Journal: Brain communications

Article Title: Spatial selectivity of ATase inhibition in mouse models of Charcot-Marie-Tooth disease.

doi: 10.1093/braincomms/fcae232

Figure Lengend Snippet: Figure 4 Phenotypic assessment of C3-PMP22 mice. (A) Phenotypic severity score represented as a sum of ledge, gait and hindlimb clasping at different ages (n = 7/group). **P < 0.005 and

Article Snippet: Cells were transiently transfected with Lipofectamine 2000 (Invitrogen #11668019) using the following constructs: mCherry-ER-3 plasmid, a gift from Michael Davidson (Addgene plasmid #55041); PMP22 human-tagged ORF clone (OriGene #RC216500); and MPZ human-tagged ORF clone (OriGene #RC202450).

Techniques:

Journal: Journal of Pharmaceutical Analysis

Article Title: New applications of clioquinol in the treatment of inflammation disease by directly targeting arginine 335 of NLRP3

doi: 10.1016/j.jpha.2024.101069

Figure Lengend Snippet: Clioquinol (CQ) affects the interaction between NOD-like receptor protein 3 (NLRP3) and flag-(never in mitosis gene a)-related kinase (NEK)/apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC). Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs)/human monocyte leukemia cells (THP-1) cells were incubated with different doses of CQ for 1 h and then stimulated with nigericin for 1.5 h. (A, C) Immunofluorescence assay of ASC speck. BMDMs were stained with ASC antibody (green) and 4′,6-Diamidino-2-phenylindole (DAPI) (Blue). ASC speck formation was observed by immunofluorescence. BMDMs (A), and THP-1 cells (C). (B, D) The percentage of ASC specks was measured by ImageJ (relative to LPS + nigericin). BMDMs (B), and THP-1 cells (D). (E) Western blot analysis of ASC oligomerization level after cross-linking with disuccinimidyl suberate in NP-40-insoluble pellets from BMDMs. (F) NLRP3 self-oligomerization level was measured by native-polyacrylamide gel electrophoresis (PAGE). (G, H) Immunoprecipitation analysis of the interaction between NLRP3 and NEK7 in BMDMs. IP:NLRP3 (G), and IP:NEK7 (H). (I, J) Transfected HEK-293T with plasmid overnight and treated with CQ for 8 h, and then CO-immunoprecipitation (IP) with flag antibody and Western blot was used to evaluate the interaction between green fluorescent protein (GFP)-NLRP3 and FLAG-NEK7 (I) or FLAG-ASC (J). Data are representative of n = 5 (B, D), and are expressed as mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, ns: not significant, determined by Mann–Whitney test (relative to LPS + nigericin). Glyceraldehyde phosphate dehydrogenase (GAPDH) served as a loading control in (E–G).

Article Snippet: Then, 500 μL of disuccinimidyl suberate (S0657, Selleck) solution (2 mM) were added to the pellets and cross-linked for 60 min at room temperature on a shaker.

Techniques: Activation Assay, Derivative Assay, Incubation, Immunofluorescence, Staining, Western Blot, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection, Plasmid Preparation, Standard Deviation, MANN-WHITNEY, Control

Journal: PLoS ONE

Article Title: Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons: Implications for Parkinson’s disease

doi: 10.1371/journal.pone.0261730

Figure Lengend Snippet: Panels (a) and (b) respectively show the effects of the WNT modulators and NEUROD1 overexpression upon NR0B1 . Panels (c) and (d) show effects upon NR0B2 expression. Results are expressed as mean ± SEM of three (biological) replicate experiments. Analysis of panel (a) was via one-way ANOVA with post-hoc Dunnett’s test. For panels (b) and (d) analysis was via Student’s t-tests (at each time point as in ). For panel (c) analysis was via a non-parametric Kruskal-Wallis test with post-hoc Dunn’s test to accommodate undetectable levels of NR0B2 in two out of three samples, both undetectable values were assigned to 0.0028 (equal to the lowest value detected).

Article Snippet: At this time, cultures were transfected with NEUROD1 (SC118625, Origene), NR0B1 (SC319624, Origene), NR0B2 (SC122920, Origene) or blank plasmids (PCMV6XL5, Origene) according to manufacturer’s instructions.

Techniques: Over Expression, Expressing

Journal: PLoS ONE

Article Title: Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons: Implications for Parkinson’s disease

doi: 10.1371/journal.pone.0261730

Figure Lengend Snippet: Expression patterns following transfection with NR0B1 (green), NR0B2 (red) or blank (blue) plasmids show that NR0B1 transfection increases both NR0B1 and NR0B2 and impacts expression of NEUROD1 , TH , KCNJ6 , LMX1A and TUBB3 . Control wells underwent transfection with an empty plasmid. Results are expressed as mean ± SEM of three (biological) replicate experiments. Analysis was performed with One-way ANOVA followed by post-hoc Dunnett’s test where each post transfection timepoint represented a repeated measure (t = 0 was not included in analyses). * and ** indicate P<0.05 and P<0.01, respectively. shows increased NR0B1 and NR0B2 immunolabelling up to six days after transfection.

Article Snippet: At this time, cultures were transfected with NEUROD1 (SC118625, Origene), NR0B1 (SC319624, Origene), NR0B2 (SC122920, Origene) or blank plasmids (PCMV6XL5, Origene) according to manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Plasmid Preparation