CD59-mediated Ca²⁺ signaling requires CD3ζ expression.

<p>(A) Cluster distribution of Ca²⁺ time traces in WT and TCR^high cells is shown upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (91.4±2.1% and 92.9±3.9% upon anti-CD3 stimulation, 34.4±16.3% and 72.0±16.6% upon anti-CD59 stimulation in WT and TCR^high cells, respectively). Mean values from two independent experiments, each with three technical replicates are shown (n ≥ 167 per cell type and condition). (B) Cluster distribution of Ca²⁺ time traces in WT, TCR^-, and cells expressing CD8-ζ fusion protein is shown upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (90.1±1.4%, 1.9±0.7% and 7.4±2.3% upon anti-CD3 stimulation, 25.2±6.8%, 1.6±0.4% and 13.4±1.6% upon anti-CD59 stimulation for WT, TCR^-, and CD8-ζ cells, respectively). Mean values from four independent experiments, each with three technical replicates, are shown (n ≥ 343 per cell type and condition). Multiple comparison tests for the fractions showing Ca²⁺ release patterns in (A) and (B) were assessed by one-way ANOVA, significances are shown where applicable, * p < 0.05, ** p < 0.01, *** p < 0.001.</p

TCR/CD3- and CD59-mediated Ca²⁺ signaling are dependent on Lck and LAT.

<p>Cluster distribution of Ca²⁺ time traces in differently treated cells upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. (A) Ca²⁺ measurements were performed with WT cells transiently transfected with negative control siRNA (siNeg), Lck-specific siRNA (siLck), WT cells treated with 10 µM PP2 (PP2), and Lck-deficient J.CaM1.6 cells. Clusters representing Ca²⁺ release patterns are framed in black (89.4±10.1%, 76.5±10.4%, 28.3±39.1%, and 23.4±9.9% upon anti-CD3 stimulation, 36.9±13.2%, 12.7±6.1%, 2.6±2.0%, and 1.2±1.2% upon anti-CD59 stimulation for siNeg, siLck, PP2-treated, and J.CaM1.6 cells, respectively). Mean values from at least two independent experiments, each with three technical replicates, are shown (n ≥ 204 per cell type and condition). (B) Cluster analysis of Ca²⁺ time traces in WT cells transiently transfected with negative control siRNA (siNeg), LAT-specific siRNA (siLAT), and LAT-deficient J.CaM2.5 cells. Clusters representing Ca²⁺ release patterns are framed in black (89.4±10.1%, 73.5±5.8%, and 3.0±2.3% upon anti-CD3 stimulation, 36.9±13.2%, 13.0±6.6%, and 1.7±1.6% upon anti-CD59 stimulation for siNeg, siLAT, and J.CaM2.5 cells, respectively). Mean values from at least three independent experiments, each with three technical replicates, are shown (n ≥ 208 per cell type and condition). Multiple comparison tests for the fractions showing Ca²⁺ release patterns in (A) and (B) were assessed by one-way ANOVA, significances are shown where applicable, ** p < 0.01, *** p < 0.001. (C) Knock-down of target proteins was tested by Western blotting. 48 h after transfection cell lysates from siRNA treated cells were probed with anti-Lck, anti-LAT, and anti-β-actin. J.CaM1.6 cells, J.CaM2.5 cells, and WT cells served as controls.</p

Reconstitution of Lck by forced interaction of CD3ζ and Lck facilitates TCR/CD3- but not CD59-mediated Ca²⁺ signaling.

<p>(A) Lck expression levels in WT cells, J.CaM1.6 cells and J.CaM1.6 cells expressing mEGFP-tagged Lck fused to CD3ζ (CD3ζ-Lck) were tested by Western blotting, the same blot was reprobed using anti-β-actin as a control. (B) Plasma membrane localization of CD3ζ-Lck-mEGFP in J.CaM1.6 cells was imaged by fluorescence microscopy. CD3ζ-Lck-mEGFP fluorescence and a bright field (BF) image of a transfected J.CaM1.6 cell are shown (scale bars  =  10 µm). (C) Cluster distribution of Ca²⁺ time traces in Lck-deficient J.CaM1.6 cells and J.CaM1.6 cells stably expressing mEGFP-tagged Lck fused to CD3ζ (CD3ζ-Lck) is shown upon anti-CD3 or anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (35.7±4.9% and 71.8±2.3% upon anti-CD3 stimulation, 1.4±2.3% and 1.0±1.3% upon anti-CD59 stimulation for J.CaM1.6 and CD3ζ-Lck cells, respectively). Mean values from three independent experiments, each with three technical replicates, are shown (n ≥ 323 per cell type and condition). Multiple comparison tests for the fractions showing Ca²⁺ release patterns were assessed by one-way ANOVA, significances are shown where applicable, ***p < 0.001.</p

Single-cell Ca²⁺ measurements reveal differential heterogeneity upon anti-CD3 and anti-CD59 stimulation.

<p>Jurkat cells (WT) were loaded with Indo-1/AM followed by stimulation with anti-CD3-, anti-CD59-, or anti-CD71-coated surfaces. Individual Ca²⁺ time traces were measured for 200 s after identification of initial cell-surface contact as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085934#pone-0085934-g001" target="_blank">Figure 1S</a>. (A) Box plots of individual Ca²⁺ time traces generated by analysis of the whole cell population upon anti-CD3, anti-CD59, and anti-CD71 stimulation (black line  =  median, grey area  = 50% of Ca²⁺ time traces, dotted lines  =  75% of Ca²⁺ time traces). (B) Individual Ca²⁺ time traces from single-cell measurements were grouped into 11 clusters by affinity propagation clustering as described in Materials and Methods. Each plot shows the respective Ca²⁺ time traces for a cluster, an exemplar trace for each cluster is shown in black. Clusters representing Ca²⁺ release patterns are framed in black. (C) Stimulus-dependent cluster distribution upon anti-CD3, anti-CD59, and anti-CD71 stimulation in WT cells is shown by stacked bar plots. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (88.5±4.9%, 31.8±12.0%, and 5.5±5.3% for anti-CD3, anti-CD59, and anti-CD71 stimulation, respectively). Mean values from at least three independent experiments, each with three technical replicates, are shown (n ≥ 249 per stimulatory condition). (D) CD3ε and CD59 surface expression levels in WT cells. WT cells were surface stained with Alexa Fluor 647-conjugated anti-CD3ε and FITC-conjugated anti-CD59 or isotype controls and analyzed by flow cytometry. Live cells were gated based on the Forward Scatter and Side Scatter profiles and propidium iodide exclusion. A representative dot plot of four technical replicates is shown.</p

Lck expression and anti-CD59 stimulation influence CD3 surface expression.

<p>WT cells were transfected with negative control siRNA (siNeg) or Lck-specific siRNA (siLck) and experiments were performed 48 h after transfection. (A) Testing of Lck knock-down efficiency. Cell lysates were probed by Western blotting for Lck expression and β-actin as a control. (B) Efficiency of Lck knock-down tested by ensemble Ca²⁺ measurements. siLck and siNeg cells were loaded with Indo-1/AM. Cells were incubated with anti-CD59 mAb or anti-IgG2a for 5 min at 37 °C. For antibody cross-linking, goat anti-mouse F(ab’)₂ was added to samples and Ca²⁺ mobilization of the whole cell population was measured by a microplate reader. For testing TCR surface expression levels, cells were stimulated with anti-CD59 or anti-IgG2a, as isotype control, followed by incubation with goat anti-mouse F(ab’)₂ for (C) 1 h or (D) 15 h at 37°C. Cells were surface stained at 4 °C with FITC-conjugated anti-CD3ε or isotype control and analyzed by flow cytometry. Live cells were gated based on the Forward Scatter and Side Scatter profiles and propidium iodide exclusion. Fluorescence values displayed are isotype control corrected. Representative results of two separate experiments are shown (mean ±SD, n = 4). Multiple comparison tests were assessed by one-way ANOVA, significances are shown where applicable, ***p < 0.001.</p

Effect of BGP-15 on DChol desorption from lipid monolayers to MBCD.

<p><b>A</b>) MBCD-mediated removal of DChol from lipid monolayers at constant lateral surface pressure. When indicated 10 µM BGP-15 was injected into the subphase underneath SM/DChol monolayer 5 min before MBCD was administered. Surface area was measured before (A₀) and at the indicated time points after (A_t) MBCD injection. <b>B</b>) Effect of BGP-15 concentration on MBCD-mediated DChol desorption from SM/DChol monolayers. The monolayers were equilibrated with BGP-15 for 5 min before MBCD was injected into the subphase.</p

Effect of BGP-15 treatment on heat-induced HSF1 acetylation in HEK293T cells and on <i>in vitro</i> SIRT1 activity.

<p><b>A</b>) HEK293T cells transiently co-transfected with mouse HSF1-FLAG and p300 were heat shocked for different lengths of time at 42°C or for 60 min at 42°C followed by 60 min recovery (R). After immunoprecipitation by FLAG, samples were probed for acetylated lysin by western blotting [n = 3, error bars represent standard error of the mean (SEM)]; <b>B</b>) Samples were or were not treated with 10 µM BGP-15 for 60 min before and during 60 min heat shock or 60 min recovery following heat shock (R) Acetylated HSF1 was determined as above. [n = 4, p<0.05, error bars represent standard error of the mean (SEM)]; <b>C</b>) In vitro activity of SIRT1 using activators, inhibitors and BGP-15 [n = 3, error bars represent standard error of the mean (SEM)].</p

The effect of BGP-15 on the temperature-induced change in cluster fraction of mGFP-GPI.

<p>CHO cells stably expressing mGFP-GPI were subjected to 39.5°C with or without 10 µM BGP-15. TOCCSL experiments were performed and at indicated times the cluster fraction (two or more fluorophores per domain) was calculated [n = 2 error bars represent standard error of the mean (SEM)].</p

<i>Hsp25</i> mRNA expression in B16-F10 cells modified by Rac1 inhibitor and BGP-15 administration.

<p>B16-F10 cells were pretreated or not by Rac1 inhibitor NSC23766 for 2 hours before 1 h 41.5°C heat shock with or without 10 µM BGP-15. RNA was isolated and the expression of <i>hsp25</i> was tested by RT-PCR. [n = 3, error bars represent standard error of the mean (SEM)].</p

The effect of BGP-15 on cholesterol-rich membrane domains and the heat shock inducibility of <i>hsp25</i> in B16-F10 cells with different cell density.

<p><b>A</b>) Fluorescence intensity of fPEG-Chol in B16-F10 cells seeded with low (LCN, 1.5×10⁶/10 cm plate) or high cell number (HCN, 6×10⁶/10 cm plate with or without 10 µM BGP-15). Student's t-test was used for statistical analyses, and p = 0.05 was set as a significance threshold. <b>B</b>) The size distribution profile of membrane microdomains labeled by fPEG-Chol in HCN (with or without 10 µM BGP-15) and LCN cells and imaged by TIRF microscopy. <b>C</b>) HCN (with or without 10 µM BGP-15) and LCN cells were heated at 42°C for 1 hour and the expression of <i>hsp25</i> was tested by RT-PCR.</p