(A) Schematic diagram illustrating the area in IKAP responsible for FD. The FD mutation at position six of exon 20 splice donor site is shown by an arrow. In FD patients, two mRNA isoforms, one containing exon 20 and one without, can be present. (B) RT-PCR analysis of IKAP mRNA. RNA was extracted from control, heterozygous and FD cells and the endogenous splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exon 19 and 21. Isoforms were quantified using ImageJ. GAPDH was used as control for cDNA amounts. (C) QPCR analysis of the IKAP mRNA. Left side: Level of exon 20 inclusion isoform (wt). Relative quantity represents normalization to control cells. Right side: Level of exon 20 skipped isoform (mut). Relative quantity represents normalization to heterozygous (Htz) cells. All values were normalized to HPRT mRNA. QPCR experiments were amplified in triplicate; results shown are mean values ± SD. (D) Analysis of IKAP protein levels. Western blotting of extracts from the indicated cell lines using an anti-IKAP antibody (Santa Cruz Biotechnology, D-17). Band intensities were quantified using ImageJ.

(A) Chemical structure of PS. (B) FDA cells or (C) FDB cells were treated with 0, 5, 10, 100, 200 and 300 µg/ml PS. RNA was extracted after 24 hr for FDA cell line and after 48 hr for FDB cell line. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to levels in untreated control cells. Right side: RT-PCR analysis of the splicing of the endogenous IKAP mRNA in FD cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.

PS was added to FDB cells at a concentration of 100 µg/ml. Every two days the medium was replaced and fresh PS was added. RNA was extracted 3, 7 and 14 days following the initial addition of PS. All experiments were repeated independently three times, and the results shown are representative of an average experiment. (A) QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to levels in untreated control cells harvested on the same day. QPCR experiments were amplified in triplicate; results shown are mean values ± SD. (B) RT-PCR analysis of the splicing of the endogenous IKAP in FDB cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. (C) Western blotting using an anti-IKAP antibody (BD-Bioscience) of FDB cells treated with PS for two weeks. The exposure of the analysis of 293T cells was reduced to one fourth of its original amount in order to avoid intense background and to better visualize the effect of PS. Band intensities were quantified using ImageJ.

FDB cells were treated with 100 µg/ml PS. RNA was extracted 24 hr following the addition of the supplement. A portion of each RNA sample was used for microarray analysis, and an aliquot was saved for experimental validation. QPCR analysis of genes shown by the gene expression microarray analysis to be (A) up-regulated or (B) down-regulated as a result of PS treatment. Each panel shows the levels in FDB cells after PS treatment relative to untreated FDB cells. (C) All values were normalized to a control, the LZIC gene transcript, that did not change as a result of PS treatment. QPCR experiments were amplified in triplicate; results shown are mean values ±S.D.

One day prior to treatment 750,000 cells were seeded. The cells were treated with 100 µg/ml PS for 24 hr and then fixed and stained with PI according to the flow cytometry protocol. The percentage of cells (A) at the G1 stage and (B) at the growth stages, S+G2, of untreated control, heterozygous, FDA and FDB cells. The percentages of cells at the growth stages, S+G2, of (C) FDA or (D) FDB cells following PS treatment compared to untreated FD cells. All experiments were repeated independently four times, and the results shown are mean values ± SD. P-values of significant results are indicated.

(A) Sharp·PS GOLD4508P (Gold), (B) SharpGPC 85F (GPC), or (C) Krill oil+4225F (Krill oil) were added to FDB cells at the indicated concentrations. RNA was extracted 24 hr following the addition of the supplement. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to that of untreated control cells. Right side: RT-PCR analysis of the splicing of IKAP in FDB cells. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.

(A) Kinetin or (B) tocotrienol (Toco) were added to FDB cells at the indicated concentrations. RNA was extracted 24 hr following the addition of the substance. Left side: QPCR analysis of the level of exon 20 inclusion isoform (wt). Data were normalized to that of untreated control cells. Right side: RT-PCR analysis of the splicing of the endogenous IKAP. All splicing products were separated on a 2% agarose gel after RT-PCR reaction using primers to exons 19 and 21. The PCR products were eluted and sequenced. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.