Journal: Redox Biology

Article Title: Involvement of the mitochondrial nuclease EndoG in the regulation of cell proliferation through the control of reactive oxygen species

doi: 10.1016/j.redox.2020.101736

Figure Lengend Snippet: Analysis of the impact of Endog expression in cell proliferation and ROS abundance in diverse mouse, rat and human cell types. A) Cardiomyocyte (CM) number was counted from 5 to 9 hearts of Endog +/+ and Endog −/− of 4-days-old (P4) male and female mice (representative images shown at right), after digestion of the ventricular tissue that was previously weighted (B). C) Quantification of the expression of the proliferation-related Ki-67 nuclear antigen in histological ventricular tissue preparations from P4 Endog +/+ and Endog −/− mice. Ki-67+ nuclei were counted in 3 different slices from 3 hearts/genotype and data are expressed as Ki-67 + nuclei/total nuclei in the slice. All individual quantifications are plotted (1000–2000 nuclei/genotype). D) MitoSOX™ fluorescence was quantified by flow cytometry in preparations of ventricular cardiomyocytes from P4 Endog +/+ and Endog −/− mice cultured in the presence or absence of 0.2 mM N -Acetyl-Cysteine (NAC; n = 6). E) Neonatal rat ventricular cardiomyocyte cultures were left untreated (NT, not transduced), transduced with a scrambled sequence (Scr) or an Endog -specific silencing sequence (shRNA) and then treated or not with 0.2 mM NAC for 48 h. Cells were fixed and stained with muscle-specific α-actinin (cardiomyocytes) and Hoechst dye (nuclei). Cardiomyocytes were counted in 10 different microscopic fields/treatment in 4 independent experiments. All 40 values/treatment are plotted. F) Skin fibroblasts were obtained from P4 Endog +/+ and Endog −/− mice, plated and amplified. Equal number of cells were seeded in 2 plates/genotype and counted after 72 h. Data are expressed as the number of cell cycles completed in 72 h.Cells in replicate plates were counted at time zero to confirm equal initial cell number. All values from 4 independent experiments are plotted. G) MitoSOX™ fluorescence was quantified as in (D) in preparations of Endog +/+ and Endog −/− fibroblasts treated or not with NAC. H) Endog +/+ and Endog −/− fibroblasts were seeded in presence or absence of NAC and counted after 48 h. Conditions are as in F. I) Strategy for the CRISPR-Cas9-dependent ENDOG gene truncation by insertion of the puromycin resistance cassette (PuroR), using homologous recombination in 4 different selected sites within the ENDOG gene in HEK293 human cells. Expression of ENDOG was analyzed in control cells (C) and several clones from each of the four PuroR insertion sites (see Material and methods section). J) MitoSOX™ fluorescence was quantified as in (D) in preparations of control cells and cells from CRISPR-Cas9-treated clones 1.1 and 4.3 treated or not with 0.2 mM NAC (n = 7). K) HEK293 cells were counted in control and ENDOG-deficient cultures from clones 1.1 and 4.3 cultured for 48 h, treated or not with NAC. Proliferation was expressed as cell cycles completed in 48 h. Data plotted are from four experiments performed in duplicates. Graphs show experimental values plus mean ± SD, except D, G and J where median ± interquartile range is depicted. Analysis of the effect of Endog expression in the experimental values was performed with the Student's t-test (B, C and F). 2-way ANOVA was used to analyze the influence of Endog expression and gender (A) or NAC treatment (E, H and K) and the interaction between them in cell number. The Kruskal-Wallis test was used to compare the effects of Endog expression and NAC addition on MitoSOX™ fluorescence followed by the Dunn's test for selected post hoc comparisons (D, G and J). ns = not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Immunohistochemistry was performed in paraffin-included heart slices as previously described [ ] using primary antibodies against Ki67 (MAB4190, Merck) and Cyclin D (SC-20044), biotinylated secondary antibodies (Jackson) and developed with deaminobenzidine (DAB, DAKO) contrasted with hematoxylin.

Techniques: Expressing, Fluorescence, Flow Cytometry, Cell Culture, Transduction, Sequencing, shRNA, Staining, Amplification, CRISPR, Homologous Recombination, Clone Assay

Journal: Scientific Reports

Article Title: The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

doi: 10.1038/s41598-021-92529-w

Figure Lengend Snippet: Protection of the primary hippocampal neurons by ATF6β. ( A ) Primary hippocampal neurons were treated with Tg (300 nM) or Tm (1 µg/ml) for 24 h, and cell survival/death was evaluated by the LIVE/DEAD viability assay. Representative fluorescent microscopic images from four independent experiments are shown. The graph depicts the percentages of dead cells. n = 4 experiments. Data are shown as mean ± SEM. ***p < 0.001 by a two-way ANOVA followed by the Bonferroni test. Scale bar: 20 μm. ( B ) Apoptosis was evaluated by immunocytochemical staining of cleaved caspase-3 (red), βIII tubulin (green) and DAPI staining (blue). Representative fluorescent microscopic images from four independent experiments are shown. The graph depicts the percentages of cleaved caspase-3-positive cells. n = 4 experiments. Data are shown as mean ± SEM. ***p < 0.001 by a two-way ANOVA followed by the Bonferroni test. Scale bar: 20 μm. ( C ) Apoptosis was evaluated by immunocytochemical staining of cleaved caspase-3 (red) and GFP (green) using primary hippocampal neurons transfected with vector, ATF6β cDNA or ATF6α cDNA together with GFP cDNA, followed by Tm treatment for 24 h. Representative fluorescent microscopic images are shown. Arrows indicate caspase-3-positive and GFP-positive cells. The graph depicts the percentages of cleaved caspase-3-positive cells out of GFP-positive cells. n = 400 cells per condition from two independent experiments. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001 by a two-way ANOVA followed by the Bonferroni test. Scale bar: 10 μm. Note that transfection of ATF6β cDNA, but not ATF6α cDNA, rescued Atf6b −/− neurons.

Article Snippet: Cultured hippocampal neurons and Neuro 2a cells were fixed in 4% paraformaldehyde for 15 min at room temperature, and permeabilized in 0.3% Trinton-X100 for 10 min. Primary antibodies against GFP (MBL598; Merck; 1:500), Myc (sc-40; Santa Cruz Biotechnology; 1:200), cleaved caspase-3 (Asp175, Cell Signaling Technology, Inc; 1:800) andβIII tubulin (MAB1637; Merck; 1:500) were used.

Techniques: Viability Assay, Staining, Transfection, Plasmid Preparation