Journal: Scientific Reports

Article Title: Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish

doi: 10.1038/s41598-021-87879-4

Figure Lengend Snippet: Inhibition of autophagy prevents abnormal development and improves survival in larsb- knockout larvae. (A) Morphology of larsb + / + and larsb −/− embryos injected with either control MO or atg5-MO (72 h post fertilization (hpf)). Scale bars: 500 µm. (B) Western blot analysis of Lc3b protein expression at 72 hpf for wild-type embryos injected with either control MO or atg5-MO. β-actin levels served as the loading control. (C) Morphological abnormality at 72 hpf in the livers of larsb −/− larvae under Tg[ fabp10 :mcherry] background injected with either control MO or atg5-MO. Scale bars: 200 μm. (D) Quantification of liver size in larsb −/− larvae under Tg[ fabp10 :mcherry] background (72 hpf). Liver sizes were evaluated using ImageJ software version 1.52a ( https://imagej.nih.gov/ij/ ). n = 4 fish/group. Error bars indicate SEM. Student’s t-test; ***P < 0.001. (E) Western blot analysis of Lc3b protein expression at 72 hpf for wild-type embryos treated with DMSO or bafilomycin A1. β-actin levels served as the loading control. (F) Morphological abnormality at 72 hpf in the livers of larsb −/− larvae under Tg[ fabp10 :mcherry] background treated with DMSO or bafilomycin A1. Scale bars: 200 μm. (G) Quantification of liver size in larsb −/− larvae under Tg[ fabp10 :mcherry] background (72 hpf). Liver sizes were evaluated using ImageJ software version 1.52a ( https://imagej.nih.gov/ij/ ). n = 10 fish/group. Error bars indicate SEM. Student’s t-test; *P < 0.05. (H) Kaplan–Meier survival curve of larsb + / + (n = 23) and larsb −/− (n = 15) larvae treated with DMSO and larsb + / + (n = 11) and larsb −/− larvae (n = 21) treated with bafilomycin A1. Statistics were calculated and the figure was produced in GraphPad software version 8 ( https://www.graphpad.com/scientific-software/prism/ ). Larsb: leucyl-tRNA synthetase b, MO: morpholino, n.s.: non-significant, DMSO: dimethyl sulfoxide, Dpf: days post fertilization.

Article Snippet: Western blotting was performed with antibodies against Lars (#13868; Cell Signaling Technology, Beverly, MA, USA), p62 (PM045; Medical & Biological Laboratories, Nagoya, Japan), LC3B (PM036; Medical & Biological Laboratories), ATG5 (NB110-53818; Novus Biologicals, Littleton, CO, USA), β-actin (A3854; Sigma-Aldrich, St. Louis, MO, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G9295; Sigma-Aldrich).

Techniques: Inhibition, Knock-Out, Injection, Control, Western Blot, Expressing, Software, Produced

Journal: Autophagy

Article Title: Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy

doi: 10.1080/15548627.2023.2165313

Figure Lengend Snippet: Autophagy flux is induced in microglia after tMCAo and OND. ( A ) Representative confocal images of the DG of 2 mo fms -EGFP mice exposed to sham and tMCAo surgery at 6 h and 1 d. LC3 present in autophagosomes was immunostained and observed as puncta (yellow). Microglia were visualized by the transgenic expression of fms -EGFP (cyan) and cellular nuclei were identified by DAPI (white). SQSTM1 ( Ai ) and LAMP1 ( Aii ) were immunostained and visualized as puncta (magenta). ( B ) Number of LC3 puncta normalized to microglial cytoplasmic area (LC3 puncta number/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( C ) Total area of LC3 puncta normalized to microglial cytoplasmic area (LC3 puncta area/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( D ) Number of LC3 and SQSTM1 puncta that colocalize normalized to microglial cytoplasmic area (LC3-SQSTM1 puncta number colocalization/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( E ) Number of LC3 and LAMP1 puncta that colocalize normalized to microglial cytoplasmic area (LC3-LAMP1 puncta number colocalization/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( F ) Number of SQSTM1 puncta normalized to microglial cytoplasmic area (SQSTM1 puncta number/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( G ) Number of LAMP1 puncta normalized to microglial cytoplasmic area (LAMP1 puncta number/µm 2 ) in sham and tMCAo (6 h and 1 d) mice. ( H ) Quantified total SQSTM1 puncta area normalized by microglial cytoplasmic area (SQSTM1 puncta area/µm 2 ) ( Hi ), the colocalized area of LC3 and SQSTM1 puncta normalized by microglial cytoplasmic area (colocalized LC3-SQSTM1 puncta area/µm 2 ) ( Hii ), total LAMP1 puncta area normalized by microglial cytoplasmic area (LAMP1 puncta area/µm 2 ) ( Hiii ), and the colocalized area of LC3 and LAMP1 puncta normalized by microglial cytoplasmic area (colocalized LC3-LAMP1 puncta area/µm 2 ) ( Hiv ). ( I ) Experimental design used to transfect BV2 microglia-like cells with the fluorescent tandem mRFP-GFP-LC3 to assess autophagy flux in control conditions (C) and after OND (3 h) or rapamycin (Rapa, 100 nM, 6 h) treatments. Representative confocal images of control, OND and rapamycin treated microglia. Nuclei are stained with DAPI (white), autophagosomes and autolysosomes are differentiated according to the tandem expression (yellow and red, respectively). ( J ) GFP:RFP mean fluorescence intensity ratio, indicative of autophagy flux. ( K ) Mean RFP fluorescence intensity ( Ki ), mean GFP fluorescence intensity ( Kii ), total number of puncta ( Kiii ), and area occupied by puncta ( Kiv ) per cell and normalized to control conditions (expressed as % change versus control conditions). Bars show mean ± SEM ( B-H ). Violin plots show the data distribution, including extreme values; lower and upper hinges correspond to the first and third quartile, respectively ( J, K ). n = 4 mice per experimental condition ( B-H ); n = 12–17 cells from 3 independent experiments ( J, K ). Data were analyzed by one-way ANOVA followed by Holm-Sidak post hoc test ( B-H ), Bonferroni post hoc test ( J ); by Kruskal-Wallis one- way ANOVA on ranks followed by Dunn’s multiple comparisons ( K ). * represents significance between sham or control and tMCAo, OND or rapamycin: one symbol represents p < 0.05, two symbols represent p < 0.01 and three symbols represent p < 0.001. Scale bars: 20 µm, z = 20 µm ( A ); 10 µm, z = 1.9 µm (control), 3.3 µm (OND), and 3.9 µm (rapamycin) ( I ).

Article Snippet: SQSTM1 (rabbit) , 1:200 , Novus Biologicals , NBP1-48,320.

Techniques: Transgenic Assay, Expressing, Control, Staining, Fluorescence

Journal: Autophagy

Article Title: Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy

doi: 10.1080/15548627.2023.2165313

Figure Lengend Snippet: List of reagents and/or resources used in this article.

Article Snippet: SQSTM1 (rabbit) , 1:200 , Novus Biologicals , NBP1-48,320.

Techniques: Western Blot, Activity Assay, Software, Laser-Scanning Microscopy, Microscopy, Irradiation

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Venn diagram demonstrating the overlap of orthologues proteins expressed in HuH7 and Vero cells based on the NCBI gene IDs corresponding to majority protein IDs. (B) The overlap of virus- and thapsigargin-regulated proteins common to HuH7 and Vero E6 cells was calculated based on gene IDs. This analysis identifies 180 proteins with higher and 61 proteins with lower expression in thapsigargin-treated infected cells compared to virus infection alone (ratio > 0, p ≥ −log 10 1.3). (C) Heatmaps showing individual mean ratio values of log 2 -transfomed normalized protein intensities for the top 50 up- or downregulated proteins in virus-infected and thapsigargin-treated cells. Ratio values of infected or thapsigargin-treated conditions compared to untreated cells (-) cells are shown for comparison. Note that ratios are sorted and color coded according to the virus plus thapsigargin conditions with infection alone conditions set as denominator. Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2-regulated protein in an independent study ( Stukalov et al ., 2020 ). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone. Gene ID lists were analyzed using Metascape software ( Zhou et al ., 2019 ). (E) Experimental evidence, co-occurrence, co-expression and confidence scores from the STRING database ( Szklarczyk et al , 2019 ) were used to identify protein:protein interactions (PPI) amongst the 180 up- and 61 downregulated thapsigargin-sensitive proteins. As shown, based on experimental evidence and combined STRING score criteria, 59 and 26 coregulated proteins are engaged in defined PPI networks; the remaining proteins are not known to interact. (F) Validation of thapsigargin-induced HERPUD1 and CTH upregulation by immunoblotting of HuH7 or Vero E6 whole cell extracts from cells treated as described above. BiP and IRE1α levels are shown for comparison. (G) Quantification of thapsigargin-mediated re-expression of HERPUD1 and CTH in cells infected with HCoV-229E, MERS-CoV or SARS-CoV-2 from two independent immunoblot experiments. Error bars show s.d.. (H) Heatmap showing thapsigargin-reprogrammed proteins of KEGG 04141 (mean ratio ≥ 1.5 fold) along with p values. See also for projection of thapsigargin-mediated protein changes on the KEGG pathway map. (I) Venn diagram showing the intersection of thapsigargin- / virus-regulated proteins with all novel ERAD components (FDR of 1 %) identified by ( Leto et al ., 2019 ). The regulation of 31 overlapping components is shown as a heatmap displaying mean ratio values in thapsigargin-treated or infected cells. Red colors highlight UBA5 and ZNF622 as discussed in the text. (J) Summary of the main findings of our study. Abbreviations: M, MERS-CoV; S, SARS-CoV-2; T, thapsigargin.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Virus, Expressing, Infection, Comparison, Software, Protein-Protein interactions, Biomarker Discovery, Western Blot