Journal: The Journal of General Physiology

Article Title: X-ray irradiation triggers immune response in human T-lymphocytes via store-operated Ca 2+ entry and NFAT activation

doi: 10.1085/jgp.202112865

Figure Lengend Snippet: In resting Jurkat cells, STIM1 and Orai1 are located in the ER and PM, respectively. (A and C) Confocal images of cellular distribution of endogenous STIM1 and Orai1 in Jurkat cells (A) and PBMCs (C). PM and ER of cells (first row) were stained with CellMaskOrange and ER-tracker red, respectively. Images shown as false color in blue. The second row shows immunostaining of STIM1 (green) and Orai1 (magenta) and secondary antibody tagged with Alx488 and Alx647, respectively. An overlay of both channels is shown in right column. (B and D) Line plots for each marker were taken in positions report in merged images. Fluorescence intensity of either Orai1/PM (B) or STIM1/ER (D) were normalized to the highest value of each signal; the colors of line plots correspond to those in images. All scale bars, 10 μm. The antibodies for detecting STIM1 and Orai1 are specific. (E) Jurkat cells in which Orai1 was knocked out generate no more signal in immunostaining with Orai1 antibody, while still producing signal with STIM1 antibody. (F) Staining of WT Jurkat cells in which either the primary STIM1 or Orai1 antibodies (top row) or the respective secondary antibodies (bottom row) were left out generated no appreciable fluorescent signals.

Article Snippet: Jurkat cells were fixed on BSA/poly-L-lysine–coated glass coverslips 15, 30, 45, 60, or 90 min after treatment using 4% paraformaldehyde and stained with primary antibodies for STIM1 (PA1-46217; Thermo Fisher Scientific), Orai1 (NBP1-75523; Novus Biologicals, or O8264; Sigma-Aldrich), and NFATc2 (MA1-025; Thermo Fisher Scientific).

Techniques: Staining, Immunostaining, Marker, Fluorescence, Generated

Journal: The Journal of General Physiology

Article Title: X-ray irradiation triggers immune response in human T-lymphocytes via store-operated Ca 2+ entry and NFAT activation

doi: 10.1085/jgp.202112865

Figure Lengend Snippet: IR triggers Ca 2+ regulated STIM1/Orai1 CRAC channel formation. (A) Distribution of endogenous Orai1 (magenta, first column) and STIM1 (green, second column) in Jurkat cells immune-stained with Alx647 and Alx488, respectively. Overlays of green and magenta images with magnification of indicated areas are shown in third and fourth columns. Fixed cells were obtained from untreated/nonirradiated control cells (top row), cells treated for 15 min with 2 µM Tg (central row), or cells 15 min after 5-Gy x-ray exposure (bottom row). (B) Probability of finding, in a population of Jurkat cells, positive clustering of STIM1/Orai1 (P STIM1/Orai1+ ) after irradiation with 1.5 Gy (squares) or 5 Gy (circles). Criteria for cluster detection are specified in Materials and methods. For each condition, ≥282 cells were analyzed. (C) Representative confocal images of same cells with fluorescent donor molecule Orai1::eCFP (magenta, first column), acceptor molecule STIM1::eYFP (green, second column), and heatmaps of the resulting FRET signals (third column) 15 min after treatment. Images are from untreated cells (control), cells incubated with 2 µM Tg, 25 μl/ml ImmunoCult Human CD3/CD28/CD2 T-Ac, or irradiated with 5 Gy. All three treatments generate a visible FRET-signal in the PM. Scale bars, 10 μm. (D) Mean FRET signal (±SD, n ≥ 5) from PM of cells as in C: untreated/nonirradiated control cells (crtl), cells 5 min in 2 μM Tg, 15 min in 25 μl/ml T-Ac, or 20 min after irradiation with 5 Gy. Statistical differences between treatments were analyzed by unpaired Student’s t test, and respective P values are given in the figure.

Article Snippet: Jurkat cells were fixed on BSA/poly-L-lysine–coated glass coverslips 15, 30, 45, 60, or 90 min after treatment using 4% paraformaldehyde and stained with primary antibodies for STIM1 (PA1-46217; Thermo Fisher Scientific), Orai1 (NBP1-75523; Novus Biologicals, or O8264; Sigma-Aldrich), and NFATc2 (MA1-025; Thermo Fisher Scientific).

Techniques: Staining, Control, Irradiation, Incubation

Journal: The Journal of General Physiology

Article Title: X-ray irradiation triggers immune response in human T-lymphocytes via store-operated Ca 2+ entry and NFAT activation

doi: 10.1085/jgp.202112865

Figure Lengend Snippet: Time course of stimulus-induced STIM1/Orai1 colocalization. (A) Fluorescent images of a representative Jurkat cell cotransfected with STIM1::eYFP (green) and Orai1::eCFP (magenta) with focus on PM/cytosol interface. Images were taken before (0 min) and 4 and 10 min after treating cells with 10 μM Tg. White boxes show ROIs in membrane/cytosol interface for calculating PCC value of the two fluorescent markers. (B) Change in PCC (∆PCC) for colocalization of STIM1::eYFP and Orai1::eCFP. Data obtained from confocal live-cell real-time acquisition of Jurkat cells as in A, heterologously expressing the two proteins. In five independent experiments a mean PCC of 3.8 ± 0.08 was estimated from 18 untreated control cells (triangle). Changes in PCC values over time from untreated and treated cells (circles) are shown as deviation from this control value (∆PCC). The PCC values of untreated cells remain at the same level (ctrl, black) but increase with different kinetics in cells stimulated with 2 µM Tg (orange) or 5 Gy x rays (5 Gy, magenta). The data were fitted with logistic equation ( , solid lines) yielding the following times for half-maximal increase in STIM1/Orai1 colocalization: 2 min for Tg and 12 min for x ray. Data for the three conditions are mean values ± SD from N ≥ 4 independent experiments with n ≥ 4 cells each. Scale bars, 2.5 μm.

Article Snippet: Jurkat cells were fixed on BSA/poly-L-lysine–coated glass coverslips 15, 30, 45, 60, or 90 min after treatment using 4% paraformaldehyde and stained with primary antibodies for STIM1 (PA1-46217; Thermo Fisher Scientific), Orai1 (NBP1-75523; Novus Biologicals, or O8264; Sigma-Aldrich), and NFATc2 (MA1-025; Thermo Fisher Scientific).

Techniques: Membrane, Expressing, Control

Journal: The Journal of General Physiology

Article Title: X-ray irradiation triggers immune response in human T-lymphocytes via store-operated Ca 2+ entry and NFAT activation

doi: 10.1085/jgp.202112865

Figure Lengend Snippet: Calcium-dependent SOCE/NFAT pathway is activated by IR in naive T-lymphocytes. (A) Distribution of endogenous Orai1 (magenta, first column) and STIM1 (green, second column) in fixed PBMCs immunostained with secondary antibodies Alx488 and Alx647, respectively. A merge of the two channels is shown in the third column, with higher magnification of marked areas in the fourth column. The merge of untreated control cells additionally shows the nucleus stained with Hoechst DNA dye (blue). Images show cells that were fixed as untreated/nonirradiated control cells (ctrl, top row) and cells fixed 15 min after treatment with 2 µM Tg (second row) or after exposing cells to 5 Gy (third row). (B) Mean ratio (± SD, number of cells in brackets) of green fluorescence in ROI (inset image, red circle) in cytosol divided by fluorescence in ROI in direct vicinity over PM (black circle). Data from untreated control cells (ctrl) as well as cells exposed to 2 μM Tg or 5 Gy x rays 60 min after treatment. (C) Confocal images of PBMCs showing nucleus stained with Hoechst DNA dye (blue, first column) and endogenous NFATc2 (green, second column) stained with Alx488. Overlay of both columns is shown in third column. Cells were fixed immediately (untreated/nonirradiated control, top row), 15 min after 2 µM Tg Ca 2+ store depletion (second row) or 60 min after x-ray exposure with 5 Gy (third/fourth row). All scale bars, 10 μm. (D) Mean ratio (± SD, number of cells in brackets) of GFP fluorescence in nucleus (inset image, magenta circle) divided by fluorescence of total cell (white circle). Statistical differences between treatments in B and D were analyzed by unpaired Student’s t test, and respective P values are given in the figure. Source data are available for this figure: .

Article Snippet: Jurkat cells were fixed on BSA/poly-L-lysine–coated glass coverslips 15, 30, 45, 60, or 90 min after treatment using 4% paraformaldehyde and stained with primary antibodies for STIM1 (PA1-46217; Thermo Fisher Scientific), Orai1 (NBP1-75523; Novus Biologicals, or O8264; Sigma-Aldrich), and NFATc2 (MA1-025; Thermo Fisher Scientific).

Techniques: Control, Staining, Fluorescence