Relative mRNA levels of FISP and FISP-sp in Th2 cells. The mRNA expression levels of FISP and FISP-sp were analyzed by quantitative real time PCR in Th2 D10 cells and in 5-day differentiated primary Th2 cells. In quantitative real time PCR amplification, the normalized reporter signal represents the fluorescence changes adjusted to base-line levels before amplification and the threshold cycle (Ct) of amplified products. A, designed primer sets are as follows: common to FISP and FISP-sp (set 1), FISP (set 2), FISP-sp (set 3), and specific for both FISP and FISP-sp but yielding amplicons of different sizes because of splicing of 29 bases (set 4). B, ratio of FISP to FISP-sp mRNA was shown to be ∼10:1 in D10 and Th2 cells. C, threshold cycle of amplified products, which were amplified using primer sets 1, 2, and 3. D, melting curve graph for PCR products amplified by primer set 4 demonstrates the presence of two different species of amplified PCR products. Arrows indicate the relative peaks of FISP and FISP-sp.

FISP-sp inhibits FISP-induced cell growth inhibition and apoptosis. A, effect of the control vector, FISP, FISP-sp, and FISP + FISP-sp upon the growth of B16F10.9 cells, analyzed 24, 48, and 72 h post-transfection. B, apoptotic changes in B16F10.9 cells were analyzed after transfection with mock vector, FISP, FISP-sp, or FISP + FISP-sp. At 72 h post-transfection, photomicrographs were taken of adherent cells in each sample (upper panel). The cells were then washed and stained with 0.1 μg/ml Hoechst 34580 (Invitrogen) or propidium iodide (Sigma). The slides were then examined by fluorescence microscopy and photographed. Cells showing signs of apoptosis (fragmented nuclei) and membrane blebbing are shown using Hoechst and propidium iodide treatment, respectively (bottom and middle panels, respectively). C, overexpression of FISP induces expression of the GADD family of genes in B16F10.9 melanoma cells, in a time-dependent manner. Total RNA was extracted, and real time PCR analysis was performed using specific primers. All of the GADD genes examined were found to be up-regulated in the FISP-transfected samples but not in the samples transfected with FISP-sp or FISP + FISP-sp. D, B16F10.9 cells were transfected with either mock vector, FISP, FISP-sp, or FISP + FISP-sp. At 48 and 72 h post-transfection, DNA was extracted from the cells, and fragmentation was analyzed. E, FISP activates caspase-3 in tumor cells. Activation of procaspase-3 is indicated by the generation of its cleavage products, as observed in FISP-transfected B16F10.9 mouse melanoma cells, but not in FISP-sp-treated cells. β-Actin was used as a loading control.

Generation of FISP-sp under conditions of ER stress. A, schematic representations of protein structures resulting from the +29-SP-ΔATG-frame3-GFP and +29-SP-ΔATG-frame3-LUC vectors. B, HEK293 and EL-4 cells were transfected with the +29-SP-ΔATG-frame3-LUC vector or with the pXPG vector as a control (mock). Higher levels of luciferase activity were observed in tunicamycin-treated samples, compared with untreated samples. NIH3T3 cells were transiently transfected with the +29-SP-ΔATG-frame3-GFP vector. Anti-GFP monoclonal antibodies were used for Western blot analysis. Bands matching the size of the expressed FISP-sp-GFP protein were more pronounced in their intensity, in cells treated with tunicamycin. C, cDNA from +29-SP-ΔATG-frame3-LUC or vector only. Transfected NIH3T3 and EL-4 cells left untreated or treated with tunicamycin were prepared and analyzed by quantitative real time PCR analysis. Expression levels in untreated (w/o) samples were assigned a value of 1. The fold change in FISP-sp expression upon tunicamycin treatment was determined by comparison of treated and untreated samples. D, transient transfections in mouse primary Th2 cells were carried out, and luciferase activity was analyzed. Cells were collected, and lysates were assessed using the dual luciferase assay system. Analysis of mRNA levels revealed a 25-fold increase upon induction of ER stress. Expression levels in untreated (w/o) samples were assigned a value of 1. The data are representative of at least two independent experiments. E, transient transfections in B16F10.9 and HeLa cells were performed, and analysis of luciferase activity was performed as described above. Analysis of mRNA levels revealed a 75% decrease upon induction of ER stress in B16F10.9 cells. The data are representative of at least two independent experiments.

Cloning of a novel FISP splicing variant, FISP-sp. A, splicing variant of FISP, marked with an arrow, was cloned from a D10 cell cDNA library. The difference in the length of FISP-sp and FISP transcripts was confirmed by PCR. B, DNA sequence alignment of FISP and FISP-sp shows that FISP-sp lacks 29 bp from the 5′-end of FISP exon 4. Boxed regions indicate the site of alternative splicing in FISP. Boxed nucleotide sequences correspond to the sequencing histogram of FISP and FISP-sp. C, FISP and FISP-sp amino acid sequences are identical at the N-terminal region but are different at the C-terminal region, beginning at the boxed area, because of a frameshift caused by splicing. Schematic representations of FISP and FISP-sp peptide structures are shown below the amino acid sequence.

FISP-sp is not secreted but is specifically localized in the ER. A, protein expression of FISP and FISP-sp. HEK293 cells were transiently transfected with 3′-Myc/His-tagged FISP (FISP-Myc/His), 3′-HA-tagged FISP-sp (FISP-sp-HA), or empty vectors (pcDNA 3.1 Myc-His A, and pcDNA 3.1-HA). Protein lysates were collected from cells after transfection with FISP-Myc/His, FISP-sp-HA, or vector alone. Samples (30 μg) were used for Western blot analysis using mouse anti-Myc and anti-HA antibodies. To detect the secreted forms of FISP and FISP-sp protein, cells were cultured for 12 h in serum-free DMEM. Conditioned medium was collected and concentrated 60-fold. A volume of 20 μl conditioned medium was subjected to SDS-PAGE. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. B, subcellular localization of FISP and FISP-sp in COS-7 cells. Protein localization was analyzed by immunocytochemistry. FISP was detected using anti-His or anti-Myc antibodies, and FISP-sp was detected using an anti-HA antibody. The Golgi and ER were visualized using Golgi-BODIPY TR ceramide and an anti-PDI antibody. Images of different cellular compartments exhibiting FISP and FISP-sp staining were merged. FISP protein staining partially overlapped with ER and Golgi apparatus staining. However, staining of FISP-sp protein matched exactly with ER staining and was not detected in the Golgi apparatus. C, subcellular localization of FISP and FISP-sp in HeLa cells.

Physical interaction of FISP and FISP-sp. A, FISP-sp blocks FISP secretion. HEK293 cells were transiently transfected with 3′-Myc/His-tagged FISP (FISP-Myc/His) or 3′-HA-tagged FISP (FISP-HA), or their respective empty vectors (pcDNA 3.1 Myc-his A, and pcDNA 3.1-HA). To detect the secreted forms of these proteins, cells were cultured for 12 h in serum-free DMEM. Conditioned medium was collected, concentrated 100-fold, precipitated with 100% acetone, and subjected to SDS-PAGE and Western blot analysis using mouse anti-Myc and anti-HA antibodies. IB, immunoblot. B, FISP-sp physically interacts with FISP. HEK293 cells were transiently transfected with 3′-Myc/His-tagged FISP (FISP-Myc/His) or 3′-HA-tagged FISP-sp (FISP-sp-HA) expression vectors. Immunoprecipitation analysis was performed 24 h post-transfection using anti-Myc, anti-HA, or anti-IgG (control) antibodies, and the same samples were further analyzed using Western blot analysis as indicated. FISP and FISP-sp bands are indicated by arrows. C, subcellular localization of FISP and FISP-sp upon expression vector co-transfection in COS-7 and HeLa cells. Protein localization was analyzed by immunocytochemistry. FISP was detected using anti-His or anti-Myc antibodies, and FISP-sp was detected using an anti-HA antibody. The ER was visualized using an anti-PDI antibody. Images of ER compartments and FISP or FISP-sp staining were merged. The pattern of FISP protein staining overlapped exactly with that of FISP-sp staining in the ER compartment, in both in COS-7 and HeLa cells. D, HEK293 cells were transfected with 3′-Myc/His-tagged FISP (FISP-Myc/His) or 3′-HA-tagged FISP-sp (FISP-sp-HA), or were co-transfected with both expression vectors. MG132 (10 μm) was added to cells 24 h post-transfection or cells were left untreated. Cells were analyzed 12 h later by Western blot analysis using anti-Myc or anti-HA antibodies. Bands corresponding to FISP and FISP-sp are indicated by arrows.