Fig 2: The therapeutic efficacy of EP4 antagonist-induced MSC EVs/exosomes requires CNP. A, The scheme of animal experiments, indicating the time points of damage induction, EV/exosome administration, and examination of behavior. B, The time spent by Dox-withdrawn DTA mice (UC) and Dox-withdrawn Camk2a/DTA mice treated with control MSC GWEVs, shGFP control GWEVs (shGFP), scrambled control GWEVs, and CNP-knockdown MSC GWEVs (shCNP-1, shCNP-2) on exploring the new location. The plot shows percentages of the time spent on the new location over the total time. Data are mean ± SEM (n = 5). *P = .05, **P = .005, compared with shGFP. C, The time spent by UC mice and Dox-withdrawn Camk2a/DTA mice treated with GWEVs (shGFP, shcontrol, shCNP-1, and shCNP-2) on exploring the new object. The plot shows percentages of the time spent on the new object over the total time. Data are mean ± SEM (n = 5). **P = .005, compared with shGFP. D, The time spent by UC mice and Dox-withdrawn Camk2a/DTA mice treated with GWEVs, (shGFP, shcontrol, shCNP-1, and shCNP-2) on finding the platform in the Morris water maze. Data are mean ± SEM (n = 5 mice for each group). **P = .005. CNP, cyclic nucleotide 3'-phosphodiesterase; DTA, diphtheria toxin A; EV, extracellular vesicle; GWEV, GW EP4 antagonist-induced MSC EVs/exosome; MAP2, microtubule-associated protein 2; MSC, mesenchymal stem cell

Fig 3: Elevated CNP in EP4 antagonist-induced MSC EVs contributes to their ability to convert 2',3'-cAMP to adenosine and to promote ß3-tubulin polymerization. A, Protein lysates from control (vehicle DMSO-treated) and GW-treated MSCs, and from EVs prepared from these cells, were subjected to Western blot analyses. The proteins elevated in EP4 antagonist-induced MSC EVs are labeled in red. B, CNP protein levels, normalized with exosomal GAPDH in the different batches of EVs prepared from MSCs (EV) or GW-treated MSCs (GWEV), are compared on a per-vesicle basis. Bars are means ± SEM (n = 6). ***P = .001. C, CNP protein levels in the MSCs constitutively expressing shRNAs against no target (shcontrol), GFP (shGFP), and CNP (shCNP-1, sh-CNP-2). GAPDH was used as a loading control. D, Levels of CNP and GAPDH proteins in the same number of control MSC GWEVs (shGFP and shcontrol) and shCNP knockdown MSC GWEVs (shCNP-1 and shCNP-2). E, The abilities of intact control MSC GWEVs (shGFP and shcontrol) and CNP-knockdown MSC GWEVs (shCNP-1, shCNP-2) to convert 2',3'-cAMP into adenosine. Data are means of adenosine production ± SEM (n = 3). **P = .005, ***P = .001. F,G, The effect of MSC EV, MSC GWEV, control MSC GWEV (shGFP and shcontrol), and CNP-knockdown MSC GWEVs (shCNP-1, shCNP-2) lysates on in vitro ß3 tubulin polymerization. Data are means ± SEM (n = 3). *P = .05, **P = .01, ***P = .001. CNP, cyclic nucleotide 3'-phosphodiesterase; EV, extracellular vesicle; GWEV, GW EP4 antagonist-induced MSC EVs/exosome; MSC, mesenchymal stem cell