Fig. 4.

Characterization of cells expressing shRNA against human IP6K1. (A) Immunoblot to detect IP6K1 in lysates from HeLa and HCT116 cell lines that stably express the indicated shRNA (NT, non-targeting control; shIP6K1–1 and shIP6K1–4, two different shRNA sequences directed against human IP6K1). (B, C) The bar graphs represent IP6K1 knockdown levels in HeLa (B) and HCT116 (C). Data represents mean ± SEM from three independent experiments. (D, E) HPLC profile of [³H] inositol labeled HeLa NT and shIP6K1–4 (D) and HCT116 NT and shIP6K1–1 (E) cell lines. Soluble inositol phosphate counts were normalized to the total lipid inositol count for each sample. Peaks corresponding to IP₅, PP-IP₄, IP₆ and IP₇ are indicated. Data are representative of two independent experiments. (F) Growth analysis by MTT assay was used to determine the doubling time for HeLa and HCT116 cells stably expressing the indicated shRNA. Data represents mean ± SEM from three independent experiments and was analyzed using one-way ANOVA with Tukey's multiple comparison test. The P value was determined to be > 0.05 across all comparisons, indicating no significant difference in doubling time in cells expressing shIP6K1 compared with NT.