renilla control plasmid phrl-tk  (Promega)

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca 2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/eLife.72051

Figure Lengend Snippet: ( A ) Averaged fura-2 responses, their peak amplitude (middle graph bar) and slope (right graph bar) evoked by Ca 2+ re-addition in Tg treated (1 µM, 8 min) Jurkat cells lines generated by CRISPR with control or ORAI1-targeted guiding sequences and stably re-expressing either WT or C143A ORAI1-YFP. Data are mean ± SEM of 210 (Control), 242 (KO), 189 (WT), and 203 (C143A) cells from three independent experiments ( B ) Western blot showing the amount of biotinylated GFP immunoreactivity in the PM in Jurkat CRISPR ORAI1 cells reconstituted with WT or C143A ORAI1-YFP. Representative of 2 independent experiments. ( C ) Individual (thin lines) and averaged (thick line) fura-2 recordings of Jurkat CRISPR ORAI1 cells reconstituted with WT or C143A ORAI1-YFP, exposed to CD3/CD28-coated beads in Ca 2+ containing solution (left). Averaged peak and integrated responses evoked by CD3/CD28 beads in individual cells during the recording period (right). Data are from 52 cells (WT) and 72 cells (C143A) from three independent experiments. ( D ) Relative changes in NFATC-Luciferase vs. housekeeping- Renilla luminescence evoked in 4 h by Tg (1 µM) and PMA (100 nM) in the indicated cell lines. Data are mean ± SEM of 8–12 biological replicates from three independent experiments. ( E ) Endogenous NFATC1 translocation evoked in 4 hr by Tg (1 µM) in the indicated cell lines, measured by immunofluorescence. Data are mean ± SEM of the nuclear to cytosol NFATC1 intensity ratio of 58–161 cells from four independent experiments. ( F ) Time-course of NFATC1 translocation evoked by plates coated with CD3 (OKT3 1 µg/ml). Data are mean ± SEM of 86–161 cells from five independent experiments. ( G ) IL-2 production evoked by untreated (NT) or surface coated CD3 (OKT3 1 µg/ml). Left, Representative density dot plots of Jurkat cells stained for IL-2. Graph bars represent the mean ± SEM of three independent experiments. One-way ANOVA Dunnett’s multiple comparisons test ( A ), Sidak multiple comparisons test ( D and E ), two-tailed unpaired Student’s t -test (C and G).

Article Snippet: A total of 2 × 10 6 Jurkat cells were electroporated with Amaxa nucleofector (Kit V) with 1 µg of firefly luciferase encoding plasmid 9NFAT-luc together with 0.2 µg of control plasmid encoding the Renilla luciferase (phRL-TK-luc, Promega).

Techniques: Generated, CRISPR, Stable Transfection, Expressing, Western Blot, Luciferase, Translocation Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca 2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/eLife.72051

Figure Lengend Snippet: ( A ) Conditions used to study synapse formation between Raji and Jurkat T cells. Raji are pulsed with SEE prior to co-culture with Jurkat cells, IS formation evaluated in living (25 min) and fixed cells (1 H) and Jurkat activation at 24 hr. ( B ) Relative changes in NFAT-Luciferase vs. housekeeping- Renilla luminescence evoked by coculture of the indicated cell lines for 24 hr with naïve or SEE (1 µg/ml) pulsed Raji. Data are mean ± SEM of 15–20 biological replicates in cells from four independent experiments. ( C ) Representative images of ORAI1-deficient Jurkat T cells reconstituted with WT or mutant ORAI1-YFP co-cultured with pulsed RAJI cells stained with CellMask deep red. (Scale bar = 10 µm). ( D ) ORAI distribution in IS vs. opposite pole in IS forming between SEE-pulsed Raji and ORAI1-deficient Jurkat T cells reconstituted with WT (29 IS) or mutant (26 IS) ORAI1. Chi-square p value: 0.0014, two-sided Fisher’s exact test. ( E ) Quantification of ORAI1 enrichment in time-lapse images from C. IS accumulation was measured using kymographs of 20 pixels wide lines spanning the IS-distal cap axis, drawn on stable (not moving) IS forming in WT (12) and C143A (8) cells from three independent experiments. Graph bar shows mean ± SEM area under the curve of the kinetic enrichment graphs. ( F ) Representative confocal images of IS forming between SEE-pulsed Raji labeled with CellMask (Magenta, APC white labelling) and ORAI1-deficient Jurkat T cells reconstituted with WT or mutant ORAI1-YFP (Green), labelled with Phalloidin (Blue) and anti-TCR-PE (Red). Scale bar = 10 µm. Arrows indicate IS formation and asterisks accumulation of ORAI1 in the opposite pole. ( G ) IS enrichment for ORAI1-YFP, Phalloidin and TCR fluorescence in Jurkat T cells reconstituted with WT or C143A ORAI1-YFP. Fluorescence levels at the IS and opposite pole were extracted from 10 pixel wide line profiles. Graph bars shows mean ± SEM in WT (n = 25 IS) and C143A (n = 20 IS). Two-tailed unpaired Student’s t -test ( B, E and G ); Chi square ( D ). ( H ) Proposed model: S-acylation by PAT20 targets ORAI1 to lipid rafts, enabling the coordinated recruitment of ORAI1 and TCR to the immune synapse for efficient signalling.

Article Snippet: A total of 2 × 10 6 Jurkat cells were electroporated with Amaxa nucleofector (Kit V) with 1 µg of firefly luciferase encoding plasmid 9NFAT-luc together with 0.2 µg of control plasmid encoding the Renilla luciferase (phRL-TK-luc, Promega).

Techniques: Co-Culture Assay, Activation Assay, Luciferase, Mutagenesis, Cell Culture, Staining, Labeling, Fluorescence, Two Tailed Test