Cell Transfection, Plasmids, and Reagents
HeLa (human epithelial) cells and Jurkat (human T lymphocyte) E6-1 line were purchased from ATCC. DNA plasmids and siRNA were cotransfected into HeLa or Jurkat cells with Genesilencer reagent (Gene Therapy Systems, San Diego, CA). Full-length human STIM1 cDNA was isolated by PCR, sequenced, and cloned in to pDS_XB-YFP vector (ATCC). Enhanced yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) (Clontech, Palo Alto) was inserted immediately downstream of the predicted signal-peptide region of human STIM1. The YFP-conjugated EF-hand mutant of STIM1, YFP-STIM1(D76A), was made by site-directed mutagenesis with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Nucleotide sequences of constructs were verified by sequencing. NF-ATc1-YFP was provided by Dr. Won Do Heo. pECFP-Nuc, pECFP-ER, and pEYFP-Nuc plasmids were obtained from Clontech. pECFP-CAAX plasmid was provided by Dr. Mary Teruel. Thapsigargin, histamine, and BHQ (2,5-di-(t-Butyl)-1,4-benzohydroquinone) were purchased from Calbiochem. Anti-human CD3 (T cell receptor) antibody (BD BioSciences) was used at 20 μg/ml. SKF 96365 (Sigma) was used at 20 μM.
siRNA Library of Signaling Proteins
Two thousand, three hundred and four human signaling-related proteins were selected from the NCBI (RefSeq database) on the basis of the presence of signaling domains, such as protein kinase, SH2, SAM, EF, and PH domains, as well as by text searches of signaling-related terms. Gene-specific primers for the selected signaling proteins were designed with an in-house primer program and were used to generate ~600 bp cDNA fragments immediately upstream of the stop codon of each mRNA by PCR. An additional set of nested primers was designed to add T7 promoters at both ends of the final cDNA fragment. Nested PCR products were subjected to in vitro transcription, in vitro dicing, and purification to produce siRNA as described previously [24
]. The siRNA signaling set was sorted according to the NCBI RefSeq Protein accession number and was stored in 24 96-well plates. The screen for SOC influx regulators was done by transfecting HeLa cells with the siRNA signaling set at an average concentration of 10 nM for 2 days in the 96-well format. Twenty-four siRNAs present in duplicates were screened at a time in an experimental microplate. The whole screen was performed twice.
Ca2+ measurements were made with a fluorescence microplate reader (FlexStation, Molecular Devices). HeLa cells were loaded with 2 μM Fura-2-AM in extracellular buffer (125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 20 mM HEPES, 10 mM glucose, and 1.5 mM CaCl2 [pH 7.4]) for 30 min at room temperature. Fura-2 fluorescence was measured by illuminating the cells with an alternating 340/380 nm light every 5 s. Fluorescence intensity was measured at 510 nm. Changes in intracellular Ca2+ concentration are presented as the change in the ratio of fluorescence intensity for excitation at 340 and 380 nm. For Ca2+ add-back experiments, 3 mM EGTA was added together with histamine and thapsigargin to remove extracellular Ca2+, and 10 mM Ca2+ was added back after Ca2+ store depletion. Imaging-based single-cell Ca2+ measurements of HeLa or Jurkat cells were performed with a 4× (HeLa) or 10× (Jurkat) objective on an automated fluorescent microscope ImageXpress 5000A (Molecular Devices) by loading cells with 0.5 μM Fura-2-AM. Fluorescence intensities of single cells were measured with the ImageXpress analysis software.
Mn2+ Quench Assays
Two millimolars Mn2+ was added to cells immediately before image acquisition. Histamine and thapsigargin were added 50 s after, and image acquisition was continued for another 90 s. Quenching of Fura-2 fluorescence was measured by illuminating cells with 360 nm light every 4 s, and fluorescence intensity was measured at 510 nm with ImageXpress.
NF-ATc1 translocation was monitored in HeLa cells cotransfected with NF-ATc1-YFP and pECFP-Nuc plasmids with ImageXpress 5000A with a 10× objective. Live-cell confocal imaging experiments were performed with transfected HeLa cells with a 40× objective on a spinning-disk confocal microscope (Nipkow Wallac system). Live-cell TIRF imaging was done with transfected HeLa cells with a 60× objective on a Nikon TIRF microscopy system. Images were analyzed with MetaMorph software (Universal Imaging).