Figure 2.
RAD51 antigene PNA downregulates RAD51 mRNA expression in vitro. (A) PNA targeting RAD51 transcription. The top line shows the sense strand of the RAD51 gene, which is homologous to the active PNA; “+1” indicates the transcriptional start site. Thus, PNArad51_nls (second line) is complementary to the antisense strand of the RAD51 promoter, spanning bases −9 to +6. PNArad4μ_nls (third line) is a quadruple-mismatch control PNA with the same base composition as PNArad51_nls. (B) H929 and (C) MM.1S cells were treated with 10-μM PNArad4μ_nls or 5-μ and 10-μM PNArad51_nls for 48 h prior to harvest. RAD51 gene expression was determined by RT-qPCR, for 6 biological replicates per group. Results depict relative change normalized to control-PNA-treated samples, after standardization against GAPDH transcripts as internal control. **, ***, **** indicate P < 0.01, 0.001 or 0.0001, respectively, relative to control. (D, E) H929 cells were exposed to 10-μM PNArad4μ_nls or 10-μM PNArad51_nls for 24 h; then vehicle (H2O) or 10 μM Melphalan (Mel) was added for a further 24 h. Cells were harvested and RAD51 gene expression was determined as in (B) and (C) using either GAPDH (D) or β-actin (E) as the internal control (reference point). ** indicates P < 0.01, relative to control.