Journal: Scientific Reports

Article Title: Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess

doi: 10.1038/s41598-020-64521-3

Figure Lengend Snippet: Derivation and characterization of cell lines lacking Atp7a , MtI and MtII genes. ( a ) Primary fibroblasts were isolated from the lungs of Atp7a fl/Y ;MtI +/+ /MtII +/+ and Atp7a fl/Y ;MtI −/− /MtII −/− mice and then immortalized by transfection with a plasmid expressing the SV40 large T antigen (SV40 Tag) resulting in WT and MT- cells, respectively. An adenoviral vector encoding CRE recombinase was used to delete Atp7a in WT and MT- cells to obtain ATP7A- and ATP7A-/MT- cells, respectively. ( b ) PCR analysis of genomic DNA was used to confirm deletion of MtI and MtII genes in both the MT- and ATP7A-/MT- cell lines. Expected PCR product sizes: MtI gene (WT = 161 bp; knockout = 176 bp); MtII gene (WT = 282 bp; knockout = 299 bp). ( c ) Immunoblot analysis was used to confirm the loss of ATP7A protein in both ATP7A- and ATP7A-/MT- cell lines. Tubulin was detected as a loading control. Images of full-length gels and immunoblots are provided in the supplementary data.

Article Snippet: Expression plasmids for human MTI and MTII genes were purchased from Origene (RC205942 and RC202748, respectively).

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Knock-Out, Western Blot

Journal: Scientific Reports

Article Title: Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess

doi: 10.1038/s41598-020-64521-3

Figure Lengend Snippet: Relative contributions of ATP7A and metallothioneins to Cu tolerance. ( a ) Cu sensitivity of WT, ATP7A-, MT- and ATP7A-/MT- cells. For each cell line, 10 3 cells/well were seeded in 6-well plates containing basal medium, or basal medium containing 1 µM BCS with or without the indicated concentrations of CuCl 2 . After 6 days, cell survival was determined using the Crystal Violet assay and imaged. ( b ) Quantification of Crystal Violet staining. Data are expressed as percent cell survival for each cell line normalized against its growth in BCS (mean ± SEM). ( c ) Complementation of ATP7A-/MT- cells with plasmids encoding human cDNAs for ATP7A ( + ATP7A), MTI ( + MTI) or MTII ( + MTII). Cells were transfected with each plasmid and then selected in basal medium supplemented with 1 µM CuCl 2 . Equal numbers of surviving cells (10 3 cells/well) were then seeded into 6-well plates containing basal medium, 1 µM BCS or 1 µM BCS plus the indicated concentrations of Cu. After 6 days, cell survival was determined using the Crystal Violet assay and imaged. ( d ) Quantification of Crystal Violet staining. Data are expressed as percent cell survival for each cell line normalized against its growth in BCS (mean ± SEM).

Article Snippet: Expression plasmids for human MTI and MTII genes were purchased from Origene (RC205942 and RC202748, respectively).

Techniques: Crystal Violet Assay, Staining, Transfection, Plasmid Preparation

Journal: Cells

Article Title: Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction

doi: 10.3390/cells10081871

Figure Lengend Snippet: Mechanism of action of triazinetriones on TrkA. ( a ) Full-length TrkA with a HA-tag (TrkA-HA) fused to the C -terminus was purified through immunoprecipitation using anti-HA agarose beads. Purified TrkA-HA was incubated with DMSO (blue circles and hatched line), ACD855 (5 µM) (black triangles and solid line), or ACD856 (1 µM) (red squares and solid line) for approx. 5 min. Thereafter, ATP was added to yield the indicated concentration. Each data point is the mean ± SEM ( n = 3). The solid lines are the curve-fit using the Michaelis–Menten equation used to calculate the apparent Vmax(app) and km(app), and the dotted lines are the 95% confidence band for each curve fit. ( b – d ) Affinity labeling and streptavidin adsorption of Trka. ( b ) Western blot of streptavidin adsorbed TrkA-HA non-covalent labeled with NHS-biotinylated triazinetrione compound (lane 2) or covalent labeled by UV-crosslinking of 100 µM sulfo-SBED biotinylated compound (lane 3). Detection of immunoreactive band was performed with anti-TrkA antibody. Supernatant loaded to the left (lane 1) was used as positive control for the Western blot. Arrows on the left indicate the migration of molecular weight markers corresponding to 198, 98, and 62 kDa. ( c ) Anti-HA agarose immunoprecipitation of cross-linked or non-crosslinked sulfo-SBED compound (AC27019-SBED) from cell lysate incubated with 100 µM AC27019-SBED. Lane 1, non-UV-crosslinking, and lane 2, UV-crosslinking of sulfo-SBED labeled TrkA-HA, both detected with streptavidin-HRP. Lanes 3 and 4 are loading controls of lanes 1 and 2, respectively, were TrkA-HA was detected by immunoblotting using the anti-TrkA antibody. Both blots were part of the same gel, but the membrane was cut in two pieces, and the proteins were detected by streptavidin-HRP (left panel) or by an anti-TrkA antibody (right panel). ( d ) Streptavidin adsorption of biotinylated compound bound to TrkA-HA. Lane 1, cell lysate used for positive control of immunodetection; lane 3, cell lysate without biotinylated compound was adsorbed to streptavidin-agarose as negative control; lanes 5 and 7, cell lysates containing sulfo-SBED compound UV-crosslinked to TrkA (from two different experiments) were adsorbed to streptavidin-agarose and immunoblotted using anti-TrkA antibody. Lanes 2, 4, and 6 are empty lanes to avoid cross-contamination between lanes.

Article Snippet: Recombinant intracellular domain (ICD) of TrkA (08-186) and single site biotinylated activated (08-486-20N) or non-activated TrkA (08-486-23N) were obtained from Carna Biosciences, Odense, Denmark.

Techniques: Purification, Immunoprecipitation, Incubation, Concentration Assay, Labeling, Adsorption, Western Blot, Positive Control, Migration, Molecular Weight, Membrane, Immunodetection, Negative Control

Journal: Frontiers in Aging Neuroscience

Article Title: Surgery, Anesthesia and Intensive Care Environment Induce Delirium-Like Behaviors and Impairment of Synaptic Function-Related Gene Expression in Aged Mice

doi: 10.3389/fnagi.2020.542421

Figure Lengend Snippet: Effects of Anesthesia/Surgery/ICU on expression of synuclein alpha, neurotrophic receptor tyrosine kinase 1 and syntaxin 1a genes. (A) Snca α, Ntrk1, and STX1a mRNA levels were significantly impaired in A/S/I mice relative to controls following ICU environment (–3.785, –1.498, and –2.268 fold decrease, respectively). Hippocampal total RNA was extracted from A/S/I animals (pooled N = 3) and controls (pooled N = 3). (B–E) Protein levels of STX1a ( B: P < 0.012), Ntrk1 ( C: P = 0.039), and Snca α ( D: P = 0.017) were significantly decreased in A/S/I mice relative to controls. Snca phosphorylated at serine residue 129 was also significantly down-regulated in insult-exposed mice ( E: P = 0.008). Two-tailed unpaired t -test ( N = 7 A/S/I and N = 7 control mice). STX1a, syntaxin 1a; NtrK1, neurotrophic tropomyosin receptor kinase 1; Snca α, synuclein alpha; p-Snca α, synuclein alpha phosphorylated at serine residue 129. ∗ P < 0.05 and ∗∗ P < 0.01, respectively.

Article Snippet: Membranes were incubated for 1 h in SuperBlock (TBS) Blocking Buffer (Thermo Scientific, United States) at room temperature, followed by overnight incubation at 4°C with primary antibodies (Cell-signaling): Snca α (D37A6) Rabbit mAb # 4179 (1:3000); phospho-Snca α (Ser129, D1R1R) Rabbit mAb # 23706 (1:3000); STX1a (D4E2W) Rabbit mAb # 18572 (1:3000); Ntrk1 Antibody # 2505 (1:1000); SYP Antibody Rabbit pAb #4329 (1:1000); PSD-95 Rabbit mAb #3450 (1:1000); MAP2 Rabbit pAb #4542 (1:1000) and beta-tubulin Mouse mAb # 66240 (Proteintech, United States).

Techniques: Expressing, Residue, Two Tailed Test, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Surgery, Anesthesia and Intensive Care Environment Induce Delirium-Like Behaviors and Impairment of Synaptic Function-Related Gene Expression in Aged Mice

doi: 10.3389/fnagi.2020.542421

Figure Lengend Snippet: Effects of Anesthesia/Surgery/ICU on synaptophysin, microtubule-associated protein 2 and postsynaptic density protein 95 levels. (A–C) Protein levels of Syp ( A: P = 0.002), MAP2 ( B: P = 0.013), and PSD-95 ( C: P = 0.003) were significantly decreased in A/S/I mice relative to controls. Two-tailed unpaired t -test ( N = 5 A/S/I and N = 5 control mice). Syp, synaptophysin; MAP2, microtubule-associated protein 2; PSD-95, postsynaptic density protein 95. (D) Representative 20× magnification images of the distribution of PSD-95 immunostaining in sagittal CA1 and CA3 hippocampal sections from two control and two A/S/I mice. Arrows highlight differences in PSD-95 immunofluorescent staining. (E) Plot of STX1a, Ntrk1, Snca α, Syp, MAP2, and PSD-95 protein levels against individual mouse Z scores. Z scores from behavioral assessments obtained at the 24 h time point were used, since tissue collection for protein studies was performed immediately after this behavioral assessment. The vertical dotted line represents a protein level of 85% (expressed as change from control). ∗ P < 0.05 and ∗∗ P < 0.01, respectively.

Article Snippet: Membranes were incubated for 1 h in SuperBlock (TBS) Blocking Buffer (Thermo Scientific, United States) at room temperature, followed by overnight incubation at 4°C with primary antibodies (Cell-signaling): Snca α (D37A6) Rabbit mAb # 4179 (1:3000); phospho-Snca α (Ser129, D1R1R) Rabbit mAb # 23706 (1:3000); STX1a (D4E2W) Rabbit mAb # 18572 (1:3000); Ntrk1 Antibody # 2505 (1:1000); SYP Antibody Rabbit pAb #4329 (1:1000); PSD-95 Rabbit mAb #3450 (1:1000); MAP2 Rabbit pAb #4542 (1:1000) and beta-tubulin Mouse mAb # 66240 (Proteintech, United States).

Techniques: Two Tailed Test, Control, Immunostaining, Staining