Journal: Cell Death & Disease

Article Title: Targeting the CALCB/RAMP1 axis inhibits growth of Ewing sarcoma

doi: 10.1038/s41419-019-1372-0

Figure Lengend Snippet: a Analysis of cell viability (normalized Resazurin signal to DMSO control) in A673 Ewing sarcoma (EwS) cells treated for 72 h with the indicated concentrations of MK-3207. The graph shows the dose-dependent relative Resazurin signal. Data are represented as mean and SEM ( n = 12); unpaired two-tailed Student’s t test. b Comparison of relative Resazurin signal of A673 cells carrying a dox-inducible shRNA against RAMP1 treated with 150 µM of MK-3207 with/without knockdown of RAMP1 by additional addition of 1 µg/ml dox to the growth medium. Data are represented as mean and SEM ( n = 3); unpaired two-tailed Student’s t test. c Analysis of colony-forming capacity of A673 (left panel) and RDES (right panel) EwS cells under treatment with the small molecule CGRP receptor inhibitors MK-3207 (20 µM) or BIBN-4096 (Olcegepant; 100 µM). DMSO served as control for treatment. Representative images of the colonies are shown below. Data are represented as mean and SEM ( n = 3); unpaired two-tailed Student’s t test. d Analysis of sphere-formation capacity of A673 (left panel) and RDES (right panel) EwS cells under treatment with the small molecule CGRP receptor inhibitors MK-3207 (20 µM) or BIBN-4096 (Olcegepant; 100 µM). DMSO served as control for treatment. Sphere index was calculated by addition of diameters of all existing spheres in one well divided by diameter of spheres in the control well. Data are represented as mean and SEM ( n = 3); unpaired two-tailed Student’s t test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Article Snippet: Forty-eight hours after seeding, inhibitors were added at final concentrations of 100 µM for BIBN-4096 (Olcegepant; R&D systems, Minneapolis, MN, USA) and 20 µM for MK-3207 (AdooQ Bioscience).

Techniques: Two Tailed Test, shRNA

Journal: Nature Communications

Article Title: Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice

doi: 10.1038/s41467-022-28204-z

Figure Lengend Snippet: a pH-responsive DIPMA-MK-3207. b Transmission electron micrograph image of DIPMA-MK-3207 (Scale: 0.1 µm), from two different nanoparticle preparations (3 images captured per sample). c Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. d Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40–60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n = 5 independent experiments (Scale: 10 µm). e – g Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK-rCLR/RAMP1 cells. e Time course and f , g integrated response (AUC) before (1st phase) and after (2nd phase) washing to remove extracellular CGRP ( n = 6 independent experiments). h Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca 2+ response to CGRP in HSCs (DIPMA-MK-3207: −9M, n = 145; −8M, n = 361; −7M, n = 213: −6.5 M, n = 150; or free MK-3207: −8M, n = 83; −6M, n = 106; −5M, n = 87; −4M, n = 127: −3M, n = 127 cells). i PMA, expressed as AUC, after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6 J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle ( n = 8 mice per group). Mean±SEM. *** P < 0.001 vs. DIPMA-Ø/Veh, ### P < 0.001 vs. MK-3207 0.3 pmol and MK-3207 1 pmol. f , g , i 1-way ANOVA, Bonferroni correction. Source data are provided as a Source Data file.

Article Snippet: Assemblies of DIPMA-MK-3207 and DIPMA-Ø were dialyzed against PBS for 24 h (MWCO 3500, Membrane Filtration Products).

Techniques: Transmission Assay, Expressing, Incubation, Concentration Assay, Inhibition, Injection