Figures
Figure 1. Responses of MRGPRX2-Expressing HEK293 Cells to
Elamipretide
A. Representative Fluo-4 fluorescence traces from HEK293 cells stably
expressed with MRGPRX2 or its mouse ortholog Mrgprb2. Arrows indicate
when elamipretide was added. Each trace is from a different cell in the
same imaging field. A rise in fluorescence indicates receptor
activation. B. Responses of MRGPRX2-expressing HEK293 cells to
elamipretide (labeled as “SBT”) at the indicated concentrations.
Figure 2: Elamipretide, but not M1 and M2 metabolites, elicits
intracellular calcium mobilization in a dose- and MRGPRX2-dependent
manner
Panel A: Elamipretide induced calcium mobilization in MRGPRX2
cells. Panel B: Elamipretide does not elicit calcium
mobilization in the parental cell line, Chem-1. Panel C:Quercetin inhibited elamipretide activation of MRGPRX2. Panels D
and E: Neither M1 (D) or M2 metabolites (E) elicited
calcium mobilization in MRGPRX2 cells.
Figure 3. Mast cells are activated by elamipretide in an
Mrgprb2-dependent manner
A. Representative images of Fluo-4 fluorescence changes in response to
elamipretide (labeled “SBT”). Fluorescence signals were converted to a
heat map for ease of visualization. B. Average responses of wild type or
Mrgprb2 knockout primary mouse peritoneal mast cells to 500
micrograms/ml elamipretide. (n=3 mice for each genotype, >
200 mast cells counted for each genotype, p < 0.01, t-Test,
Two-Sample Assuming Unequal Variances).
Figure 4: In vivo inflammation in mice is induced by
elamipretide in an Mrgprb2-dependent manner
Left panel: Evans Blue extravasation in wild type (WT) and Mrgprb2
knockout (KO) mice. Left paws injected with 0.5 mg/mL elamipretide/right
paws injected with a saline control. Right, quantification of Evans Blue
accumulation, measured as specific optical density (OD) per gram of
tissue, in the footpads. Responses were calculated as the OD in
elamipretide-injected paws, divided by the OD in saline-injected paws.
(n=7 for wild type and n=8 for knockout mice. P < 0.01,
t-Test, Two-Sample Assuming Unequal Variances) Elamipretide induced
strong Evans Blue labeling and saline induced much less (left),
dependent on Mrgprb2, as elamipretide induced almost no Evans Blue
accumulation in Mrgprb2 knockout mice (left). Measurements of Evans Blue
accumulation in footpads revealed that accumulation was
~ 80% less in Mrgprb2 knockout mice, demonstrating that
at this concentration, most of the inflammation was dependent on
Mrgprb2.