Journal: Aging Cell

Article Title: Piezo1 exacerbates inflammation‐induced cartilaginous endplate degeneration by activating mitochondrial fission via the Ca 2+ / CaMKII /Drp1 axis

doi: 10.1111/acel.14440

Figure Lengend Snippet: Piezo1 modulated mitochondrial morphology and function by influencing CaMKII activity. (a, b) western blot analysis showed that the level of CaMKII phosphorylation was regulated by Yoda1 and BAPTA‐AM. (c, d) TUNEL staining showed that KN‐93 partially reversed Yoda1‐induced cartilaginous endplate (CEP) cell apoptosis. Scale bar, 100 μm. (e, f) Flow cytometry with Annexin V‐FITC/PI verified the protective effects of KN‐93 on CEP cell apoptosis. (g) Double immunofluorescence staining indicated the colocalization of p‐Drp1 and MitoTracker Red. Scale bar, 25 μm. (h, k) Flow cytometry using JC‐1 to examine the MMP in CEP cells. (i, j, l, m) MitoSOX Red and DCFH‐DA staining were used to detect the production of mitochondrial and cellular reactive oxygen species (ROS) in CEP cells. Scale bars, 50 μm (I) and 100 μm (j). ( n = 3 biological replicates, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: To further study the downstream signal transduction of Piezo1, CEP cells were treated with 5 μM Yoda1 plus 10 μM BAPTA‐AM (MedChemExpress, USA), 10 μM KN‐93 (MedChemExpress, USA) or 10 μM Mdivi‐1 (Selleck Chemicals, USA) for 12 h.

Techniques: Activity Assay, Western Blot, Phospho-proteomics, TUNEL Assay, Staining, Flow Cytometry, Double Immunofluorescence Staining

Journal: Aging Cell

Article Title: Piezo1 exacerbates inflammation‐induced cartilaginous endplate degeneration by activating mitochondrial fission via the Ca 2+ / CaMKII /Drp1 axis

doi: 10.1111/acel.14440

Figure Lengend Snippet: Piezo1 induced mitochondrial fission and dysfunction via the Ca 2+ /CaMKII/Drp1 axis in LPS‐treated cartilaginous endplate (CEP) cells. (a–e) The effects of KN‐93 on the phosphorylation and mitochondrial translocation of Drp1 were assessed using western blotting. (f, g, l, m) TUNEL staining and flow cytometry with Annexin V‐FITC/PI demonstrated that Mdivi‐1 partially rescued Yoda1‐induced CEP cell apoptosis. Scale bar, 100 μm. (h) The colocalization between p‐Drp1 and MitoTracker Red was verified by double immunofluorescence staining. Scale bar, 25 μm. (i, n) The MMP in CEP cells was evaluated by flow cytometry using JC‐1. (j, k, o, p) The production of mitochondrial and cellular reactive oxygen species (ROS) in CEP cells was measured by MitoSOX Red and DCFH‐DA staining, respectively. Scale bars, 50 μm (j) and 100 μm (k). ( n = 3 biological replicates, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: To further study the downstream signal transduction of Piezo1, CEP cells were treated with 5 μM Yoda1 plus 10 μM BAPTA‐AM (MedChemExpress, USA), 10 μM KN‐93 (MedChemExpress, USA) or 10 μM Mdivi‐1 (Selleck Chemicals, USA) for 12 h.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, TUNEL Assay, Staining, Flow Cytometry, Double Immunofluorescence Staining

Journal: bioRxiv

Article Title: Pharmacological modulation of AMPA receptor surface diffusion restores hippocampal synaptic plasticity and memory in Huntington’s disease

doi: 10.1101/297069

Figure Lengend Snippet: Impaired BDNF-TrkB-CaMKII signaling through the interaction between stargazin and PSD95 contributes to the deregulation of AMPAR surface diffusion in HD models ( a ) Schematic diagram showing that BDNF can be modulated at synthesis, transport and secretion level. ( b ) Hippocampal BDNF protein level determined by ELISA in R6/1 and Hdh Q111/Q111 mice; values are mean ± s.e.m (% of WT); n = 21 and 14 mice for WT and R6/1; n = 6 and 9 mice for WT and Hdh Q111/Q111 , respectively. ( c ) Representative kymographs of intracellular transport of BDNF-containing vesicles (white trajectories) in a neurite (50 μm from soma) over 75 seconds (s) in wHTT- and polyQ-HTT-expressing rat hippocampal neurons. The velocity of BDNF transport was reflected by the slope of trajectories (moving distance against time). ( d, e, f ) Anterograde and retrograde BDNF transport velocity in all neurites of wHTT- and polyQ-HTT-expressing rat hippocampal neurons ( d ), and hippocampal neurons from R6/1 mouse line ( e ), and in the axon of hippocampal neurons from Hdh Q111/Q111 mouse line ( f ); values are mean ± s.e.m; n = 5569, 5656, 5227 and 5706 trajectories for anterograde and retrograde wHTT and polyQ-HTT, respectively; n = 1424, 1710, 1376, and 1487 trajectories for anterograde and retrograde WT and R6/1, respectively; n = 236, 261, 194 and 256 trajectories for anterograde and retrograde WT and Hdh Q111/Q111 , respectively. ( g, h, i ) GluA2-AMPAR diffusion coefficients in rat hippocampal neurons co-expressing FL-wHTT/polyQ-HTT and GFP, or FL-polyQ-HTT and CamKII-GFP; n = 656, 685, and 349 trajectories, respectively ( g ), in neurons co-expressing FL-polyQ-HTT and GFP and treated with Vehicle, BDNF, TrkB-Fc plus BDNF, or kn93 plus BDNF; n = 1649, 1742, 480, and 1380 trajectories, respectively ( h ), and in vehicle- or BDNF-treated neurons co-expressing FL-polyQ-HTT and GFP or GFP fused wild-type stargazin (Wt-stg-GFP), or ΔC stg, in which the binding domain to PSD95 was deleted; n = 495, 568, 376, 300, 573 and 498 trajectories, respectively ( i ). Diffusion coefficients were shown as median ± 25-75% IQR; significance was determined by unpaired two-tailed Student’s t -test ( b, d, e, f ), and Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test ( g, h, i ); * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Tianeptine was purchased from T & W group and MedChemexpress CO.,Ltd; BDNF from Sigma-Aldrich; TrkB-Fc from R&D Systems; kn93 from Tocris.

Techniques: Diffusion-based Assay, Enzyme-linked Immunosorbent Assay, Expressing, Binding Assay, Two Tailed Test, Comparison

Journal: Bone

Article Title: Regulation of Mesenchymal Stem Cell Differentiation on Microstructured Titanium Surfaces by Semaphorin 3A

doi: 10.1016/j.bone.2020.115260

Figure Lengend Snippet: Downstream activation of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase (CaMKII), and phospholipase A2 (PLA2) were inhibited by treating cultures with 10μM W7 (A – E), 10μM KN93 (F – J), or 10μM AACOCF3 (K – O) respectively with or without the addition of 1μg/mL Sema3A. Protein kinase C (PKC) was inhibited using either 1μM chelerythrine chloride (P – T) or 1μM GF109203X (U – Y) with or without the addition of 1μg/mL Sema3A for 7d. Cells were then treated with fresh media for 24h. After 24h, media were collected, and cell lysates were assayed for DNA content. Media were assayed for osteocalcin, BMP2, osteoprotegerin, and Semaphorin3A. Data shown are the mean ± standard error (SE) of six independent samples. Groups not sharing a letter are statistically significant at α=0.05.

Article Snippet: Downstream activation of calmodulin (CaM), Ca 2+ /calmodulin-dependent protein kinase (CaMKII), and phospholipase A 2 (PLA2) were inhibited by treating cultures with 10μM W7 (Tocris Bioscience, Bristol, United Kingdom), 10μM KN93 (Tocris Bioscience), or 10μM AACOCF 3 (Tocris Bioscience) respectively with or without the addition of 1μg/mL Sema3A.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Gut microbiota confers host resistance to obesity by metabolizing dietary polyunsaturated fatty acids

doi: 10.1038/s41467-019-11978-0

Figure Lengend Snippet: HYA contributes to host metabolic condition via GPR40 and GPR120. Mobilization of [Ca 2+ ]i induced by LA-derived gut microbial metabolites was monitored in Flp in a hGPR40 or b hGPR120 T-REx HEK293 cells. Data are presented as Ca 2+ intensity. Cells were cultured for 24 h and then treated with or without 10 μg/mL doxycycline ( n = 8 independent cultures with doxycycline from three biological replicates; n = 6 independent cultures without doxycycline from two biological replicates). Closed symbols represent values from cells treated with doxycycline, and open symbols denote untreated groups. c – f The inhibitory effects of c Gpr40 and Gpr120 siRNA, d MEK inhibitor (U0126), e PLC inhibitor (U73122), and f CaMKII inhibitor (KN-62) on GLP-1 secretion following LA, HYA, or HYB treatment ( n = 4 independent cultures from two biological replicates). ** P < 0.01 vs. None (Tukey–Kramer test). ## P < 0.01; # P < 0.05 vs. LA (Tukey–Kramer test). $$ P < 0.01 vs. HYA (Tukey–Kramer test). (−) represents untreated cells with siRNA or antagonist. Results are presented as means ± SE. g GLP-1 concentration and h OGTT in wild-type (left, n = 10 animals per group), Gpr40 -deficient (middle, n = 8 animals per group), and Gpr120 -deficient (right, n = 9, 10, and 9 animals per group) mice were analyzed 2 h after FA administration. ** P < 0.01 vs. Control (Tukey–Kramer test). # P < 0.05 vs. LA (Tukey–Kramer test). (−) represents the mice without FA administrations. Results are presented as means ± SE. Source data are provided as a Source Data file 6

Article Snippet: For inhibitor treatment, STC-1 cells were pretreated with the MEK inhibitor U0126 (10 μM; Wako), the PLC inhibitor U73122 (1 μM; Wako), or the CaMKII inhibitor KN-62 (10 μM, Wako) for 30 min prior to the addition of FAs.

Techniques: Derivative Assay, Cell Culture, Concentration Assay