Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca 2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse / rat / human Substance P (rabbit polyclonal), Cy3 , Bioss USA , Cat # bs-0064R-Cy3.

Techniques: Functional Assay, Recombinant, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Inhibition, Fluorescence, SYBR Green Assay, Activation Assay, Bicinchoninic Acid Protein Assay, In Situ, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Software

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1α expression precedes IL-1β after ischemic brain injury and is localised to areas of focal neuronal loss and penumbral tissues

doi: 10.1186/1742-2094-8-186

Figure Lengend Snippet: IL-1α is expressed by microglia localized to focal neuronal and BBB injury 24 h after MCAo . Images are coronal sections from brains of C57BL6/H and CX3CR1-GFP +/- mice 60 min MCAo and 24 h reperfusion. Widefield images show IL-1α-expressing (red), GFP positive (green) microglia in ipsilateral (Ai), not contralateral (ii) amygdala 24 h after MCAo in a CX3CR1-GFP +/- mouse. IL-1α immunohistochemistry with cresyl violet co-staining localises IL-1α expressing microglia to the peri-infarct zone in thalamus (Bi) and cortex (Bii) of a C57BL6/H mouse. Focal IgG staining (red) co-localized with IL-1α positive microglia (green) in the ipsilateral cortex of a C57BL6/H mouse (Ci). No IgG or IL-1α staining detected in the contralateral cortex (Cii). IL-1α positive microglia detected in larger areas of IgG staining in the ipsilateral (Ciii), but not contralateral (Civ) hemisphere. Co-localization of IL-1α positive microglia (red) with areas of neuronal loss (blue) in a C57BL6/H mouse (D). Occasional IL-1α positive microglia also found in areas where neurons were morphologically intact (D, inset). Confocal images (E) are maximum Z projections (Ei, iii) and confocal slices at the level of the nucleus (Eii, iv) of IL-1α expressing, GFP positive microglia in a CX3CR1-GFP +/- mouse. Cells with (Ei, ii), and without (Eiii, iv) nuclear IL-1α. Nuclear fluorescence intensities for IL-1α and GFP were quantified from confocal images, and the fold enrichment of IL-1α and GFP in microglial nuclei was calculated in comparison to whole cell fluorescence (F). All images are representative of n ≥ 3 mice. Quantification is of n = 4 CX3CR1-GFP +/- mouse brains, with each data point representing an individual cell, n ≥ 30 cells per brain.

Article Snippet: Staining of coronal brain sections from C57BL6/H mice (Figure ) and CX3CR1-GFP+/- mice (not shown) 24 h after MCAo for IL-1α and IgG (BA-2000, Vector Labs, biotinylated horse anti-mouse IgG, 2 μg/mL; S-32356, Invitrogen, Alexa 594 conjugated streptavidin, 5 μg/mL) revealed that IL-1α expressing cells co-localized to areas of focal BBB damage, mainly near the penumbral regions of the ipsilateral hemisphere (Figure ).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence

Journal: Cells

Article Title: Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications

doi: 10.3390/cells12030354

Figure Lengend Snippet: Correlative Confocal-dSTORM Analysis on Image-Cytometry re-localized cells. ( a ) Representative re-localized G1 cells acquired by the correlative Confocal-STORM protocol described in the text. Confocal Z-Stacks were first acquired of the Tubulin-Atto425 and γH2A.X-Alexa488 fluorescence channel, followed by acquisition of the 53BP1-Cy3 and DynLL1-Alexa Fluor647 channels in dSTORM. The dSTORM plane is identified in the Z-Stack, aligned and merged in the plane (merged image in the second row on the left) and then inserted into the Z-stack (3D projection on the right). ( b ) The described correlative microscopy procedure was applied to cells in the different stages of mitosis, isolated according to the image-cytometry analysis. Z-stacks merged with the dSTORM plane of some representative cells are shown. Scale bar: 5 µm.

Article Snippet: Cells were then rinsed 3 times in PBS and incubated for 1 h with donkey anti-Rat IgG (H + L) biotin (a18749, ThermoFisher Scientific, Waltham, MA, USA) and for 45 min at room temperature with streptavidin Atto425 conjugated (S000-51, Rockland Immunochemicals, Pottstown, PA, USA).

Techniques: Cytometry, Fluorescence, Microscopy, Isolation

Journal: Science Advances

Article Title: Promoting the activation of T cells with glycopolymer-modified dendritic cells by enhancing cell interactions

doi: 10.1126/sciadv.abb6595

Figure Lengend Snippet: ( A ) Schematic of HTP transfection with a photo perforation transfection system (PTS) was used. ( B ) Representative images showing HTP transfection. Nuclei were stained by DAPI (blue), and the HA-tagged HTP was stained by FITC-avidin (green). Scale bar, 100 um. ( C ) Quantification of transfection efficiency compared with Lip2000. *** P < 0.001 compared with Lip2000. Data are means ± SEM ( n = 3). ( D ) Schematic of DC modification with glycopolymers. ( E ) Representative images showing green fluorescence on the DC cell surface. Nuclei were stained by DAPI (blue) and biotin-labeled poly-(MAG) (pMB) by FITC-avidin (green). ( F ) Representative images showing the modified DCs incubated in complete medium for specified times (1, 3, and 7 days). ( G ) Viability of engineered DC over the 7-day period. Data are means ± SEM ( n = 3). N.D., not determined.

Article Snippet: Primary antibody hemagglutinin (HA) and FITC-conjugated secondary antibody were from Wuhan Boster Biological Technology Ltd. (Wuhan, China).

Techniques: Transfection, Staining, Avidin-Biotin Assay, Modification, Fluorescence, Labeling, Incubation

Journal: Science Advances

Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

doi: 10.1126/sciadv.1600760

Figure Lengend Snippet: ( A ) BSP binds to the BRD acetyl-lysine cavity, allowing for further functionalization toward the front channel within the ZA loop (Ra vector annotated in orange) or the back of the pocket (Rb vector annotated in orange). The vectors are shown in the complex of BSP with BRD4(1). ( B ) Two variants of biotinylated BSP (BSP-a and BSP-b) were prepared to explore binding to human BRDs in cells by pull-down experiments. ( C ) Biotinylated BSP (BSP-a or BSP-b) immobilized on magnetic beads was used to pull down human CECR2 from Flp-In T-REx HEK293 cells stably expressing 3×FLAG CECR2. The protein captured from whole-cell lysate was identified using anti-FLAG. ( D ) Cell lysate from HEK293T cells was incubated with biotinylated BSP (BSP-a or BSP-b) immobilized on magnetic streptavidin beads in the presence or absence of 30 nmol of BSP for 2 hours at 4°C. After pull-down and tryptic digestion with trypsin, proteins were identified in a TripleTOF 5600 mass spectrometer. (Top) Normalized abundance of each BRD-containing protein in HEK293 cells (data from Proteomics DB; https://www.proteomicsdb.org/ ). (Bottom) Ratio of peptide to peptide abundance in the presence and absence of competing BSP, shown as a bar graph. BRD families are annotated with roman numerals. ( E ) FRAP evaluation of full-length GFP-tagged BRD4 dissociation from chromatin in U2OS cells. Nuclei of DMSO-treated (top) or BSP-treated (1 μM; bottom) cells. Target regions of photobleaching are indicated with a white circle. Scale bar, 10 μm. FL-BRD4, full-length BRD4; FL-BRD9, full-length BRD9. ( F ) Quantitative comparison of time to half-maximal fluorescence recovery for BRD4 FRAP studies using BSP (red bars) as a function of ligand concentration. ( G ) FRAP evaluation of full-length GFP-tagged BRD9 dissociation from chromatin in U2OS cells. Nuclei of DMSO-treated (top) or BSP-treated (1 μM; bottom) cells in the presence of 10 μM SAHA (added to increase the experimental window). Target regions of photobleaching are indicated with a white circle. Scale bars, 10 μm. ( H ) Quantitative comparison of time to half-maximal fluorescence recovery for BRD9 FRAP studies using BSP (red bars) as a function of ligand concentration. Data in (F) and (H) represent means ± SEM ( n = 30) and are annotated with P values obtained from a two-tailed t test (* P < 0.05 and *** P < 0.001).

Article Snippet: Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates.

Techniques: Plasmid Preparation, Binding Assay, Magnetic Beads, Stable Transfection, Expressing, Incubation, Mass Spectrometry, Comparison, Fluorescence, Concentration Assay, Two Tailed Test

Journal: Science Advances

Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

doi: 10.1126/sciadv.1600760

Figure Lengend Snippet: Δ T m shifts (°C) of biotinylated BSP (BSP-a and BSP-b) tested against a panel of BET and other diverse BRDs. Compounds (final concentration, 10 μM) were added to the proteins (final concentration, 2 μM); the temperature was increased from 25° to 96°C at a step of 3°C/min; excitation and emission filters for the SYPRO Orange dye were set to 465 and 590 nm; and experiments were performed in triplicate. Values are means ± SD.

Article Snippet: Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates.

Techniques: Concentration Assay

Journal: Science Advances

Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

doi: 10.1126/sciadv.1600760

Figure Lengend Snippet: Relative abundance of BRD-containing proteins in HEK293 cells (data taken from Proteomics DB; https://www.proteomicsdb.org/ ). Pull-down of human BRD-containing proteins with biotinylated BSP (BSP-a and BSP-b), followed by competitive elution with BSP and MS, resulted in enrichment of BSP-targeted BRDs.

Article Snippet: Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Intracellular Accumulation of Amyloid-β (Aβ) Protein Plays a Major Role in Aβ-Induced Alterations of Glutamatergic Synaptic Transmission and Plasticity

doi: 10.1523/JNEUROSCI.1201-14.2014

Figure Lengend Snippet: Twenty minute injection of Aβ42 does not significantly affect dendritic spine density of CA1 hippocampal neurons. A, B, Representative examples of CA1 neurons filled with biocytin and revealed with avidin conjugated to Alexa Fluor 488. Neuron shown in A was injected with vehicle, whereas neuron shown in B was injected with 200 nm Aβ42 for 20 min. Bottom boxes in A and B show high-magnification images of dendritic segments of cells in A and B, respectively. Scale bar, 3 μm. Alexa Fluor 488 fluorescence intensity (8-bit depth) was represented according to the color scale on the right (bottom = 0, top = 256). C, Bar graphs showing the mean number of dendritic spines per 100 μm. n.s.: p > 0.05.

Article Snippet: Subsequently, biocytin was revealed by incubating slices with Alexa Fluor 488-conjugated avidin (1:500 in blocking solution; Life Technologies) for 90 min at RT.

Techniques: Injection, Avidin-Biotin Assay, Fluorescence

Journal: Bioconjugate chemistry

Article Title: Multifunctional α v β 6 Integrin-Specific Peptide–Pt(IV) Conjugates for Cancer Cell Targeting

doi: 10.1021/acs.bioconjchem.7b00421

Figure Lengend Snippet: (a) Integrin β6 expression of SW480 cells and transfected cells SW480 ITGB6 low (before sorting) and SW480 ITGB6 high (after sorting) given as fold fluorescence intensity relative to autofluorescence after binding of an antihuman integrin β6-allophycocyanin antibody and measured by flow cytometry in at least three independent experiments. (b) Integrin β6 and αv expression of SW480, SW480 ITGB6 high, and endogenously ITGB6-expressing A431 cells measured of total protein lysates by Western blot. β-actin levels were used as a loading control. Integrin β6 (MW 97 kDa) and integrin αv (MW 135−140 kDa). Full gels in Figure S2. (c) Binding of biotinylated Y-1 and nontargeting scrambled version biotinylated Y-sc1 (4 nM) to SW480 ITBG6 high compared to non-expressing SW480 cells. Fluorescence of peptide-binding avidin−FITC was measured by flow cytometry and normalized to samples without biotin-labeled peptide. Data is shown as the ratio between SW480 ITGB6 high compared to non-expressing SW480 cells, measured in three independent experiments. (d) Quantification of fluorescence intensity per cell area of confocal microscopy images of SW480 and SW480 ITGB6 high cells incubated with no peptide, 1 μM biotinylated Y-1, 1 μM biotinylated Y-mono-1, or 1 μM biotinylated Y-sc1. Before imaging, cells were fixed, permeabilized, and stained using avidin−FITC (1:200). At least 30 cells of each condition were quantified using ImageJ. (e, f) Confocal microscopy images representing (e) living SW480 and (f) SW480 ITGB6 high cells exposed to 0.5 μM Cy5−Y-1 for 10 min at 37 °C (scale bar: 50 μm). Values given in panels a, c, and d are the mean ± standard deviation. Significance was established using one-way ANOVA with Bonferroni’s multiple comparison test (***, p ≤ 0.001; **, p ≤ 0.01; and *, p ≤ 0.05).

Article Snippet: For further selection of highly integrin β 6 -expressing cells, 10 6 transfected cells were sorted by fluorescence-activated cell sorting (FACS) for the highest integrin β 6 expression using a commercially available integrin β 6 antibody conjugated to allophycocyanin (APC, R&D Systems Antihuman Integrin β 6 -Allophycoycanin, no. FAB4155A).

Techniques: Expressing, Transfection, Fluorescence, Binding Assay, Flow Cytometry, Western Blot, Avidin-Biotin Assay, Labeling, Confocal Microscopy, Incubation, Imaging, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: Paired crRNA library screening identified that SHROOM1 is an HDR suppressor of dual-cut BFP (blue fluorescence protein) reporter. ( a ) Schematic of paired crRNA library screening strategy in the main text; ( b ) multiple genes were enriched in the HDR population. Representative genes are highlighted in red. Data were generated from n = 4 independent experiments. The p -value was calculated using a two-sided Student’s t -test. ( c ) Relative HDR ratio of dual-cut BFP reporter cells treated with individual siRNA of top eight genes enriched in the screening. Data were generated from n = 3 independent experiments. Error bars, ± SD * p < 0.05; ** p < 0.01; *** p < 0.001 by two-sided Student’s t -test. ( d ) Relative HDR ratio-time curve of dual-cut BFP reporter cell lines expressing mono-paired crRNAs. Data were collected every two days from day 20 to day 36 after cell sorting. Data were generated from n = 3 independent experiments. ( e ) Genes enriched in the HDR population of simulated screening. Data were generated from n = 4 independent experiments. Error bars, ±SD * p < 0.05; ** p < 0.01; *** p < 0.001 by two-sided Student’s t -test.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: Library Screening, Fluorescence, Generated, Expressing, FACS

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: Knockdown of SHROOM1 enhances the knock-in efficiency of cells in vitro. ( a ) Schematic overview of gene-targeting strategies with siRNA and different types of donor at the FBL locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; IF/IR, inserted forward/reverse primer. ss, single strand; ds, double strand; dc, double cut. ( b ) Experimental scheme for targeted FBL -2A-GFP knock-in in HEK293T cells. Representative visual fields and sorting charts ( c ) and relative knock-in efficiency. Scale bar, 200 μm. ( d ) of ss, ds, and dc donor-based strategies with SHROOM1 siRNA or not at the FBL locus in HEK293T cells. Data were generated from n = 3 independent experiments. Error bars, ± SD ** p < 0.01; *** p < 0.001 by two-sided Student’s t -test ( e , f ). Relative knock-in efficiency of ds and dc donor-based strategies with siRNA in HEK293T, HCT116, or Hepa1-6 cells. Data were generated from n = 3 independent experiments. Error bars, ± SD * p < 0.05; ** p < 0.01 by two-sided Student’s t -test.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: Knock-In, In Vitro, Generated

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: Knockdown of SHROOM1 enhances the knock-in efficiency of mouse embryos. ( a ) Experimental design of micro-injection. Cas9-Avidin mRNA, sgRNA, biotin-ss donor, and siRNA were injected into mouse zygotes and the injected zygotes were transferred to pseudo-pregnant mice for genotyping analysis. Genotyping of Ddx4 locus ( b ) and Icos locus ( c ) in mice treated with SHROOM1 siRNA or NC siRNA after incision by CRISPR/Cas9; ( d ) Summary of SHROOM1 siRNA-mediated HDR at the Ddx4 and Icos loci. Results with significant differences are highlighted in red.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: Knock-In, Injection, Avidin-Biotin Assay, CRISPR

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: SHROOM1 is a potent suppressor of HDR progress. ( a ) Schematic of SHROOM1 deletion using CRISPR/Cas9 and two sgRNAs in HEK293T cells. PAM, highlighted in red; protein codon, highlighted in blue or green, KO for knockout; ( b ) Western blot of different types of cells or treatment. KO, for knockout; p SHROOM1 for SHROOM1 cDNA contained plasmid; relative knock-in efficiency ( c ) and representative visual fields and sorting charts ( d ) in SHROOM1 knockout or wild-type HEK293T cells with treatments. YU238259, an HR inhibitor; Scr7, a NHEJ inhibitor; dc, double-cut sites contained donor. Data were generated from n = 3 independent experiments. Error bars, ± SD *** p < 0.001; n.s., no significance; by two-sided Student’s t -test. Scale bar, 200 μm.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: CRISPR, Knock-Out, Western Blot, Plasmid Preparation, Knock-In, Generated