Journal: Cellular and molecular life sciences : CMLS

Article Title: The TRPM7 kinase limits receptor-induced calcium release by regulating heterotrimeric G-proteins

doi: 10.1007/s00018-018-2786-z

Figure Lengend Snippet: HEK293-TREx cells overexpressing either human wild-type TRPM7 (TRPM7 WT), a TRPM7 mutant deficient in phosphotransferase activity (M7-K1648R), or human wild-type TRPM4 (TRPM4 WT) were investigated for Ca2+ signals through protease-activated receptor stimulation using thrombin or trypsin, and metabotrophic purinergic-receptor stimulation using NECA, ADP or ATP. Ca2+ release was assessed using a fluorescent kinetic plate reader in the 384 well format (see methods). The 340 nm/380 nm ratio was normalized to one to the average of the 10 data points acquired before compound application in each of the 384 wells. The normalized ratio was then plotted against time of the experiment. The shaded areas indicate exposure to the respective agonists in extracellular solution absent of Ca2+: either thrombin (30 U), trypsin (100 nM), NECA (3 mM), ADP (3 mM) or ATP (3 mM) on (A) hTRPM7-WT (n = 4-8), p(B) hTRPM7-K1648R (n = 4-8), (C) hTRPM4 (n = 4). Cells were either non-induced (tet−; black line) or induced for 20 hrs with tetracycline (tet+, red line).

Article Snippet: We thank Dr. Clay Wakano from the Queen’s High-Throughput Drug Screening Laboratory for providing access to and support for the 96/384-well kinetic plate reader assay partially supported by Hamamatsu/Queen’s PET Imaging, LLC (AF).

Techniques: Mutagenesis, Activity Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: The TRPM7 kinase limits receptor-induced calcium release by regulating heterotrimeric G-proteins

doi: 10.1007/s00018-018-2786-z

Figure Lengend Snippet: hTRPM7-HEK293-TREx cells were investigated for thrombin-induced Ca2+ release using a fluorescent kinetic plate reader in a 96 well format (see methods). Data capture proceeded at 1 Hz over 300 s. (A) Ratiometric raw data traces of Fura-2 emission signals (in red) measured in 24 individual wells on a 96 well plate over 300 s. Excitation wavelengths were 340 nm and 380 nm. The white dashed vertical bar in each well indicates time of application of either control (0 U), 10 U, 20 U or 30 U of thrombin, repeated in three wells each (1-3 for non-induced cells, 4-6 in tetracycline-induced cells for 20 hrs). (B) The 340 nm/380 nm ratio in non-induced hTRPM7-HEK293-TREx cells was normalized to one using the data points acquired before compound application in each of the 96 wells. Data were averaged and plotted against time of the experiment. Wells were exposed to either 30 U, 20 U, or 10 U thrombin in the absence of extracellular Ca2+, or superfused with external solution without thrombin (control) (n = 3, each condition). (C) Same experimental setup as in (B), however, cells had been induced with tetracycline for 20 hrs to overexpress hTRPM7 (n = 3, each concentration). (D) The graph plots the maximal change in the 340 nm/380 nm ratio induced by thrombin exposure. Data taken from panel (B) and (C) (*p < 0.05).

Article Snippet: We thank Dr. Clay Wakano from the Queen’s High-Throughput Drug Screening Laboratory for providing access to and support for the 96/384-well kinetic plate reader assay partially supported by Hamamatsu/Queen’s PET Imaging, LLC (AF).

Techniques: Control, Concentration Assay

Journal: eLife

Article Title: Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules

doi: 10.7554/eLife.33864

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , AMAXA huvecs nucleofector kit , Lonza , Lonza:VPB-1002 , .

Techniques: Cell Culture, Recombinant, Sequencing, Amplification, Software