Figures & data
Figure 1 . LMK-235 induces apoptosis of DLBCL cells in a time and dose manner. (A) The DLBCL cell lines OCI-LY10 and OCI-LY3 cells were plated in triplicate and treated with 0, 0.5, 1.0, 1.5, 2.0 and 2.5 μmol/l LMK-235 for 12 hours, 24 hours, 36 hours and 48 hours, respectively. Flow cytometry was used to determine apoptosis.(B) OCI-LY10 and OCI-LY3 cells were treated with different concentration of LMK-235 (0.5, 1.0, 1.5, 2.0 and 2.5 μmol/l) for 24 hours,24 hours, 36 hours and 48 hours, and HDAC4 and HDAC5 protein levels were examined by Western blot .All experiments were conducted three times. Data are represented as mean±SD.*P<0.05,**P<0.01 versus the control group.
Figure 2 . LMK-235 upregulated BCLAF1 expression. (A) DLBCL cell lines OCI-LY10 were treated with different concentrations of LMK-235 (0, 0.5, 1.0, 1.5, 2.0, 2.5 μmol / l)for 24 hours or vehicle alone, and the mRNA expression of BCLAF1 was analyzed with q-PCR. Each value represents the average of three independent experiments. (B) DLBCL cell lines were treated with the indicated concentrations of LMK-235 at different times. The mRNA expression of BCLAF1 was analyzed with q-PCR. (C) DLBCL cell lines were treated with LMK-235, and the relative protein expression of the BCLAF1 were detected by Western blot. All experiments were performed three times. The data is expressed as mean ± SD. * P <0.05 compared with the control group.
Figure 3 LMK-235 inhibited the NF-kB signaling pathway. (A)Western blot analysis showed that phosphorylation of IκB-α and p65 (p-p65 and pIκB-α) was inhibited after LMK-235 treatment in OCI-LY10 cells, but total-p65 or total-IκB- α were not affected. The cells were treated for the indicated drug concentration and sampled at the different time points with the same result.(B) The protein levels of p-p65 and pIκB-α in OCI-LY3 cellswere assessed by Western blot.
Figure 4 HDAC4 gene silencing caused apoptosis of DLBCL cells and increased expression of BCLAF1.(A) Relative HDAC4 mRNA expression in OCI-LY10 cells treated with or without si-NC and si-HDAC4.(B) The protein levels of HDAC4, BCLAF1, p-IκB-α and p-p65 were assessed by Western blot. (C)The protein levels of p-IκB-α and p-p65 were assessed by Western blot. (D)Cell apoptosis rates were tested by flow cytometry. The expression of BCLAF1 at the mRNA level was also analyzed by q-RT-PCR after si-HDAC4 and Bay11-7082 treatment. All experiments were performed three times.Data are represented as mean±SD.*P<0.05,**P<0.01 versus the control group.
Figure 5 Down-regulation of HDAC4-induced upregulation of BCLAF1 was mediated through inhibition of NF-kB activity. Western blot detected the protein expression of HDAC4, BCLAF1, phosphorylated P65 and total P65, phosphorylated IκB-αS32 / S36 and total IκB-α. Western blot bands were quantified with Quantity One software. All experiments were repeated three times.
