Fig. 2: BAP1 deficiency inhibits NB cell growth in vitro.

A Immunoblot (IB) analyses of whole-cell lysate (WCL) derived from the BE2C cells infected with indicated lentiviral sgRNAs. The infected cells were selected with 2–3 μg/ml puromycin for 72 h to eliminate non-infected cells before they were harvested. B Quantification of the MYCN and BAP1 protein intensities in (A). C IB analysis of WCL derived from BE2C cells infected with the indicated BAP1 shRNAs. Cells were treated with or without indicated concentration of MG132 for overnight before they were harvested. D Representative images for the wound-healing assays of shBAP1 BE2C cells. The wound edges are indicated by yellow lines. Scale bar, 100 μm. E The quantitative results of (D) (n = 8). The y axis represents the relative wound area. Error bars represent s.d. from eight repeats. t test. F Representative images for the wound-healing assays of shBAP1 SH-EP Tet21/N cells (MYCN amplification). The wound edges are indicated by yellow lines. Scale bar, 100 μm. G The quantitative results of (F) (n = 10). The y axis represents the relative wound area. Error bars represent s.d. from ten repeats. t test. H, I Growth curves of BE2C (H) cells and SH-EP Tet21/N cells (I) with shRNA-mediated BAP1 knockdown (shBAP1). **P < 0.01. J Colony-formation assay. Depletion of endogenous BAP1 in BE2C cells displays moderate decrease in colony-formation ability. The number of colonies were counted and quantified. Data are shown as mean ± SD for three independent experiments. P value was indicated in figure, t test. BE2C cells were infected with indicated lentiviral shRNAs. The infected cells were selected with 2 μg/ml puromycin for 72 h to eliminate non-infected cells before they were used for colony-formation assay. K Colony-formation assay. Depletion of endogenous BAP1 in SH-EP Tet21/N cells decreases the colony-formation ability. However, upon depletion of MYCN by the Dox treatment, the colony-formation ability of SH-EP Tet21/N+Dox cells is not changed after depletion of endogenous BAP1. The number of colonies were counted and quantified. Data are shown as mean ± s.d. for three independent experiments. P value was indicated in the figure, t test. SH-EP Tet21/N cells were infected with indicated lentiviral shBAP1. The infected cells were selected with 2 μg/ml puromycin for 72 h to eliminate non-infected cells before they were used for colony-formation assay. L Representative images (n = 5) of migrated SH-EP Tet21/N cells infected with the indicated lentiviral shBAP1. The SH-EP Tet21/N cells were treated with or without Dox before plated for the transwell migration assay. Scale bar, 100 μm. M Quantification of the migrated cells in (L). In all plots, data are shown as mean ± s.d. for five independent experiments.