Fig 1: Detection of AChE by Western blot in pellets from different Jurkat cell-conditioned medium (CCM) and non-conditioned medium (NCM). Left panels = representative WB, right panels, quantification of the AChE signals in 3 to 6 independent Western blots. AU = AChE band intensity in a given pellet/sum (AChE band intensity in all 6 pellets). (a) RPMI with 10% FBS. (b) Serum-free TexMACSTM medium. (c) RPMI-10% FBS depleted from serum EVs by overnight ultracentrifugation. (d) RPMI-10% FBS extra-depleted of serum EVs by overnight ultracentrifugation leaving out 5ml above pellet. 2 K, 10 K100 K = pellets recovered from 20ml of CCM or NCM. AChE is clearly detected in non-conditioned serum-containing medium (a), and is only partially depleted by EV depletion (c). (e-g) Representative images of immunogold labelling for AChE of 2 K, 10 K and 100 K pellets recovered from conditioned medium (RPMI-10% extra-depleted of serum EVs). Close-ups of the areas inside white squares are shown. (h) CD63/AChE double stain of 100 k pellets recovered from Jurkat conditioned medium (extra-depleted from FBS).
Fig 2: Effect of HLJG0701-β on the contents AChE (A) and ACh (B) in mice brain tissue. Data are presented as mean ± standard deviation. Error bar: standard deviation. ##, significant difference compared with control by Student’s t-test, p < 0.01; $, significant difference compared with OVX + d-galactose group by Student’s t-test, p < 0.05; *, significant difference compared with OVX + d-galactose group by one-way ANOVA, p < 0.05.
Fig 3: Most AChE activity in serum-containing medium is not pelletable. (a) AChE activity was not detected in serum-free media formulations (AIM Vand RPMI-1640 NCM), but was found at high levels in serum-containing medium (R10 = RPMI 10% FBS NCM). Differential ultracentrifugation of AIM Vand RPMI recovered no activity in differentially centrifuged pellets (2 K, 10 K, 100 K). From serum-containing medium, only a very small fraction of total AChE activity could be recovered by differential centrifugation, and values in complete NCM. (b) After conditioning medium with 72 h culture of PM1 lymphocytic cells, similar amounts of AChE activity as in (a) could be pelleted from serum-containing medium. Comparatively smaller amounts were recovered when cells were switched to AIM V medium.
Fig 4: Biomolecular analysis of memorial dysfunction and learning enhancement. AChE activities analysis (a), BDNF contents (b), gene expression analysis of mAchR1, CBP, CREB1, and BDNF (c). Analysis of IHC against Ki67 (d) and its immunoreaction density (e). Data are expressed as the mean ± SD (n = 8 to 10 for AChE activities analysis and BDNF concentration; n = 4 to 5 for gene expression analysis; n = 2 for ICH analysis, resp.). p# < 0.05, p## < 0.01, and p### < 0.001 versus normal group and p∗ < 0.05 and p∗∗ < 0.01 versus scopolamine only injection group. AChE: acetylcholinesterase, mAchR1: muscarinic acetylcholine receptor 1, BDNF: brain-derived neurotrophic factor, CREBP: cAMP response element-binding protein, and CBP: CREBP binding protein.
Fig 5: Simplified model of cholinergic synaptic dysfunction in DYT1 dystonia. (Left) Cartoon showing normal signaling between ChIs (cholinergic interneurons) and MSNs, leading to the expression of normal LTD (long‐term depression). In DYT1 dystonia (right), synaptic vesicles contain more VAChT (vesicular acetylcholine transporter), which allows to store significant amounts of ACh. During LTD induction, activation of an abnormal D2R (dopamine 2 receptor) increases the release of these vesicles fully loaded with ACh into the synaptic cleft. Despite the increase in AChE (acetylcholinesterase) activity, which helps degrading ACh, the excessive cholinergic tone disrupts the expression of synaptic plasticity in MSNs. [Color figure can be viewed at wileyonlinelibrary.com]
Supplier Page from Abcam for Acetylcholinesterase Assay Kit (Colorimetric)