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

Figure 6.

Figure 6

YBX1-mediated regulation of AR and ARv7 in C4-2 cells. (A) YBX1 does not regulate AR or AURKA mRNA levels in C4-2 cells. YBX1, AR and AURKA mRNA levels were analyzed as described in Materials and Methods in C4-2, WT YBX1-C4-2 and 2A-YBX1-C4-2 cells. (B) YBX1 upregulation increases ARv7 mRNA levels in 22Rv1 cells. YBX1, AR, ARv7 and AURKA mRNA levels were analyzed in 22Rv1, WT YBX1-22Rv1 and 2A-YBX1-22Rv1 cells. (C) AR levels positively correlates with YBX1 levels in transfected C4-2 cells. Different amounts of YBX1 DNA was transfected in C4-2 cells. YBX1 and AR levels were analyzed after 30 h. (D) AR levels positively correlates with YBX1 levels in vector, YBX1 and 2A-YBX1-expressing C4-2 cells. (E) AR levels positively correlates with YBX1 levels in vector, YBX1 and 2A-YBX1-expressing 22Rv1 cells. (F) ARv7 levels positively correlates with YBX1 levels in vector, YBX1 and 2A-YBX1-expressing 22Rv1 cells. (G) AR binds YBX1 in vector, YBX1 and 2A-YBX1-expressing C4-2 and 22Rv1 cells. YBX1 was immunoprecipitated from vector, YBX1 and 2A-YBX1-expressing C4-2 and 22Rv1 cells, followed by AR immunoblotting (top panel). Middle panel shows IgG IP in the same six cell lines, followed by AR IB. Bottom panel shows YBX1 levels in the input. (H) YBX1 prevents AR degradation. AR levels were analyzed in C4-2, YBX1-C4-2 and 2A-YBX1-C4-2 cells following treatment with cycloheximide for 6 and 12 h. (I) Graphical representation of AR half-life. The results of densitometric scanning are shown graphically with YBX1 signal normalized to actin signal. * p < 0.05. (J) YBX1 stabilizes AR by inhibiting its ubiquitylation. 6x-His-Ubiquitin expressing C4-2 cells were treated with either scrambled or YBX1 shRNA lentivirus for 30 h, followed by MG132 treatment for 12 h. AR was isolated and ubiquitylation analyzed using 6x-His antibody. (K) YBX1 stabilizes AR by inhibiting its ubiquitylation. Ubiquitylation was performed as described above, except ubiqitylated proteins were isolated using Ni-NTA beads followed by AR IB. Each experiment was done at least three independent times. Representative data are shown. (L) AURKA-mediated phosphorylation of YBX1 promotes enzalutamide-resistance. C4-2, YBX1-C4-2 and 2A-YBX1-C4-2 cells were plated overnight, enzalutamide (1 µM) was added and cells grown for additional 24, 48 or 72 h, followed by MTT assay. (M) YBX1 overexpression increases docetaxel resistance in C4-2 cells. C4-2, YBX1-C4-2 and 2A-YBX1-C4-2 cells were plated overnight, followed by docetaxel (100 nM) treatment. The cells were grown for additional 24, 48 or 72 h, followed by MTT assay. (N) 2A-YBX1 overexpression sensitizes C4-2 cells to AURKA inhibitor MLN8237 and enzalutamide, whereas YBX1 overexpression confers resistance. C4-2, YBX1-C4-2 and 2A-YBX1-C4-2 cells were plated overnight, followed by enzalutamide (1 µM) or MLN8237 (1 µM) treatments either independently or in combination. The cells were grown for additional 48 h, followed by MTT assay.