stably transfected with or without GDF1 (two-tailed independent Student’s t-test, data are presented as mean values ± SEM, n = 3 independent experiments). Scale bars represent 100 µm. c–e Representative hepatic markers (ALB, ADH1, ARG1, and TTR) and liver progenitor markers (AFP, EPCAM, KRT19, SOX9, and KRT7) were examined by qPCR in PLC-8024 cells stably transfected with or without GDF1 (c), coculture of wild-type PLC-8024 cells with PLC-8024-GDF1 cells (d), or addition of recombinant GDF1 protein for 15 days (e) (*P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant, two-tailed independent Student’s t test, data are presented as mean values ± SD, n = 3 independent experiments). f, g The retrodifferentiation of HCC cells induced by GDF1 was examined at the protein level by western blot in both PLC-8024 cells and Huh7 cells (f) (n = 3 independent experiments), and by IHC staining of subcutaneous and orthotopic liver tumours (g) (n = 5 independent experiments). Scale bars represent 100 µm. h Immunofluorescent costaining of GDF1 (green) with liver progenitor markers and hepatic markers (red) was examined in HCC clinical tissues. Scale bars represent 100 µm (n = 3 independent experiments). rGDF1, recombinant human GDF1 protein. Source data are provided as a Source Data file.