Figure 5

DZIP1 knockdown and overexpression do not affect the accumulation or stability of mRNAs associated with DZIP1-containing complexes but modify the quantity of stress granules per cell. (A) Protein extracts from DZIP1 knockdown (siDZIP1) and negative control (siNC1) cells after 72 h of transfection were analyzed by western blotting with antibodies against DZIP1 and GAPDH. (B) Western blots shown in (A) were used for quantitation and are representative of results from two experiments. (C) Quantitative RT-PCR analysis of DZIP1, GLI1, PTCH1, BRD8, SNX2 and IFT80 mRNA levels in DZIP1 knockdown cells. (D) Quantitative RT-PCR analysis of DZIP1, GLI1, PTCH1, BRD8, SNX2 and IFT80 mRNA levels in HeLa cells transfected with pDZIP1-GFP or pGFP alone. GAPDH was used as an internal housekeeping gene control. (E) Mean half-life for some of the mRNA targets of DZIP1 (IFT80, SNX2, BRD8 and PTCH1) in HeLa cells transfected with siDZIP1 or siNC1. Triplicates were performed for qRT-PCR and the mean half-life was obtained from two independent experiments. (F) DZIP1 knockdown cells and control (NC1) were subjected to oxidative stress and the formation of stress granules was determined by TIA1 staining. The granules were counted and statistically analyzed. At least 5 fields per slide (technical triplicate) were counted. *P ≤ 0.05.