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. 2020 Mar 12;12(3):660. doi: 10.3390/cancers12030660

Figure 3.

Figure 3

YBX1 stabilizes AURKA protein in C4-2 and 22Rv1 cells. (A) Overexpression of YBX1 enhances AURKA levels. (B) Histogram shows relative band intensities of YBX1 and AURKA normalized to the corresponding tubulin level from three independent experiments. * signifies significance at p < 0.05 compared to control. (C) YBX1 knock-down using YBX1 shRNA decreases AURKA in C4-2 cells. Cells were infected with control shRNA lentivirus (lane 1), or YBX1-shRNA lentivirus (lane 2), and AURKA and YBX1 levels were analyzed after 30 h. (D) Histogram shows relative band intensities of YBX1 and AURKA normalized to the corresponding tubulin level from three independent experiments. (E) YBX1 overexpression enhances AURKA levels in 22Rv1 cells. (F) Histogram shows relative band intensities of YBX1 and AURKA normalized to the corresponding tubulin level from three independent experiments. (G) YBX1 knockdown depletes AURKA protein levels in 22Rv1 cells. Cells were infected with control shRNA lentivirus (lane 1), or YBX1-shRNA lentivirus (lane 2), and AURKA and YBX1 levels were analyzed after 30 h. (H) Histogram showing relative band intensities of YBX1 and AURKA normalized to the corresponding tubulin level from three independent experiments. (I,J) YBX1 does not regulate the mRNA levels of AURKA in C4-2 and 22Rv1 cells, respectively. YBX1 was overexpressed and AURKA mRNA levels analyzed. (K,L) YBX1 does not regulate the mRNA levels of AURKA in C4-2 and 22Rv1 cells, respectively. YBX1 was knocked-down and AURKA mRNA levels analyzed. (M) YBX1 inhibits AURKA degradation. YBX1-C4-2 and C4-2 cells were treated with cycloheximide and AURKA levels evaluated. (N) Graphical representation of AURKA half-life in C4-2 cells. The results of densitometric scanning are shown with AURKA levels normalized to tubulin levels. (O) YBX1 prevents AURKA degradation in 22Rv1 cells. YBX1-22Rv1 and 22Rv1 cells were treated with cycloheximide and AURKA levels evaluated. (P) Graphical representation of YBX1 degradation rate in 22Rv1 cells. * p < 0.05. (Q) YBX1 stabilizes AURKA by inhibiting its ubiquitylation. 6x-His-Ubiquitin-expressing C4-2 cells were infected with either scrambled or YBX1 shRNA lentivirus for 30 h and then treated with MG132 for 12 h. AURKA was isolated and ubiquitylation analyzed using 6x-His antibody. (R) YBX1 stabilizes AURKA by inhibiting its ubiquitylation. Ubiquitylation was performed as described above, except that ubiqitylated proteins were isolated using Ni-NTA beads followed by AURKA IB. (S,T) YBX1 depletion does not impact the subcellular localization of AURKA in C4-2 and 22Rv1 cells, respectively. Cells were exposed to control or YBX1 shRNA for 30 h, fixed and stained with AURKA antibody (green) or DAPI (blue). >100 cells were analyzed from multiple random frames. Each experiment was done at least three independent times. Representative data are shown.