Figure 7.

LKB1 regulates Tribbles 3 expression through DBC1.A, graphical representation of microarray data from control (−dox) and PyST expressing U2OS cells (+dox) for Tribbles 3 (TRB3). These experiments were carried out as biological triplicates. B, fold change for TRB3 mRNA in PyST expressing cells (+dox, 24 h) as compared to control cells (−dox), as shown by RT-qPCR. The data were normalized by using actin as control. C, PyST increases protein levels of TRB3. pTREX-PyST cells were induced for expression of PyST for different time periods as indicated (0 to 52 h), and cell lysates were then analyzed by Western blotting for DBC1, TRB3, and PyST using antibodies against respective proteins. D and E, confirmation of DBC1 expression in U2OS stable cells at protein and mRNA levels by Western blotting (using anti DBC1 antibody) and RT-qPCR, respectively. F and G, effect of the overexpression of DBC1 and LKB1 respectively, on TRB3 mRNA levels using RT-qPCR. For all these experiments, total cellular RNA was extracted from respective cell lines. After cDNA synthesis, RT-qPCR was carried out using appropriate primers and normalization was carried out using actin as control. The experiments were carried out in duplicates and the gene expression normalized to actin expression and fold change was calculated using ΔΔCq method. p-values were calculated using student’s t test. Shown are ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. The error bars represent mean ± SEM. DBC1, deleted in breast cancer 1; PyST, polyoma small T antigen.