Journal: Neurobiology of Disease

Article Title: Neuroprotective activity of ursodeoxycholic acid in CHMP2B Intron5 models of frontotemporal dementia

doi: 10.1016/j.nbd.2020.105047

Figure Lengend Snippet: UDCA Identifies GCLC As a Novel Regulator of CHMP2B Intron5 Toxicity. A. The mRNA abundance of gclc from pan-neuronally expressing CHMP2B Intron5 third instar larval brains shows a significant upregulation when raised on UDCA (600 μM) supplemented food compared to vehicle (ethanol). Mean ± SEM, n = 5 per genotype, fold change values in log 2 scale. ⁎⁎⁎ q < 0.001. B. Representative images of the Drosophila eye phenotype caused by CHMP2B Intron5 expression with eye-specific GMR -Gal4 driver ( GMR -Gal4, UAS-CHMP2B Intron5 ) and amelioration by co-expression of UAS- GCLC . C. Quantification of the eye phenotype from (B) genotypes. n = 100. D, E. Co-expression of GCLC (UAS- GCLC 6 ) ameliorates unregulated synaptic growth characterised by increased bouton number (D) and NMJ length (E) at the third instar larval NMJ (Muscle 6/7, hemisegment A3) in CHMP2B Intron5 ( nSyb -Gal4) expressing larvae. ANOVA with post-hoc Tukey comparison between groups ⁎ p < .05, ⁎⁎ p < .01. F. Pan-neuronal ( nSyb -Gal4) expression of GCLC rescues aberrant crawling behaviour in 3rd instar wandering larvae pan-neuronally expressing CHMP2B Intron5 . ANOVA with post-hoc Dunnett's comparison to wild type controls ⁎⁎⁎ p < .001 and Tukey comparison between groups ### p < .001. G. Representative micrographs of mature neurons expressing FLAG-tagged CHMP2B Wildtype or CHMP2B Intron5 ± plasmids expressing the catalytic (GCLC) and modifying (GCLM) subunits of glutamate cysteine ligase (GCL). H, I Quantification of total arbour size (H) and cumulative number of intersections per unit distance (I) in CHMP2B Wildtype or CHMP2B Intron5 ± GCLC/M. Data represents mean ± SEM analysed using one-way ANOVA and Tukey's multiple comparisons post hoc test ( ## p < .01, ### p < .001). Scale bar = 50 μm. 20 neurons analysed, per condition, across 3 biological replicates.

Article Snippet: Neurons were transfected at 12 days in vitro (DIV) with Lipofectamine 2000 (11,668,019, Thermo Scientific) in transfection medium ( ) with either FLAG-tagged CHMP2B Wildtype or CHMP2B Intron5 for 5 h. The cDNAs for catalytic (GCLC, MC203908) and modifying (GCLM, MR225622) subunits of GCL were obtained from Origene.

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

doi: 10.1101/2020.05.14.090738

Figure Lengend Snippet: a. Resazurin-based viability assay for HCT116 (left) and Nalm6 (right) Wild type (WT) and Δ ADH5 cells at 100 and 50 μM L-buthionine-sulfoximine (L-BSO), respectively (s.e.m., n=5). b. Western blot analysis of GCLM expression in clones edited by CRISPR/Cas9. c. ADH5 gene showing the exon targeted by CRISPR/Cas9 and the genetic modifications of the Δ ADH5 clone on which GCLM was inactivated. d. GCLM gene showing the exon targeted by CRISPR/Cas9 and the genetic modifications of the Δ GCLM clones used in this work.

Article Snippet: After 20 days, cells were expanded, and inactivation of the desire gene confirmed by western blot using GCLM (Atlas antibodies, #HPA023696) or ADH5 antibodies .

Techniques: Viability Assay, Western Blot, Expressing, Clone Assay, CRISPR

Journal: bioRxiv

Article Title: Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

doi: 10.1101/2020.05.14.090738

Figure Lengend Snippet: a. Resazurin-based viability assay in Wild type (WT) and Δ ADH5 Nalm6 cells in presence of different concentrations of formaldehyde (FA) with or without 50 μM L-buthionine-sulfoximine (L-BSO) (mean ± s.e.m., n=6). b. HCT116 3D-spheroid formation in presence of 100 μM L-BSO and the indicated concentrations of FA. Pictures were taken 5 days after seeding cells on agarose-coated plates (scale bar 0.5 mm). c. Resazurin-based viability assay performed with Wild type, Δ ADH5 , Δ GCLM and Δ ADH5 Δ GCLM cells in response to increasing concentrations of FA (mean ± s.e.m., n=5, asterisks represent the statistical significance according to one-way ANOVA for multiple comparison using a Tukey-corrected test between Wild type and Δ GCLM ). d. Representative images of HCT116 3D-spheroid formation for the same cell lines described in (C). Pictures were taken at day 5 after seeding (scale bar 0.5 mm). e. Colony survival assay prepared seeding 600 of WT, Δ ADH5 , Δ GCLM and Δ GCLM Δ ADH5 cells in 6-well plates in presence of the indicated concentration of FA (mean ± s.e.m., n=5, one-way ANOVA using a Tukey’s multiple comparison test). f. Representative images of the colony survival assay quantified in e.

Article Snippet: After 20 days, cells were expanded, and inactivation of the desire gene confirmed by western blot using GCLM (Atlas antibodies, #HPA023696) or ADH5 antibodies .

Techniques: Viability Assay, Comparison, Clonogenic Cell Survival Assay, Concentration Assay

Journal: bioRxiv

Article Title: Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

doi: 10.1101/2020.05.14.090738

Figure Lengend Snippet: a. Scheme showing the spontaneous reaction between formaldehyde (FA) and glutathione (GSH) yielding S-hydroxymethylglutathione (HSMGSH). b. Extracted ion chromatograms for [HSMGSH + H]+ ion at m/z 338.1022 ± 0.0500 generated from a non-spiked Wild type (WT) sample (green, left axis), a 20 μM spiked WT sample (black dotted line, right axis), and a 20 μM HSMGSH standard solution (blue, left axis). c. Product ion mass spectra of [HSMGSH + H] + precursor ion for a WT sample (green), and for a 20 μM HSMGSH standard solution (blue), using a collision cell voltage of 10 V. d. Box and whiskers plot for GSH content in WT and Δ ADH5 cells calculated as normalized chromatographic peak areas relative to the number of viable cells (n=9, Mann-Whitney test, *** P<0.0001). The box and whiskers plots are represented by a line in the box corresponding to the median; the edges are the 25th and 75th percentiles and the whiskers extend to the most extreme values in data. e. Left: Box and whiskers plot for HSMGSH content relative to GSH in WT and Δ ADH5 cells (n=9, Mann-Whitney test). Right: Net HSMGSH content in WT and Δ ADH5 cells calculated as normalized peak areas relative to the number of viable cells (n=9, Mann-Whitney test). f. Box and whiskers plot for total GSH (GSH disulfide (GSSG) plus GSH) content in WT and Δ ADH5 cells calculated as normalized peak areas relative to the number of viable cells (n=9, Mann-Whitney test). g. Bar plots for GSH content in WT (n=17), Δ ADH5 (n=15), Δ ADH5 /pADH5 (n=8), Δ GCLM (n=4) and Δ ADH5 Δ GCLM (n=4) cells, and in WT (n=7) and Δ ADH5 (n=7) cells exposed to 100 μM L-BSO for 48 h. Every dot is the percentage of fluorescence intensity in a single well relative to the average fluorescence of WT samples run the same day and corrected for viability determined using resazurin (mean ± s.e.m., unpaired t-test). h. Box and whiskers plot for GSSG content in WT and Δ ADH5 cells calculated relative to the number of viable cells (n=9, Mann-Whitney test). i. Box and whiskers plot for GSH:GSSG ratio in WT and Δ ADH5 cells (n=9, Mann-Whitney test). j. Flow cytometry representative plots from WT and Δ ADH5 cells harboring the cytosolic Grx1-roGFP2 reporter. Data was recorded 48 h post-FA exposure. k. Quantitation of oxidized Grx1-GFP2 (OxD (Grx1-roGFP2)) sensor from plots depicted in (J) (mean ± s.e.m., n=4, unpaired t-test).

Article Snippet: After 20 days, cells were expanded, and inactivation of the desire gene confirmed by western blot using GCLM (Atlas antibodies, #HPA023696) or ADH5 antibodies .

Techniques: Generated, MANN-WHITNEY, Fluorescence, Flow Cytometry, Quantitation Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: The Role of Fucoxanthin as a Potent Nrf2 Activator via Akt/GSK-3β/Fyn Axis against Amyloid-β Peptide-Induced Oxidative Damage.

doi: 10.3390/antiox12030629

Figure Lengend Snippet: Figure 3. Effects of fucoxanthin on expression of Nrf2 and Keap1 in Aβ25-35-induced PC12 cells. The cells were pretreated with the indicated concentrations of fucoxanthin for 1 h and stimulated with 10 µM Aβ25-35 for 4 h. (A) The nuclear and cytoplasm expression of Nrf2 as measured by western blot. (B) Relative ratio of nuclear and cytoplasmic Nrf2. (C) The total Nrf2 and Keap1 expression. Quantification of (D) total Nrf2 and (E) Keap1 protein expression levels. NE, nuclear extract; CE, cytoplasmic extract. β-Actin and PCNA were used as the loading control for whole cells and nuclear fractions, respectively, in western blot assay. (E) Keap1 protein expression levels. NE, nuclear extract; CE, cytoplasmic extract. mRNA (F,G) and protein (H,I) levels of phase-II enzymes including NQO1, GCLm, and TrxR1. The cells were pretreated with indicated concentrations of fucoxanthin for 1 h and stimulated with 10 µM Aβ25-35 for 24 h. The mRNA expression levels of phase-II enzymes were determined by RT-PCR analysis. For the normalization of mRNA expression GAPDH was used as the loading control. Results are indicated as mean ± S.D. and represent three independent experiments with 3 replications in each experiment. ## p < 0.01 and # p < 0.05 compared with the control groups; *** p < 0.001, ** p < 0.01 and * p < 0.05 compared with the Aβ25-35-treated alone.

Article Snippet: Blotted membranes were blocked in a 5% skim milk solution in Tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 2 h, and probed overnight at 4 ◦C with the following primary antibodies: Bcl-2, Bax, NQO1, GSK-3β, p-Fyn, Fyn, β-actin (1:2000 dilution; all from Santa Cruz Biotechnology, Dallas, TX, USA), Keap1, p-Akt, Akt, p-GSK-3β (1:2000 dilutions; all from Cell Signaling Technology, Danvers, MA, USA), GCLm (1:2000 dilution; Cusabio Technology LCC, Wuhan, China), TrxR1, Nrf2, and PCNA (1:2000 dilution; all from GeneTex Inc., San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants (Basel, Switzerland)

Article Title: The Role of Fucoxanthin as a Potent Nrf2 Activator via Akt/GSK-3β/Fyn Axis against Amyloid-β Peptide-Induced Oxidative Damage.

doi: 10.3390/antiox12030629

Figure Lengend Snippet: Figure 4. Effects of fucoxanthin on Akt/GSK-3β/Fyn signaling in Aβ25-35-induced PC12 cells. The cells were pretreated with the indicated concentrations of fucoxanthin for 1 h and stimulated with 10 µM Aβ25-35 or 1 h. The expression of (A) p-Akt (Ser 473)/Akt, (B) p-GSK-3β (Ser 9)/GSK-3β, and (C) p-Fyn/Fyn. Western blotting results of (D) p-Akt (Ser 473)/Akt, (E) p-GSK-3β (Ser 9)/GSK-3β, (F) p-Fyn/Fyn, (G) Nrf2, and (H–K) phase-II enzymes (NQO1, GCLm, and TrxR1) expression co- treatment of the PI3K inhibitor LY294002 and fucoxanthin. After pretreatment with the PI3K inhibitor LY294002 for 30 min, the cells were treated with 5 µM fucoxanthin for 1 h, followed by Aβ25–35 treatment. Results are indicated as mean ± S.D. and represent three independent experiments with 3 replications in each experiment. ## p < 0.01 compared with the control groups; *** p < 0.001 and ** p < 0.01 compared with the Aβ25-35-treated alone; §§ p < 0.01 compared with the Aβ25–35 -treated alone; s p < 0.01 and R p < 0.05 compared with the fucoxanthin group without LY294002.

Article Snippet: Blotted membranes were blocked in a 5% skim milk solution in Tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 2 h, and probed overnight at 4 ◦C with the following primary antibodies: Bcl-2, Bax, NQO1, GSK-3β, p-Fyn, Fyn, β-actin (1:2000 dilution; all from Santa Cruz Biotechnology, Dallas, TX, USA), Keap1, p-Akt, Akt, p-GSK-3β (1:2000 dilutions; all from Cell Signaling Technology, Danvers, MA, USA), GCLm (1:2000 dilution; Cusabio Technology LCC, Wuhan, China), TrxR1, Nrf2, and PCNA (1:2000 dilution; all from GeneTex Inc., San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control