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: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: (A) Quantification of chromosomal aberrations in HCT116 parental and ZGRF1 −/− cells. Means with 95% confidence intervals are plotted. p values were calculated using Mann-Whitney U tests. N ≥ 32 spreads per condition. (B and C) Examples of metaphase spreads from parental cells (B) or ZGRF1 −/− cells (C) untreated or treated with 20 ng/mL MMC for 24 h. White arrows mark chromosomal aberrations. The images shown highlight the types of aberrations scored rather than being representative of the number of aberrations seen per spread. (D) ZGRF1 −/− cells show a higher frequency of co-localizing γ-H2AX and FANCD2 foci under both unperturbed conditions and when treated with MMC. Quantification of co-localizing γ-H2AX and FANCD2 foci under unperturbed conditions and with two different treatments with MMC is shown. Error bars show 95% confidence intervals. p values were calculated using Mann-Whitney U tests. N > 100 cells for each condition. (E) Representative images of HCT116 parental and ZGRF −/− cells under unperturbed conditions or after treatment with 20 ng/mL MMC for 4 h. Arrows mark co-localizing foci for one cell, where the pixel intensity of FANCD2 foci was a minimum of 6,000 arbitrary units higher than background. Cells with very low or absent γ-H2AX foci were not scored, as this indicated inadequate immunostaining. (F) Colony formation assay of HCT116 parental and ZGRF1 −/− and FANCM −/− single- and double-mutant cell lines treated with the indicated doses of MMC for 24 h. The graph of the parental cell line is statistically different from each of the knockout cell lines (p < 0.05, t test), but the knockout cell lines are not significantly different from each other (n.s.). (G) Colony formation assay of HCT116 parental and ZGRF1 −/− and FANCJ single- and double-mutant cell lines treated with the indicated doses of MMC for 24 h. The graph of the parental cell line is statistically different from the ZGRF1-knockout cell line (p < 0.05, t test). The FANCJ single- and double-mutant cell lines are not significantly different from each other (n.s.). Graphs in (F) and (G) show the mean with 95% confidence interval. Statistical significance was calculated using unpaired t tests without assuming consistent SD. *p < 0.05. n.s., no significant difference. n ≥ 3 for each cell line.

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: MANN-WHITNEY, Immunostaining, Colony Assay, Mutagenesis, Knock-Out

Journal: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: (A) ZGRF1 localizes to nuclear foci during ICL repair. Cells expressing ZGRF1–2xYFP from the endogenous promoter were synchronized at the G1/S border by treatment with 2 mM thymidine for 18 h before release into S phase in Leibovitz’s L-15 medium containing 0.4 μM Hoechst 33258. Four hours before release, 20 ng/mL of MMC or vehicle was added to the cultures. Arrows indicate ZGRF1 foci. (B) Quantification of ZGRF1 foci after MMC treatment. Quantification of the experiment in (A). Error bars show 95% confidence intervals. p values were calculated using Mann-Whitney U tests. N = 90–160 cells for each condition. (C) Colocalization of ZGRF1 and FANCD2. Cells expressing ZGRF1–2xYFP from the endogenous promoter and ectopically integrated mCherry-FANCD2 were synchronized in S phase with 2 mM thymidine 18 h prior to microscopy. Four hours before microscopy, 20 ng/mL MMC or vehicle was added to the culture. Yellow arrows mark ZGRF1 foci, red arrows mark FANCD2 foci, and orange arrows mark the co-localizing foci. (D) Quantification of co-localizing FANCD2 and ZGRF1 foci in the experiment reported in (C). Error bars show 95% confidence intervals. p values were calculated using Mann-Whitney U tests. N > 400 cells for each condition.

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: Expressing, MANN-WHITNEY, Microscopy

Journal: Cell reports

Article Title: The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

doi: 10.1016/j.celrep.2020.107849

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were washed twice with PBS and incubated with primary antibodies (1:2000 anti-FANCD2 (Novus Biologicals, cat. no. NB100–182SS) and 1:500 anti-γH2AX (Millipore, cat. no. 05–636)) in 3% BSA in PBST overnight at 4°C.

Techniques: Imaging, Western Blot, Virus, Recombinant, Protease Inhibitor, In Situ, Plasmid Preparation, Software

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