Fig 2: Effects of acupuncture on the expression levels of hippocampal GluR1 receptor. These results show the changes in hippocampal GluR1 expression levels (CA1, CA2, CA3, and DG) after administration of acupuncture (AP1, AP2, or CP) or amitriptyline (10 mg/kg, i.p.) for 28 consecutive days (A–F). Free-floating coronal hippocampus sections from the three groups (Sham, PSNL, and AP1) were subjected to immunofluorescence with GluR1 (green) and NeuN (red) antibodies to label GluR1-positive NeuN in neurons (A). Representative graphs showing the expression levels of GluR1 in the hippocampus (A,B) and a representative figure showing the hippocampal regions in the mouse brain (C). n = 3–4/group. Scale bar: 100 µm. *p < 0.05, **p < 0.01 compared to the Sham group in each area. #p < 0.05, ##p < 0.01 compared to the PSNL group in each area. The expression levels of hippocampal GluR1 protein were examined (D). n = 5/group. **p < 0.01 compared to the Sham group. #p < 0.05 compared to the PSNL group. All data were analyzed with a one-way ANOVA followed by Newman–Keuls post hoc tests. All data are expressed as the mean ± SEM. The cognitive function values in the NOR test and nociceptive values in the von Frey test were correlated with the GluR1 protein levels in the hippocampus (E,F). The r-values were analyzed with the Spearman rank correlation coefficient. AP1, acupuncture 1 (GB30 and GB34); AP2, acupuncture 2 (HT7 and GV20); CP, control point.

Fig 3: The clathrin adaptor CALM is enriched at postsynaptic endocytic zones where AMPAR endocytosis occurs.(A) During LTD induction, CP-AMPARs are recruited into synaptic regions, whereas CI-AMPARs are internalized via CME by binding to AP-2. Within a few minutes of LTD progression, the increased Ca2+ concentration activates phosphatases inducing GluA1 dephosphorylation and subsequent CP-AMPAR removal by a still unknown endocytic mechanism. (B and C) Tryptic digest of synaptosomes reveals postsynaptic localization of CALM. (B) Synaptosomes were left untreated or incubated with trypsin and analyzed by immunoblotting. (C) For quantification, trypsin-treated samples were normalized to untreated controls. (D and E) Three-channel time-gated STED confirms predominant postsynaptic localization of CALM. (D) Representative image of wild-type (WT) hippocampal neurons immunolabeled with antibodies against the presynaptic marker Bassoon, the postsynaptic marker Homer 1, and CALM (scale bar, 500 nm). Yellow line indicates position of the normalized fluorescent intensity depicted in (E). (F) The majority of CALM resides outside of the PSD. WT mouse brain homogenates were subjected to subcellular fractionation, and equal protein amounts of total homogenate (T), cytosolic fraction (S2), synaptosomes (P2), synaptosomal membranes (P3), synaptic plasma membranes (SPMs), and PSD were compared by immunoblotting with the indicated markers. Statistics Table 1. ns, not significant.

Fig 4: FOXC1 enhances c-MYC expression to promote gastric cancer cell proliferation through activation of the Wnt signaling pathway. (A) TopFlash/FopFlash assays using normal gastric cells (GES-1) and gastric cancer cells (AGS, SGC-7901, MGC-803, MKN-45, HGC-27). (B) qPCR results in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells indicated that mRNA levels of ß-catenin remained unchanged. (C) The results of western blot assays in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells indicated that protein levels of ß-catenin remained unchanged (ß-catenin Antibody: CST #8480S). (D) FOXC1 KO increased ß-catenin levels in the cytoplasm but decreased them in the nucleus (ß-catenin Antibody: CST #8480S). (E) Immunofluorescence assay in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells showed that the level of ß-catenin translocation into nucleus changed with knockout and overexpression FOXC1 (ß-catenin Antibody: CST #8480S). (F) FOXC1 overexpression inhibit the phospho-ß-Catenin (Ser33/37/Thr41) in AGS and MKN-45 cells (phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #9561T). (G,H) co-IP assays and immunofluorescence assay indicated that FOXC1 binds to the ß-catenin protein in the cytoplasm (ß-catenin Antibody: CST #8084S; FOXC1 Antibody: Abcam #ab227977). (I) Western blot assays and immunoprecipitation assay were applied to confirmed the distribution of FOXC1 in the nucleus. (J) co-IP assay was applied to confirmed the distribution of phospho-ß-Catenin (Ser33/37/Thr41) in the nucleus (phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #9561T). (K) co-IP assay was applied to confirmed whether FOXC1 bound to the ß-catenin protein in the cytoplasm and nucleus. Immunoprecipitation assay results showed that FOXC1 bound to non-phospho-ß-Catenin in the cytoplasm (ß-catenin Antibody: CST #8084S; phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #9561T; Non-phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #8814T), and FOXC1 did not bound to ß-Catenin in the nucleus (ß-catenin Antibody: CST #8084S). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Overexpressing hTau in primary cultured neurons decreases synaptic proteins with reduced phosphorylation of CREB/TrkB and GluA1. (a, b) The hippocampal neurons (5 div) were infected with lenti-syn-hTau-mCherry or the empty vector and cultured for another 7 days, and the expression of hTau (110 kDa) was confirmed by Western blotting using Tau5 (reacts with total tau). (c, d) Infection of different MOI lenti-hTau virus induced a dose-dependently increased expression of tau in cultured hippocampal neurons measured by Western blotting using Tau5 (n = 6, **p < .01 vs. MOI 1). (e) Infection of 10 MOI lenti-hTau virus significantly decreased CREB phosphorylation (n = 6, **p < .01 vs. MOI 1). (f–h) Overexpressing hTau induces reduction of multiple synaptic proteins. The hippocampal neurons (5 div) were infected with lenti-syn-hTau-mCherry or the empty vector and cultured for another 7 days, and the levels of presynaptic (g) and postsynaptic (h) proteins were measured by Western blotting. The experiments were repeated three times from nine different batches of primary cultured neurons. (i) Overexpressing hTau decreases mRNA level of SYT with no significant effect on SYN1 and SYP measured by qPCR. (j–l) Overexpressing hTau decreases dendrites branches and total dendrite length. The cultured hippocampal neurons (18 div) were stained with MAP2, and then, the dendrite arborizations and total length were analyzed by Sholl (scale bar: 50 µm). The total dendrite length and the branches at 20 to 140 µm from soma were measured (n = 30 per group from six independent cultures, black line, p < .05, vs. Vec). (m, n) Overexpressing hTau decreases CREB/TrkB/GluA1 phosphorylation and reduces BDNF measured by Western blotting. The phosphorylation levels of CREB, TrkB, and GluA1 were normalized to the total protein levels or to ß-actin. The experiments were repeated three times from nine different batches of primary neurons. Data were expressed as mean or mean ± SEM, *p < .05, **p < .01 versus Vec, but **p < .01 versus MOI 1 in (d) and (e)