Figure 2. Absence of Functional Stromal Interaction Molecule 1 (STIM1) in Fibroblasts and Restoration of Ca2+ Influx with Addition of Exogenous STIM1 and STIM2.
Panel A shows the STIM1 messenger RNA (mRNA) levels in fibroblasts from controls and Patient V-1. Quantitative real-time PCR was performed on complementary DNA (cDNA) from Patient V-1’s fibroblasts and from control fibroblast cell lines (seven lines for the STIM1 experiments and one line for the STIM2 experiments), amplified with the use of primers specific for STIM1, STIM2, and the gene encoding glyceraldehyde-3-phos-phate dehydrogenase (GAPDH). Threshold cycles (CT) for STIM1 and STIM2 expression, normalized to those of GAPDH (ΔCT), are plotted as 0.5ΔCT×104 (arbitrary units). The experiments were run in triplicate; results from one representative experiment are shown. The I bars indicate the standard error. Panel B shows the STIM1 protein expression, as detected on Western blotting, in fibroblasts from Patient V-1, a control, and a previously described patient with ORAI1 deficiency (due to an R91W mutation),8 as well as Jurkat T cells that were known to express STIM1 (derived from a patient with T-cell acute lymphoblastic leukemia). Total cell lysates were separated by means of sodium dodecyl sulfate–polyacrylamidegel electrophoresis and incubated with an affinity-purified antibody against the C-terminal domain of STIM1. Actin expression (as measured after incubating separated cell lysates with anti-actin antibody) is shown as a control, indicating equal loading. In Panel C (top), store-operated Ca2+ entry in fibroblasts from Patient V-1 is shown to be restored after transduction with bicistronic retroviral vectors expressing full-length STIM1 or STIM2 (right) but not after transduction with an empty vector (no insert) or a vector expressing ORAI1 (left). All vectors contained an internal ribosome entry site green fluorescent protein (GFP) cassette for the detection of transduced cells. Ca2+ measurements were obtained, and are presented, as described in Figure 1B. Between 20 and 40 cells per experiment were analyzed. Panel C (bottom) shows representative results for fibroblasts from Patient V-1 transduced with STIM1 or STIM2 vectors promoting bicistronic GFP expression. The Fura-2 images show pseudocolored intracellular Ca2+ levels: blue represents low Ca2+, green intermediate Ca2+, and yellow–red high Ca2+. The GFP images show cells expressing GFP from an internal ribosome entry site, indicative of simultaneous STIM1 or STIM2 expression. In contrast to nontransduced cells (not visible in the GFP images), transduced cells (green) show high intracellular Ca2+ levels. Panel D shows the average (from between three and five experiments) peak Ca2+ level and initial rate of Ca2+ influx in fibroblasts from Patient V-1 containing empty vectors or vectors expressing ORAI1, STIM1, or STIM2. The data shown were measured after the addition of 20 mM of Ca2+ at 500 seconds. In Panel D, the I bars indicate the standard error.