Journal: Molecular cell

Article Title: Cancer cells employ nuclear caspase-8 to overcome the p53-dependent G2/M checkpoint through cleavage of USP28

doi: 10.1016/j.molcel.2019.12.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After blocking with 5% FCS in PBS (++) for 30 min at RT, primary antibodies against caspase-8 (#PA5-21336, Thermo Scientific), cleaved caspase-8 (#sc-166320; Santa Cruz), and myc-tag (#ab18185, Abcam), respectively, were incubated for at RT 2 h. After washing, Alexa-488 or −546 coupled isotype-specific secondary antibodies (#A11034, #A11030, Life Technologies) were incubated at RT for 1 h, coverslips washed, and mounted in Mowiol (#D2522, Sigma).

Techniques: Western Blot, Immunocytochemistry, Immunohistochemistry, Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Extraction, Imaging, Software

Journal: Circulation Research

Article Title: Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

doi: 10.1161/01.res.0000338496.95579.56

Figure Lengend Snippet: Figure 4. SOCE in HUVECs is mediated by Stim1 and Orai1. A, Quantitative polymerase chain reaction data showing decreased expression of Orai1 and Stim1 mRNA in silenced cells compared to control cells (scrambled siRNA), 72 hours after transfection (n3; 1-way ANOVA, *P0.05). B, Western blots and densitometry showing significant downregulation of Stim1 (left) and Orai1 (right) pro- teins 72 hours after siRNA transfection. C, Stim1 and Orai1 knockdown inhibits SOCE in response to thapsigargin. D, SOCE in Stim1- silenced and Orai1-silenced cells can be rescued by overexpression of eYFP-Stim1 and CFP-Orai1, respectively. All traces shown rep- resent average data from 7 to 25 cells from at least 3 independent experiments.

Article Snippet: The primary antibodies used: GOK/Stim1 (1:250, BD Biosciences); Orai1-NT (1: 1000, ProSci); TRPC1 (1:500, Alomone labs); TRPC4 (1:500, Alomone labs).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Transfection, Western Blot, Knockdown, Over Expression

Journal: Circulation Research

Article Title: Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

doi: 10.1161/01.res.0000338496.95579.56

Figure Lengend Snippet: Figure 5. Stim1 and Orai1 mediate ICRAC in HUVECs. A, siRNA against either Stim1 or Orai1 in HUVECs greatly reduced ICRAC recorded under DVF conditions compared to control scrambled siRNA-transfected cells. B, Representative ramps taken at time points indicated by * in A. C, Summary of the whole-cell data recorded from control-siRNA, Stim-1#1-siRNA and Orai1#1-siRNA transfected HUVECs. D, mRNA expression of Stim and Orai isoforms in HUVECs. E, Ca2 imaging showing thapsigargin-activated SOCE, its potentiation with 5 mol/L 2-APB, and subsequent inhibition by 50 mol/L 2-APB; trace is average of 22 cells and representative of 4 independent experiments (1-way ANOVA, *P0.05).

Article Snippet: The primary antibodies used: GOK/Stim1 (1:250, BD Biosciences); Orai1-NT (1: 1000, ProSci); TRPC1 (1:500, Alomone labs); TRPC4 (1:500, Alomone labs).

Techniques: Control, Transfection, Expressing, Imaging, Inhibition

Journal: Circulation Research

Article Title: Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

doi: 10.1161/01.res.0000338496.95579.56

Figure Lengend Snippet: Figure 8. Stim1, Stim2, and Orai1 knockdown inhibit proliferation in HUVECs. A, Four groups of cells were transfected with the follow- ing siRNA: control, Stim1, Orai1, and Stim1 plus Orai1. At time 0, 10 000 cells were seeded per well (9.6 cm2) in triplicate. At times indicated, cells were detached and total number of cells per well counted after trypan blue exclusion. B, Data in (A) are represented as fold-increase of cell number compared to time 0. Cell cycle analysis was performed 3 days after transfection with siRNA using pro- pidium iodide staining with flow cytometry in control-siRNA (C), Stim1#1-siRNA (D), Orai1#1-siRNA (E), and Stim1#1 Orai1#1-siRNA (F) transfected HUVECs. Control not stained with propidium iodide is shown in (C). G, Quantitative polymerase chain reaction showing Stim2 mRNA knockdown, 72 hours after transfection with specific siRNA. H, EC proliferation at 72 hours after transfection with indi- cated siRNA using the same protocol described in (A). Data are representative of 4 independent experiments (1-way ANOVA, *P0.05).

Article Snippet: The primary antibodies used: GOK/Stim1 (1:250, BD Biosciences); Orai1-NT (1: 1000, ProSci); TRPC1 (1:500, Alomone labs); TRPC4 (1:500, Alomone labs).

Techniques: Knockdown, Transfection, Control, Cell Cycle Assay, Staining, Cytometry, Real-time Polymerase Chain Reaction