Journal: EMBO Reports

Article Title: Interferon‐mediated repression of miR ‐324‐5p potentiates necroptosis to facilitate antiviral defense

doi: 10.15252/embr.202154438

Figure Lengend Snippet: A Schematic description of a screening for miRNAs blocking necroptosis. Human miRNA mimics, nontarget miRNA (the negative control, NC), and the positive control RIPK3 siRNA oligos (siRIPK3) were transferred into HT‐29. After 48 h, cells were treated with 40 ng/ml TNF‐α (T), 100 nM Smac mimetic (S), and 20 μM z‐VAD (Z) for 24 h, and then cell viability was determined by measuring ATP levels. Identical concentrations were used in later experiments unless otherwise stated. T + S + Z: TNF‐α, Smac mimetic and z‐VAD. B Graphical representation of the screen results with each miRNA identified from the library (842 miRNAs). Arrowheads point to siRIPK3, miR‐324‐5p, and the negative control (NC). C The RNA sequences of miR‐324‐5p and miR‐324‐3p were aligned by DNAman. HT‐29 cells were transfected with NC, siRIPK3, miR‐324‐5p, or miR‐324‐3p. After 48 h, cells were treated with T + S + Z for 24 h. Cell viability was determined by measuring ATP levels. D Human gastric carcinoma MKN45 cells were transfected with NC, siRIPK1, or miR‐324‐5p. After 48 h, cells were treated with T + S + Z for 24 h. Cell viability was determined by measuring ATP levels. E Human colon cancer 174T cells were transfected with NC or miR‐324‐5p. After 48 h, cells were treated with T + S + Z for 24 h. Cell viability was determined by measuring ATP levels. F Human glioblastoma T98G cells were transfected with NC, siRIPK1, or miR‐324‐5p. After 48 h, cells were treated with T + S for 24 h. Cell viability was determined by measuring ATP levels. G Human monocytic leukemia U937 cells were transfected with NC, siRIPK3, or miR‐324‐5p. At 48 h, cells were treated with 100 ng/ml LPS (L) and 20 μM z‐VAD (Z) for an additional 24 h. Cell viability was determined by measuring ATP levels. Data information: In (C–G), data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t ‐test. All experiments were performed at least three times, and representative data are shown.

Article Snippet: Human U937 myelomonocytic cells (ATCC Number: CRL‐1593.2) were grown in RPMI‐1640 medium (Hyclone).

Techniques: Blocking Assay, Negative Control, Positive Control, Transfection

Journal: EMBO Reports

Article Title: Interferon‐mediated repression of miR ‐324‐5p potentiates necroptosis to facilitate antiviral defense

doi: 10.15252/embr.202154438

Figure Lengend Snippet: A U937 cells were treated with 100 ng/ml IFN‐α, 100 ng/ml IFN‐β, 100 ng/ml IFN‐γ, 100 ng/ml LPS, or 25 μg/ml poly(I:C) for 24 h. Identical concentrations were used in later experiments unless otherwise stated. qPCR analysis for the expression of MLKL. B U937 cells were treated with IFN‐α, IFN‐β, IFN‐γ, LPS, or poly(I:C) for 24 h. and western blotting analysis of MLKL and β‐actin (left). Quantification of MLKL normalized to β‐actin levels (right). C U937 cells were treated with PBS, IFN‐α, IFN‐β, IFN‐γ, LPS, or poly(I:C) for 24 h. qPCR analysis for the expression of miR‐324‐5p. D, E U937 cells were transfected with NC, siMLKL, or miR‐324‐5p. After 48 h, cells were treated with IFN‐β (D) or IFN‐γ (E) for 24 h. Western blotting analysis of MLKL and β‐actin. F, G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN‐β (F) or IFN‐γ (G) treatment. After 24 h, qPCR analysis for the expression of miR‐324‐5p. After 48 h, western blotting analysis of p‐STAT1, STAT1, MLKL, and β‐actin. H, I U937 cells were transfected with NC, or STAT1 siRNA oliogs (siSTAT1). After 48 h, cells were treated with IFN‐β (Η) or IFN‐γ (Ι). qPCR analysis for the expression of miR‐324‐5p. Western blotting analysis of STAT1, MLKL and β‐actin. J HT‐29 cells were treated with IFN‐γ for 24 h. The cell lysate was collected for ChIP analysis. STAT1 binding to miR‐324‐5p promoter DNA region was determined by Chip‐qPCR. The amount of precipitated DNA was calculated as percent input. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t ‐test. All experiments were performed at least three times, and representative data are shown. Source data are available online for this figure.

Article Snippet: Human U937 myelomonocytic cells (ATCC Number: CRL‐1593.2) were grown in RPMI‐1640 medium (Hyclone).

Techniques: Expressing, Western Blot, Transfection, Incubation, Binding Assay, ChIP-qPCR

Journal: EMBO Reports

Article Title: Interferon‐mediated repression of miR ‐324‐5p potentiates necroptosis to facilitate antiviral defense

doi: 10.15252/embr.202154438

Figure Lengend Snippet: A, B U937 cells were electroporated with NC or miR‐324‐5p. After 48 h, cells were exposed to IFN‐β(Α) or IFN‐γ(Β) for 24 h. Western blotting analysis for the expression of MLKL and β‐actin. Quantification of MLKL normalized to β‐actin levels. C MiR‐324‐5p +/+ HT‐29, and miR‐324‐5p −/− HT‐29 cells were treated with IFN‐β or IFN‐γ for the indicated time. Western blotting analysis of MLKL and β‐actin. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t ‐test. All experiments were performed at least three times, and representative data are shown. Source data are available online for this figure.

Article Snippet: Human U937 myelomonocytic cells (ATCC Number: CRL‐1593.2) were grown in RPMI‐1640 medium (Hyclone).

Techniques: Western Blot, Expressing

Journal: EMBO Reports

Article Title: Interferon‐mediated repression of miR ‐324‐5p potentiates necroptosis to facilitate antiviral defense

doi: 10.15252/embr.202154438

Figure Lengend Snippet: A U937 cells were transfected with NC, siMLKL‐3′UTR, or miR‐324‐5p. After 48 h, cells were exposed to 100 ng/ml IFN‐γ plus 20 μM z‐VAD for 24 h. Identical concentrations were used in later experiments unless otherwise stated. Cell viability was determined by measuring ATP levels. B qPCR analysis for the expression of MLKL (left) and miR‐324‐5p (middle) in PBMC‐derived macrophages that were treated with PBS or IFN‐γ for 24 h. The corresponding western blotting analysis of MLKL and β‐actin (right). C, D PBMC‐derived macrophages (C) and HT‐29 cells (D) were transfected with NC, siMLKL, or miR‐324‐5p. After 48 h, cells were exposed to S + Z or IFN‐γ + S + Z for 24 h. Cell viability was determined by measuring ATP levels (left). The corresponding western blotting analysis of MLKL and β‐actin (right). E qPCR analysis for the expression of miR‐324‐5p in miR‐324‐5p knockout HT‐29 cells (miR‐324‐5p −/− cells). Altered miR‐324‐5p DNA sequences were shown in miR‐324‐5p −/− clone 19# and 36#. F MiR‐324‐5p +/+ HT‐29, miR‐324‐5p −/− −19#, and miR‐324‐5p −/− −36# cells were treated with T + S + Z for the indicated times. Cell viability was determined by measuring ATP levels. G, H MiR‐324‐5p +/+ HT‐29, and miR‐324‐5p −/− HT‐29 cells were treated with IFN‐β (G) or IFN‐γ (H) plus 20 μM z‐VAD for 24 h. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t ‐test. All experiments were performed at least three times, and representative data are shown. Source data are available online for this figure.

Article Snippet: Human U937 myelomonocytic cells (ATCC Number: CRL‐1593.2) were grown in RPMI‐1640 medium (Hyclone).

Techniques: Transfection, Expressing, Derivative Assay, Western Blot, Knock-Out

Journal: Journal of Applied Clinical Medical Physics

Article Title: Surface dose measurements with commonly used detectors: a consistent thickness correction method

doi: 10.1120/jacmp.v16i5.5572

Figure Lengend Snippet: The experimental setup of surface dose measurement with extrapolation ion chamber PTW 30‐360.

Article Snippet: Surface doses measured with these detectors were 15.81 ± 2.08 (Attix) and 16.40 ± 3.01 (pTLD) in close agreement with the extrapolation chamber ( 15.93 ± 2.10 ).

Techniques:

Journal: Journal of Applied Clinical Medical Physics

Article Title: Surface dose measurements with commonly used detectors: a consistent thickness correction method

doi: 10.1120/jacmp.v16i5.5572

Figure Lengend Snippet: Percent surface dose measurements using different dosimeters. The dotted red line is the surface dose measured with the extrapolation chamber (PTW 30‐360); dotted black lines represent the SD in those measurements. Colored and red markers represent uncorrected and corrected surface doses, respectively, for each detector.

Article Snippet: Surface doses measured with these detectors were 15.81 ± 2.08 (Attix) and 16.40 ± 3.01 (pTLD) in close agreement with the extrapolation chamber ( 15.93 ± 2.10 ).

Techniques:

Journal: Journal of Applied Clinical Medical Physics

Article Title: Surface dose measurements with commonly used detectors: a consistent thickness correction method

doi: 10.1120/jacmp.v16i5.5572

Figure Lengend Snippet: Dosimeters used for estimation of surface dose

Article Snippet: Surface doses measured with these detectors were 15.81 ± 2.08 (Attix) and 16.40 ± 3.01 (pTLD) in close agreement with the extrapolation chamber ( 15.93 ± 2.10 ).

Techniques: In Vivo

Journal: The Journal of Clinical Investigation

Article Title: Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells

doi: 10.1172/JCI123955

Figure Lengend Snippet: (A) Four-hour cytotoxicity assays with NKG2A+ NK cells transduced with anti-NKG2A PEBL or GFP only (Control). Target cell lines were transduced with GpHLA-E (see Supplemental Figure 7) and luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Data are shown as mean (± SD) of cell killing using target cells cultured without NK cells as reference. NK cells from 11 donors were tested with K562, 6 with U2OS, 8 with ES8, and 5 with EW8, all in triplicate. (B) Expression of CD107a among NK cell subsets after 4-hour coculture with K562-GpHLA-E cells. Percentages are shown as mean of triplicate measurements with NK cells from 1 donor. (C) Four-hour cytotoxicity (measured as in A) of GFP-transduced NK cells against GpHLA-E–transduced target cells in the presence of the anti-NKG2A antibody Z199 compared with that of anti-NKG2A PEBL-transduced NK cells. An isotype-matched (mIgG2b) nonreactive immunoglobulin served as a control. Data are shown as mean (± SD) of triplicate measurements with NK cells from 2 donors (K562, ES8) or 1 donor (U2OS). (D) Long-term cytotoxicity of PEBL-transduced and control NK cells against GpHLA-E+ target cells. Experiments were performed at E/T 1:8 for K562, 1:2 for U2OS, and 1:4 for ES8. Tumor cell growth was measured with IncuCyte Zoom System. Data are shown as mean (± SD) of triplicate measurements with NK cells from 1 representative donor (out of 3 tested) compared with growth of the cell line without NK cells. (E) Spheroid tumors formed with U2OS-GpHLA-E cells transduced with mCherry were cocultured with PEBL-transduced or control NK cells at a 2:1 E/T. Images were collected with the IncuCyte Zoom System. Scale bars: 300 μm. Numerical data are shown in Supplemental Figure 9. *P < 0.05; **P < 0.01; ****P < 0.0001, t test.

Article Snippet: The human cell lines NK92, K562, SK-BR-3, PLC/PRF/5, U2OS, and U937 were obtained from ATCC.

Techniques: Transduction, Luciferase, Cell Culture, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells

doi: 10.1172/JCI123955

Figure Lengend Snippet: (A) Four-hour cytotoxicity of NK cells transduced with anti-NKG2A PEBL or GFP only (Control) against cell lines expressing endogenous HLA-E (see Supplemental Figure 10). EW8 and PLC/PRF/5 were transduced with luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Cytotoxicity of U937 and OP-1 was measured by flow cytometry. Box (25th–75th percentile, median) and whiskers (minimum-maximum) plots from 3 experiments with cells from 3 donors (EW8, PLC/PRF/5), and 6 experiments with cells from 2 donors (U937, OP-1) in triplicate, at 2:1, 1:1, or 1:2 E/T. (B) Spheroid tumors of U2OS-mCherry were cocultured with NK cells at 1:2 E/T in triplicate and analyzed with IncuCyte Zoom System. Data are shown as mean (± SD) red calibrated unit (RCU)/μM2. Representative images at end of culture are shown. Scale bars: 300 μm. (C) Four-hour cytotoxicity against cell lines exposed to IFN-γ (300 ng/ml; 12 hours). Plots are from 3 experiments with NK cells from 3 donors (EW8, PLC/PRF/5), and 6 with NK cells from 2 donors (U937) in triplicate at 2:1, 1:1, or 1:2 E/T. (D) Similar experiments targeting cells exposed for 12 hours to conditioned medium (C.M.) from 24-hour cocultures of NK cells with the respective cell lines. Four-hour cytotoxicity was compared with that against cells not exposed to conditioned medium. (E) Four-hour cytotoxicity against primary AML cells from 4 patients, exposed to IFN-γ (300 ng/ml; 12 hours). Data are from 4 experiments with NK cells from 2 donors in triplicate at 2:1 and 1:1 E/T. (F) NKG2A-negative NK cells from 3 donors were stimulated with K562-mb15-41BBL for 7 days, transduced with anti-NKG2A PEBL or GFP alone, and then exposed to IL-12 (20 ng/ml) for 5 days. Percentage of NKG2A+ cells at each stage is shown. (G) PEBL-transduced and control NK cells were exposed to IL-12 and tested in 4-hour cytotoxicity assays against K562-GpHLA-E cells. Data are shown as mean (± SD) of triplicate measurements at each E/T. **P < 0.01; ***P < 0.001; ****P < 0.0001, t test.

Article Snippet: The human cell lines NK92, K562, SK-BR-3, PLC/PRF/5, U2OS, and U937 were obtained from ATCC.

Techniques: Transduction, Expressing, Luciferase, Flow Cytometry

Journal: Nature immunology

Article Title: G3BP1 promotes DNA binding and activation of cGAS

doi: 10.1038/s41590-018-0262-4

Figure Lengend Snippet: a,b, qPCR analysis of indicated mRNA expression in U937 cells (a) or primary MEF cells (b) transfected with HT-DNA for indicated time. ***P < 0.001, two-tailed t-test. c, Representative immunofluorescent staining of cGAS (green), Cy3-labeled ISD (red) and G3BP1 (gray) in human primary macrophages. d, Immunofluorescent staining of cGAS (green) and G3BP1 (red) in human primary macrophages left untreated or treated with ISD (1 μg ml−1) or poly(I:C) (1 μg ml−1) for 2 h. e, Immunofluorescent staining of G3BP1 (red) in HeLa cells transfected with non-targeting control small interfering RNA (si-NC) or CGAS-specific small interfering RNA (si-cGAS), followed by stimulation with poly(I:C) (2 μg ml−1) for 2 h or arsenite (500 μM) for 1 h. f, Representative images of SG formation, indicated by G3BP1 (red), in arsenite-stimulated U937 cells that stably expressed non-targeting control short hairpin RNA (sh-NC) or TIA1-specific short hairpin RNA (sh-TIA1). g, Knockdown of TIA1 in (f) was verified by immunoblotting. α-Tubulin, loading control. h, qPCR analysis of IFNB mRNA expression in U937 transfected with HT-DNA (2 μg ml−1) for 2 h. Hoechst (blue) (c–f), nuclear stain. Scale bars (c–f), 10 μm. Data are representative of three experiments (a–d,f–h) or two experiments (e). Data are mean ± s.e.m. of triplicate samples in a,b,h.

Article Snippet: HeLa, U937 and HEK293T cells were obtained from ATCC.

Techniques: Expressing, Transfection, Two Tailed Test, Staining, Labeling, Small Interfering RNA, Stable Transfection, shRNA, Western Blot

Journal: Nature immunology

Article Title: G3BP1 promotes DNA binding and activation of cGAS

doi: 10.1038/s41590-018-0262-4

Figure Lengend Snippet: a, cGAS–G3BP1 interaction was analyzed by immunoprecipitation with IgG or cGAS antibody in human primary macrophages treated as indicated. b, Schematic drawing of the domains of G3BP1. c,d, The G3BP1–cGAS interaction was analyzed by immunoprecipitation with anti-flag M2 beads in HEK293T cells expressing HA-tagged cGAS and flag-tagged full-length and truncated G3BP1. e, qPCR analysis of IFNB mRNA expression in HT-DNA-treated WT and the indicated rescued cells (U937) (top). The expression of rescued proteins was analyzed by immunoblotting (bottom). f, qPCR analysis of Ifnb mRNA expression in HT-DNA-treated WT and the indicated rescued cells (MEF) (top). Immunoblot analysis of the rescued proteins are shown (bottom). g, Schematic drawing of the domains of cGAS (top). Plasmids of flag-tagged human cGAS as indicated were transiently expressed in HeLa cells. The G3BP1–cGAS interaction was analyzed by immunoprecipitation with anti-flag M2 beads (bottom). h, The DNA-binding capacity of the proteins was analyzed by biotin–ISD pull-down in cell lysates of HEK293T that expressed with flag-tagged cGAS and flag-cGAS-C. WCL, whole cell lysates. IP, immunoprecipitation. β-Actin (e,f), GAPDH (h) and α-Tubulin (a,c,d,g), loading controls. α-Tubulin blots in IP samples indicate the purity of IP (a,c,d,g). *P < 0.05, ***P < 0.001, two-tailed t-test (e,f). All data are representative of three experiments, mean ± s.e.m. of triplicate samples in e,f.

Article Snippet: HeLa, U937 and HEK293T cells were obtained from ATCC.

Techniques: Immunoprecipitation, Expressing, Western Blot, Binding Assay, Two Tailed Test