Figure 5: BAP1 knockdown partially suppresses cell proliferation through KLF5.

(a) Knockdown of either BAP1 or KLF5 significantly decreased DNA synthesis in HCC1806 cells. The green dye represented the proliferative cells and the blue dye represented the total cells. Representative images are shown. Scale bar, 100 μm. (b) The quantitative results (n=6) of a. The percentages represented the proportion of green cells in the total cells. The t-test was done on raw percentages without any transformation (mean±s.d. from six photos in two independent experiments). Every experimental group (KLF5si, BAP1#1si and BAP1#3si) was compared with the Lucsi group, **P<0.01, t-test. (c) Stable knockdown of BAP1 decreased KLF5 and FGF-BP and increased p27 protein levels in HCC1806 cells. (d) Stable knockdown of either BAP1 or KLF5 inhibited HCC1806 cell growth in vitro as measured by the sulforhodamine B assay (n=4). **P<0.01, t-test. The cells stably transfected Luc, KLF5, BAP1#3 or BAP1#6 shRNAs were planted into 24 wells and cell viability was measured every day. Data points represent the mean±s.d. of three duplicates per group. Statistical significance was determined by a t-test. Every experimental group (KLF5sh, BAP1#3sh and BAP1#6sh) was compared with the Lucsh group. **P<0.01, t-test. (e) Stable knockdown of either BAP1 or KLF5 suppressed HCC1806 tumour growth in NOD-SCID mice. Xenograft tumour growth was measured twice a week. Data points represent the mean±s.d. of six mice per group (12 tumours). Statistical significance was determined by a t-test. Every experimental group (KLF5sh, BAP1#3sh and BAP1#6sh) was compared with the Lucsh group. **P<0.01, t-test. (f) Stable knockdown of either BAP1 or KLF5 generated smaller xenografts compared with the Lucsh control at day 28. (g) Stable knockdown of either BAP1 or KLF5 significantly decreased tumour weight compared with the Lucsh control at day 28 (n=12). Error bars represent s.d. *P<0.05 and **P<0.01, t-test. (h) KLF5 was transiently overexpressed in BAP1 stable knockdown HCC1806 cells. KLF5 protein levels were modestly restored in the BAP1 stable knockdown cells. (i) KLF5 transient overexpression modestly increased tumour masses in BAP1 stable knockdown HCC1806 cells (n=11). (j) KLF5 transient overexpression modestly but significantly increased tumour weight in BAP1 stable knockdown HCC1806 cells (n=11). Error bars represent s.d. **P<0.01, t-test.