cd13 pe  (Becton Dickinson)

Journal: MedComm

Article Title: Single‐cell transcriptomic analysis of glioblastoma reveals pericytes contributing to the blood–brain–tumor barrier and tumor progression

doi: 10.1002/mco2.70014

Figure Lengend Snippet: Expression of pericyte markers in previous studies of stromal cell sub clusters.

Article Snippet: The following primary antibodies were used: rabbit monoclonal antibody [EPR25414‐249] against ATP1A3 + alpha 1 sodium potassium ATPase + ATP1A2 + ATP1A4 (Abcam; ab300507, 1:100 dilution); anti‐GATA1(Ab‐142) antibody (Signalway Antibody; 21041–2, 1:100 dilution); anti‐PTHR1 antibody (Affinity; DF6589, 1:100 dilution); mouse monoclonal [JC/70A] anti‐CD31 antibody (Abcam; ab9498, 1:200 dilution); rabbit polyclonal anticollagen‐IV antibody (Abcam; ab6586, 1:100 dilution); rabbit polyclonal antifibronectin antibody (Abcam; ab2413, 1:100 dilution); rabbit polyclonal anti‐CD13 antibody (Servicebio; GB11356‐100, 1:100 dilution); and recombinant antidesmin antibody (Servicebio; GB15075‐100, 1:100 dilution).

Techniques: Expressing

Journal: bioRxiv

Article Title: NuMA mechanically reinforces the spindle independently of its partner dynein

doi: 10.1101/2024.11.29.622360

Figure Lengend Snippet: (A) Schematic diagram of NuMA and its functional domains, and point mutations used for the NuMA-SpM (from Okumura et al., 2018) and NuMA-HCC mutants (combined individual mutations from Renna et al., 2020) designed to test dynein-independent NuMA functions. (B) Representative confocal images of immunofluorescence staining of WT RPE1 cells and RPE1 cells expressing NuMA-SpM-GFP, NuMA-HCC-GFP, and NuMA-FL-GFP. Cells were stained for GFP (overexpressed NuMA), total NuMA, and dynactin (p150). (C-D) Quantification of total NuMA intensity at poles (C) and ratio of pole dynein/spindle dynein intensities (D), calculated from immunofluorescence images (example of “pole” and “spindle” selections shown on (B)). Values were normalized to mean value for WT RPE1 (control) cells. Data in (C-D) from 2 independent experiments, n=28, 23, 22, and 20 cells. *p < 0.05; **p < 0.005; n.s., not significant; two-sample t-test. Mean ± S.D. (E) Schematic and representative confocal images of immunofluorescence staining of RPE1 NuMA-KO cells as examples of turbulent and bipolar spindle architectures. (F) Percentage of RPE1 NuMA-KO spindles that remain turbulent with exogenous expression of NuMA-FL-GFP, NuMA-SpM-GFP, NuMA-HCC-GFP, or nothing (control). Data from at least 2 independent experiments, n=55, 16, 11, and 12 cells. **p < 0.005; n.s., not significant; Fisher’s Exact Test.

Article Snippet: The following primary antibodies were used: rabbit anti-NuMA (1:300, NB500-174, RRID: AB_10002562; Novus Biologicals), mouse anti-p150 [Glued] (1:400, 610474, RRID: AB_397846; BD Biosciences), mouse anti-α-tubulin (1:1000, T6199, RRID: AB_477583; Sigma-Aldrich).

Techniques: Functional Assay, Immunofluorescence, Staining, Expressing, Control

Journal: bioRxiv

Article Title: NuMA mechanically reinforces the spindle independently of its partner dynein

doi: 10.1101/2024.11.29.622360

Figure Lengend Snippet: (A) Schematic diagram of NuMA and its functional domains, and point mutations used for the NuMA-SpM (from Okumura et al., 2018) and NuMA-HCC mutants (combined individual mutations from Renna et al., 2020) designed to test dynein-independent NuMA functions. (B) Representative confocal images of immunofluorescence staining of WT RPE1 cells and RPE1 cells expressing NuMA-SpM-GFP, NuMA-HCC-GFP, and NuMA-FL-GFP. Cells were stained for GFP (overexpressed NuMA), total NuMA, and dynactin (p150). (C-D) Quantification of total NuMA intensity at poles (C) and ratio of pole dynein/spindle dynein intensities (D), calculated from immunofluorescence images (example of “pole” and “spindle” selections shown on (B)). Values were normalized to mean value for WT RPE1 (control) cells. Data in (C-D) from 2 independent experiments, n=28, 23, 22, and 20 cells. *p < 0.05; **p < 0.005; n.s., not significant; two-sample t-test. Mean ± S.D. (E) Schematic and representative confocal images of immunofluorescence staining of RPE1 NuMA-KO cells as examples of turbulent and bipolar spindle architectures. (F) Percentage of RPE1 NuMA-KO spindles that remain turbulent with exogenous expression of NuMA-FL-GFP, NuMA-SpM-GFP, NuMA-HCC-GFP, or nothing (control). Data from at least 2 independent experiments, n=55, 16, 11, and 12 cells. **p < 0.005; n.s., not significant; Fisher’s Exact Test.

Article Snippet: The following primary antibodies were used: rabbit anti-NuMA (1:300, NB500-174, RRID: AB_10002562; Novus Biologicals), mouse anti-p150 [Glued] (1:400, 610474, RRID: AB_397846; BD Biosciences), mouse anti-α-tubulin (1:1000, T6199, RRID: AB_477583; Sigma-Aldrich).

Techniques: Functional Assay, Immunofluorescence, Staining, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression

doi: 10.1093/nar/gkae921

Figure Lengend Snippet: The S protein of mutant PEDV inhibits PKR phosphorylation and SG formation by upregulating ADAR1-p150 expression, and the Zα domain plays a crucial role in exerting inhibitory effects. Vero cells were transfected with 12-S or 12S-N29T. At 24 h post-transfection, the expression levels of PACT, ADAR1, PEDV-S and β-actin were detected ( A ). The transfected cells were also fixed and incubated with anti-PEDV-S and anti-ADAR1-p150/110 or anti-ADAR1-p150 ( B and D ), and the mean fluorescence values of ADAR1 per cell were determined in 20 random cells ( C and E ). Vero cells were transfected with the recombinant plasmid pADAR1-p150. At 18 h post-transfection, the cells were transfected with poly I:C for 6 h, and then the expression levels of ADAR1-p150, PKR, pPKR and β-actin were detected ( F ). The cells were also fixed and incubated with an anti-His antibody and an anti-G3BP1 antibody ( G ). The percentages of inhibition of SG formation relative to ADAR1-p150 expressing cells were displayed in the bar graph ( H ). ( I ) A schematic diagram of the structural composition of ADAR1-p150 and the constructed mutants is shown. Vero cells were transfected with mutant ADAR1-p150 plasmids. At 18 h post-transfection, the cells were transfected with poly I:C for 6 h, then fixed and incubated with anti-His and anti-G3BP1 antibodies ( J ). The percentages of inhibition of SG formation relative to mutant ADAR1-p150 expressing cells were displayed in the bar graph ( K ). Vero cells were transfected with siADAR1 or siNC for 24 h, then all cells were transfected with 12-S. At another 18 h post-transfection, cells were transfected with poly I:C for 6 h, then the expression of ADAR1-p150 was detected by western blot ( L ). Fixed cells were incubated with anti-PEDV-S and anti-G3BP1 antibodies ( M ). The percentages of inhibition of SG formation relative to 12-S expressing cells were displayed in the bar graph ( N ). For (A) and (F), the figures are representative of two independent experiments. For (B), (D), (G), (J) and (M), the images are representative of three independent experiments. The relative expression levels of target proteins according to the grayscale of β-actin in cells were also determined by ImageJ. The bar graphs of (H), (K) and (N) showed the data of three independent experiments, presented as mean ± SD. Statistics: Student’s t -test (*** P < 0.001). Scale bars: 10 μm.

Article Snippet: Rabbit monoclonal anti-ADAR1-p150 (A11466), anti-general control nonderepressible-2 (anti-GCN2) (A2307), anti-phospho-GCN2-T899 (AP1356) and anti-heme-regulated inhibitor (anti-HRI) (A24614) antibodies were purchased from ABclonal, Inc. (Wuhan, China).

Techniques: Mutagenesis, Expressing, Transfection, Incubation, Fluorescence, Recombinant, Plasmid Preparation, Inhibition, Construct, Western Blot

Journal: Nucleic Acids Research

Article Title: Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression

doi: 10.1093/nar/gkae921

Figure Lengend Snippet: The ADAR1-p150 induced by S protein inhibits the accumulation of dsRNA, with its deaminase activity playing a pivotal role in this process. Vero cells were transfected with recombinant plasmids 12-S or 12S-N29T. At 24 h post-transfection, cells were infected with JS2008. At 12 h post-infection, cells were fixed and incubated with anti-PEDV-S antibody and anti-dsRNA antibodies ( A ), and the mean fluorescence values of dsRNA per cell were determined in 20 random cells ( B ). Vero cells were transfected with mutant ADAR1-p150 plasmids. At 24 h post-transfection, cells were infected with JS2008. At 16 h post-infection, cells were fixed and incubated with anti-His antibody and anti-dsRNA antibodies ( C ), and the mean fluorescence values of dsRNA per cell were determined in 20 random cells ( D) . Vero cells were transfected with mutant 12-S plasmid. At 24 h post-transfection, cells were infected with JS2008 and simultaneously treated with 8-Azaad or DMSO. At 16 h post-infection, cells were fixed and incubated with anti-PEDV-S antibody and anti-dsRNA antibodies ( E ), and the mean fluorescence values of dsRNA per cell were determined in 20 random cells ( F ). For (A), (C) and (E), the images are representative of three independent experiments. Data are shown as the mean ± SD. Statistics: Student’s t -test (*** P < 0.001). Scale bars: 10 μm.

Article Snippet: Rabbit monoclonal anti-ADAR1-p150 (A11466), anti-general control nonderepressible-2 (anti-GCN2) (A2307), anti-phospho-GCN2-T899 (AP1356) and anti-heme-regulated inhibitor (anti-HRI) (A24614) antibodies were purchased from ABclonal, Inc. (Wuhan, China).

Techniques: Activity Assay, Transfection, Recombinant, Infection, Incubation, Fluorescence, Mutagenesis, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression

doi: 10.1093/nar/gkae921

Figure Lengend Snippet: The S-N29T mutant strain loses ADAR1-p150 induction and produces more dsRNA and SGs. Construction of recombinant PEDV strain with the 29th amino acid mutation. ( A ) Schematic diagram of the structural composition of recombinant virus rAH2012/12 and r12S-T29N. Vero cells were infected with the mutants, and the specific fluorescence of N protein was detected using IFs at 24 hpi. The plaques of cells infected with the viruses were observed at 3 days post infection ( B ). The growth curves of viruses in Vero cells were determined by measuring the virus titers at different time points after viral infection ( C ), and the dot graph showed the data of three independent determinations, presented as the mean ± SD. Vero cells were infected with rAH2012/12 or r12-N29T at 0.1 MOI for 12 and 24 h. The infected cells were detected for the expression levels of ADAR1, PKR, p-PKR, eIF2α, p-eIF2α, PEDV-N and β-actin ( D ). Figures are representative of two independent experiments. The relative expression levels of target proteins ADAR1, p-PKR and p-eIF2α were determined by measuring grayscale of β-actin in cells using ImageJ. The cells infected for 12 h were immunostained with ADAR1-p150 and PEDV-S ( E ), and the mean fluorescence values of ADAR1-p150 per cell were determined in 20 random cells ( F ). The cells infected for 12 and 24 h were immunostained with anti-dsRNA and anti-G3BP1 antibodies ( G ), and the mean fluorescence values of dsRNA per cell were determined in 20 random cells ( H ). The cells infected for 24 h were immunostained with anti-G3BP1 and anti-PEDV-S antibodies ( I ). The percentages of SGs-positive cells relative to infected cells of three independent experiments were displayed in bar graphs ( J ). For (E), (G) and (I), the images are representative of three independent experiments. Data are shown as the mean ± SD. Statistics: Student’s t -test (** P < 0.01, *** P < 0.001). Scale bars: 10 μm.

Article Snippet: Rabbit monoclonal anti-ADAR1-p150 (A11466), anti-general control nonderepressible-2 (anti-GCN2) (A2307), anti-phospho-GCN2-T899 (AP1356) and anti-heme-regulated inhibitor (anti-HRI) (A24614) antibodies were purchased from ABclonal, Inc. (Wuhan, China).

Techniques: Mutagenesis, Recombinant, Virus, Infection, Fluorescence, Expressing

Journal: Nucleic Acids Research

Article Title: Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression

doi: 10.1093/nar/gkae921

Figure Lengend Snippet: The 25PPA27 deletion of SARS-CoV-2 S protein loses ADAR1-p150 induction. Amino acid sequence alignment of the N-terminals of SARS-CoV-2 early and recent strains ( A ). Vero cells were transfected with plasmids expressing wt-NC-S or NC-S/dPPA. At 18 h post-transfection, cells were transfected with poly I:C and immunostained with anti-G3BP1 and anti-SARS-CoV-2-S antibodies ( B ). The percentages of SGs-positive cells relative to transfected cells of three independent experiments were displayed in bar graphs ( C ). Vero cells transfected with wt-NC-S or NC-S/dPPA were analyzed for the expression levels of ADAR1-p150, SARS-CoV-2-S and β-actin using corresponding antibodies at 24 h post-transfection ( D ). The relative expression levels of target proteins ADAR1-p150 according to the grayscale of β-actin in cells were also determined using ImageJ. The transfected cells were also fixed, incubated with anti-SARS-CoV-2-S and anti-ADAR1-p150 antibodies ( E ), and the mean fluorescence values of ADAR1-p150 per cell were determined in 20 random cells ( F ). Vero cells were transfected with wt-NC-S or NC-S/dPPA. At 24 h post-transfection, cells were infected with JS2008. At 12 h post-infection, cells were fixed and incubated with anti-PEDV-S antibody and anti-dsRNA antibodies ( G ), and the mean fluorescence values of dsRNA per cell were determined in 20 random cells ( H ). For (B), (E) and (G), the images are representative of three independent experiments. Data are shown as the mean ± SD. Statistics: Student’s t -test (** P <0.01, *** P <0.001). Scale bars: 10 μm.

Article Snippet: Rabbit monoclonal anti-ADAR1-p150 (A11466), anti-general control nonderepressible-2 (anti-GCN2) (A2307), anti-phospho-GCN2-T899 (AP1356) and anti-heme-regulated inhibitor (anti-HRI) (A24614) antibodies were purchased from ABclonal, Inc. (Wuhan, China).

Techniques: Sequencing, Transfection, Expressing, Incubation, Fluorescence, Infection

Journal: Nucleic Acids Research

Article Title: Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression

doi: 10.1093/nar/gkae921

Figure Lengend Snippet: Coronavirus S transcriptionally promotes ADAR1-p150 expression via TCF7L2. The mRNA levels of ADAR1-p150 were detected in Vero cells infected with rAH2012/12 or mutant strain r12S-N29T ( A ) and in cells transfected with the S and its mutants ( B ). The protein levels of ADAR1-p150 were detected in Vero cells transfected with different doses of 12-S or wt-NC-S plasmids ( C ). Figures are representative of two independent experiments. The mRNA levels of IFN-β were detected in Marc-145 cells transfected with the S and its mutants ( D ). The ADAR1-p150 promoter activities were assessed in Vero cells transfected with 12-S ( E ) or wt-NC-S ( F ) plasmids and a series of truncated ADAR1-p150 promoter reporter plasmids. The mRNA levels of TCF7L2, E3F4 and FOXH1 were detected in Vero cells transfected the PEDV ( G ) or SARS-CoV-2 ( H ) S and its mutants. The ADAR1-p150 promoter activities were assessed in Vero cells transfected with 12-S or wt-NC-S plasmids and ADAR1-p150 promoter pGLC-500 or the promoter pGLC-500 (ΔTCF7) with the deletion of the TCF7L2-binding motif plasmids ( I ). Vero cells were pretreated with siTCF7L2, co-transfected with 12-S or wt-NC-S and ADAR1-p150 promoter reporter plasmids, and incubated for 36 h. The ADAR1-p150 promoter activity was then assessed ( J ). The bar graphs showed the data of three independent experiments, presented as mean ± SD. Statistics: Student’s t -test or two-way ANOVA multiple comparisons tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Rabbit monoclonal anti-ADAR1-p150 (A11466), anti-general control nonderepressible-2 (anti-GCN2) (A2307), anti-phospho-GCN2-T899 (AP1356) and anti-heme-regulated inhibitor (anti-HRI) (A24614) antibodies were purchased from ABclonal, Inc. (Wuhan, China).

Techniques: Expressing, Infection, Mutagenesis, Transfection, Binding Assay, Incubation, Activity Assay