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Figure 4 | Molecular Cancer

Figure 4

From: PIAS1 interacts with FLASH and enhances its co-activation of c-Myb

Figure 4

PIAS1 interacts with c-Myb on chromatin and activates the expression of c-Myb target genes. (A) AH109 yeast cells were transformed with bait vector pDBT or pDBT-c-Myb and mated with the yeast strain Y187 pretransformed with pray vector pGADT7, pGADT7-PIAS1[1-501] or pGADT7-PIAS1. Interaction was verified by activation of the HIS3 reporter gene (SC-L-W-H). Each mating was performed in duplicate. HEK 293 cells were transfected with plasmids expressing 3×FLAG-PIAS1 and a Gal4p-DBD-c-Myb-HA fusion protein. Occupancies of (B) PIAS1 and (C) c-Myb on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP-qPCR. (D) CV-1 cells were transfected with the c-Myb-responsive 3×MRE(GG)-MYC reporter plasmid (0.2 μg) and plasmids encoding c-Myb, FLASH, PIAS1, PIAS1[1-501] or PIAS1[C350S] as indicated (0.2 μg each). The results are presented as relative luciferase units (RLU) and represent the mean RLU ± SEM of three independent assays performed in triplicates. (E) K562 cells were transfected with siRNAs directed against PIAS1. Effects of PIAS1 knock-down on the endogenous c-Myb target genes MYC and LMO2 were measured by quantitative real-time PCR using specific primers for the target genes and the reference genes ACTB and POLR2A. The results are presented as PIAS1, MYC or LMO2 expression (normalized for ACTB and POLR2A expression) after PIAS1 knock-down relative to their expressions in the knock-down control. The results are presented as mean relative mRNA level ± SEM. Statistical significance was calculated using the Student's t-test (P-value indicated).

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