Fig 1.
Synthesis of InsP7s and InsP8 by IP6Ks and PPIP5Ks.
The Fig describes the synthesis of 1-InsP7, 5-InsP7 and 1,5-InsP8 in both yeasts and mammalian cells. IP6K1/2/3 = isoforms 1, 2 and 3 of inositol hexakisphosphate kinase (Kcs1 is the single yeast isoform); PPIP5K1/2 = isoforms 1 and 2 of diphosphoinositol pentakisphosphate kinase (Asp1 and Vip1 are the single isoforms in Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively).
Fig 2.
Differences in InsP8 levels between HCT116UCL and HCT116NIH cells.
Panel A: extracts of HCT116NIH and HCT116UCL cells were prepared by using TiO2 to concentrate inositol phosphates, which were then resolved by electrophoresis on a 35% polyacrylamide gel, and visualized by staining with either toluidine or DAPI. Panels B,C show Partisphere SAX HPLC analysis of extracts of [3H]inositol-labeled HCT116UCL cells and HCT116NIH cells, respectively. The DPM in each fraction were normalized to the DPM (x104) of the [3H]inositol lipids. Fractions 25–50 are re-plotted on an expanded scale (left-hand axis), to highlight the InsP7 and InsP8 peaks.
Table 1.
STR profiles of HCT116NIH and HCT116UCL cell-lines, compared with HCT116 cells curated at ATCC.
The loci for eight core short tandem repeats plus Amelogenin were derived by ATCC for their curated HCT116 cell line (catalogue number CCL-247) and the HCT116NIH and HCT116UCL cells. The HCT116NIH and HCT116UCL cells had an 83% and 89% match with the parental HCT116 line, above the 80% minimum that designates common lineage.
Fig 3.
Comparisons of HCT116NIH and HCT116UCL cells: expression of IP6Ks and PPIP5Ks, capacity to dephosphorylate InsP8, cell growth, and phalloidin staining.
The following analyses of HCT116NIH and HCT116UCL cells were performed: Panel A, Western analyses of IP6Ks and PPIP5Ks. Complete gels, and procedures used to validate the antibodies, are described in S1 and S2 Figs. Panel B, quantitative RT-PCR analysis of expression of IP6K1, IP6K2 and IP6K3. Panel C, HPLC analysis of 1 μM [3H]InsP8 dephosphorylation by 70 μg cell lysates in 100 μl medium. Panel D, counting of cell growth for the indicated number of days. Panel E, labeling of the actin cytoskeleton with FITC-phalloidin. Hoechst was used as a nuclear stain.
Fig 4.
Separation of 1-InsP7 and 5-InsP7 by CarboPac HPLC.
Standards of [3H]InsP6, 1-[3H]InsP7, 5-[3H]InsP7, and [3H]InsP8 (1 nmol of each) were chromatographed on a CarboPac HPLC column. Panels A and B show HPLC runs in which either 1-[3H]InsP7 or 5-[3H]InsP7 were added individually, while Panel C shows an HPLC run in which both [3H]InsP7 isomers were added together. Panel D, the mass amount of 5-InsP7 was increased to 20 nmol.
Fig 5.
CarboPac HPLC analysis of [3H]inositol-labeled inositol phosphates in HCT116NIH and HCT116UCL cells.
Extracts of [3H]inositol-labeled HCT116NIH cells (Panel A) and HCT116UCL cells (Panel B) were prepared in parallel and analyzed by CarboPac HPLC. The DPM in each fraction were normalized to the DPM of the [3H]inositol lipids. Fractions 30–58 are re-plotted on an expanded scale (left-hand axis), so as to highlight the InsP7 and InsP8 peaks. This experiment was performed six times. In the experiment shown, 1-InsP7 is only discernable in the HCT116NIH cells. S3 Fig shows a separate experimental pair in which 1-InsP7 was only observed in the HCT116UCL cells.