pljm1 flag gfp osbp  (Addgene inc)

Journal: Molecular Medicine

Article Title: The SARS-CoV-2 nucleocapsid protein induces microglia senescence-mediated cognitive impairment via Glycolysis

doi: 10.1186/s10020-025-01410-3

Figure Lengend Snippet: SARS-CoV-2 N protein drives the senescence of BV2 microglial cells by triggering mitochondrial dysfunction. BV2 microglial cells were treated with Mdivi-1 (100 nM) 30 min before Codon-optimized pLJM1-SARS-CoV-2 N-FLAG transfection. A Representative image of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar = 20 μm). B Mitochondrial morphology stained with TOM20 in BV2 cells (bar = 2 μm). C - D The expression levels of p-DRP1 and DRP1 proteins in BV2 microglial cells detected by Western blot ( n = 3). Bar graph data represent the mean ± SD of the target protein intensity (normalized to DRP1) in the experimental group relative to that in the control group. E Representative images of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar = 20 μm). F The expression levels of p19 , p21 , p53 , Il-1β , and Tnf-α mRNA in BV2 microglial cells detected by real-time PCR ( n = 3). G SA-β-gal staining was performed after transfection of Codon-optimized pLJM1-SARS-CoV-2 N-FLAG and treatment with Mdivi-1 for 36 h (bar = 10 μm). H The fluorescence intensity of Ki67 (green) was detected by immunofluorescence (bar = 50 μm). I - J The expression levels of p-DRP1, p53, p21, p16, and γ-H2AX proteins in BV2 microglial cells were detected by Western blot ( n = 3). Bar graph data represent the mean ± SD of the target protein intensity (normalized to α-tubulin) in the experimental group relative to that in the control group. Comparisons between the two groups were made with an unpaired t -test. Differences among multiple groups were performed using ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Codon-optimized pLJM1-SARS-CoV-2 N-FLAG was cloned from SinoBiological (cat# VG40588-NF) using BamHI and EcoRI.

Techniques: Transfection, Membrane, Staining, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence, Immunofluorescence

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 5 IL4 induces RRAGD expression and combined IL4 treatment and BCR crosslinking hyperactivates mTOR signaling. A–C Lymphoma cell line pools transduced with lentiviral CRISPR-Cas9 guide pools targeting AAVS (control) or RRAGD, were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. D Non-malignant normal human LN derived B cells were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. E, F Non-malignant normal human LN derived B cells (N = 4) were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were left untreated or treated ex vivo with IL4, anti-IgM or both as indicated. Cell lysates were prepared for immunoblotting using the indicated epitopes. G densitometry results for p-p70- S6K and RRAGD normalized to actin for all conditions for four primary human LN-derived B lymphocyte samples (# 111, 89, 94, and 95) combined. Left panel: One-way Anova with post hoc Tukey’s test. Right panel: paired nonparametric Wilcoxon test. *p < 0.05, ***p < 0.001. IL4 interleukin 4, BCR B cell receptor, AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Expressing, Transduction, CRISPR, Control, Ex Vivo, Western Blot, Derivative Assay, Virus

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 6 RRAGD is required for S6 Kinase phosphorylation in lymphoma. A The RRAGD gene or AAVS (control) was targeted with lentivirus carrying pooled CRISPR-Cas9 guides in four lymphoma cell lines and following puromycin selection the pools were analyzed for RRAGD protein expression by immunoblotting. B, C RRAGD −/−or AAVS targeted lymphoma cell line pools were treated with anti-IgM or anti-IgG for 10’, cell lysates were made, and protein prepared for immunoblotting using the indicated epitopes. Densitometry data for mean p-p70- S6K:HSP90 for AAVS control cells are shown, while signals for RRAGD −/−cells were close to background. One-Way ANOVA with post hoc Tukey’s test. Densitometry based on three cell lines and N = 2 repeats *p < 0.05, **p < 0.01. D–F RRAGD −/−or AAVS targeted lymphoma cell line pools were treated +/−IL4 for 6 h and +/−anti-IgM for 10’, cell lysates were made and protein prepared for immunoblotting using the indicated epitopes. D A representative blot for OCI-LY7. E Densitometry of mean p-4E-BP1:total 4E-BP1 for AAVS control cells +/−IL4 for 6 h and +/−anti-IgM/G. One-Way ANOVA with post hoc Dunn’s test. F Densitometry of comparative data for AAVS and RRAGD −/−cells; Mann–Whitney test. Densitometry based on three cell lines and N = 2 repeats. ns, not significant, *p < 0.05, **p < 0.01. AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, IgG immunoglobulin G, IL4 interleukin 4, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Phospho-proteomics, Control, CRISPR, Selection, Expressing, Western Blot, MANN-WHITNEY, Virus

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 7 STAT6 mutations hyperinduce RRAGD expression and IL4 and BCR induced mTOR signaling. Three lymphoma cell lines were lentivirally transduced with cDNA/ORFs for WT or MUT STAT6 and cells sorted via GFP fluorescence. Cells were stimulated with IL4 and BCR crosslinking as indicated or left untreated and detergent lysates prepared for immunoblotting using the indicated epitopes. A–C Representative immunoblotting results for N = 2 independent experiments per cell pool. D Results from densitometry for phospho-S6K normalized to HSP90 for indicated conditions across three cell lines (N = 6). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05. E Results from densitometry for RRAGD normalized to HSP90 for indicated conditions for N = 2 independent experiments across three cell lines (N = 6 measurements). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05; **p < 0.01. STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, WT wild type, MUT mutated, GFP green fluorescence protein, cDNA complementary DNA, ORFs open reading frames, IL4 interleukin 4, BCR B cell receptor, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Expressing, Transduction, Western Blot

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: ( A, B) Analysis of GBM patient versus control samples focused on the level of expression of human homologs of Drosophila ACD regulators by Hierarchical clustering ( A ) and the Gene Distance Matrix ( B ). Color-coded scale bar indicates fold level of expression. (C) Kaplan-Meier survival curves corresponding to patients in the TCGA (IDHwt) and Gravendeel (IDHwt) cohort in GBM datasets. Low RAP2A expression levels in human GBM are associated with a poor prognosis.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: (A) Different GBM cell lines show similar RAP2A mRNA levels and significantly lower levels than in control Astros. (B) RAP2A mRNA levels are significantly higher in the GB5 line after infecting this line with RAP2A (GB5-RAP2A). Data shown in the scaled bar graphs in A and B was analyzed with an ANOVA and a T-test, respectively; error bars show the SD; n= 2 (in A) and 3 (in B) different experiments. (C) Immunofluorescences of the GSC stem cell markers CD133, SOX2 and Nestin reveal a significant reduction in the protein intensity in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the box plots was analyzed with a U Mann-Whitney for CD133 and Nestin and with a T-test for Sox; the central lines represent the median and the box limits the lower and upper quartiles, as determined by R software; crosses represent sample means; error bars indicate the SEM; n= number of sample points; p values: * p <0.05, ** p <0.01, *** p <0.001, ns, not significant; scale bar: 10 μm.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Control, Expressing, MANN-WHITNEY, Software

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: RT-PCRs and Westerns blots show a significant decrease in the mRNA and protein expression levels, respectively, of the GSC markers CD133, SOX2 and Nestin in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s t test; error bars show the SD; n= 3 different experiments; p values: * p <0.05, ** p <0.01.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Expressing, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: (A) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significantly lower number of Ki67 expressing cells per neurosphere than control GB5 neurospheres. Data shown in the box plots was analyzed with a T-test; the central lines represent the median and the box limits the lower and upper quartiles, as determined by R software; crosses represent sample means; error bars indicate the SEM; n= number of sample points. (B) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significant decrease in their size compared to control GB5 neurospheres of the same stage. Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s T- test; error bars show the SD; n= total number of neurospheres of 3 different experiments; p values: * p <0.05; scale bars: 10 μm (in A) and 100μm (in B).

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Expressing, Control, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: (A) Early stage GB5 neurospheres expressing RAP2A (GB5-RAP2A) show an odd number of cells significantly more frequently than control GB5 neurospheres, which show more frequently an even number of cells. (B) GB5-RAP2A dividing cells show a significant increase in the number of asymmetric NUMB localization in the progeny, compared to control GB5 dividing cells. Data shown in the bar graphs was analyzed with a Chi-Square test with Yates correction; n= number of neurosphere cell clusters analyzed. p values: ** p <0.01; scale bar: 20 μm.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Expressing, Control