Journal: Cancer cell

Article Title: ILF2 Is a Regulator of RNA Splicing and DNA Damage Response in 1q21-Amplified Multiple Myeloma

doi: 10.1016/j.ccell.2017.05.011

Figure Lengend Snippet: shRNA Knockdown and Overexpression Experiments

Article Snippet: Rabbit polyclonal anti-Fancd2 , Novus Biologicals , Cat# NB100-182.

Techniques: shRNA, Knockdown, Over Expression, Immunoprecipitation, Immunofluorescence, Virus, Plasmid Preparation, Microarray, Recombinant, Reverse Transcription, Fractionation, Mutagenesis, Software

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: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Runx2 , NOVUS BIOLOGICALS , Cat# NBP1-77461, RRID:AB_11003000.

Techniques: Recombinant, Multiplex Assay, Staining, In Situ, Marker, Expressing, Plasmid Preparation, shRNA, Software