Journal: Frontiers in Immunology

Article Title: Single cell transcriptomic analysis reveals pathogenic cell heterogeneity and candidate inflammatory-associated markers in STZ-induced diabetic mouse retina

doi: 10.3389/fimmu.2026.1827122

Figure Lengend Snippet: hdWGCNA analysis of gene co-expression modules and functional enrichment in Müller glial subpopulations cluster 0 and 1 in STZ and Ctrl groups. (A) Soft power threshold selection for the weighted network construction in the hierarchical clustering analysis of Müller glia subpopulations. The figure showed the correlation between the soft power threshold and model fit, as well as mean connectivity, median connectivity and max connectivity, with the optimal threshold indicated for constructing the network. The selected power (β = 5) represents the minimal value satisfying the scale-free topology criterion while preserving sufficient network connectivity for module detection. (B) Hierarchical clustering dendrogram of Müller glial subpopulations based on gene expression profiles. The tree showed clustering of the different gene modules identified in the analysis, with modules labeled Müller-M1 to Müller-M6. Each module represents a set of co-expressed genes associated with specific biological functions in Müller glial subpopulation. (C) Module feature plots showing the expression patterns of each module (M1-M6) within Müller cell subpopulations. For each module, cells were colored by module expression level (low to high), illustrating the distribution of module activity across Müller cell subpopulations. (D) The module membership (kME) for all genes was calculated, and the top-ranked 10 genes within each module were visualized using PlotKMEs. (E) Dot plots respectively showing the expression of distinct gene co-expression modules in Müller glial cell subpopulation and the module expression within subpopulations under different conditions. (F) Co-expression network comprising the top five hub genes from each co-expression module. (G) Network graphs showing hub-gene networks constructed from the top 25 core genes of modules 1 and 6; the inner ring denotes the top 10 hub genes and the outer ring denotes the remaining 15. (H, I) Bar charts showing functional enrichment terms for the top 100 genes from modules 1 and 6. (J) PPI network analysis of genes in Module 6, showing the top 10 hub genes selected based on connectivity scores. (K) Western blot validation of JUNB protein expression levels in the Ctrl and STZ groups (N = 3), confirming differential expression. * indicates p < 0.05.

Article Snippet: Membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against JUNB (Abmart, T55894 , 1:2000), CEBPB (Abmart, T55276 , 1:2000), and β-actin (Affinity, AF7018, 1:20000).

Techniques: Expressing, Functional Assay, Selection, Preserving, Gene Expression, Labeling, Activity Assay, Construct, Western Blot, Biomarker Discovery, Quantitative Proteomics

Journal: bioRxiv

Article Title: CRISPR Screens Reveal Epstein-Barr Virus-activated JunB as a Key Lymphoblastoid B cell Dependency Factor that Represses Cyclin Dependent Kinase Inhibitor P18INK4c

doi: 10.64898/2026.03.31.715635

Figure Lengend Snippet: (A) Log2(fold change) of the four guides targeting JunB (red) compared to the distribution of all sgRNA guides from the GM12878 and P3HR-1 Avana dropout screen. Fold change was calculated comparing abundance of sgRNA day 21 post selection versus input. (B) JunB sgRNA abundances in Brunello and Avana dropout screens. Log 2 JunB sgRNA abundances from GM12878 (red) and P3HR-1 (blue) cells 21 days post library selection were determined via deep sequencing and compared to input library sgRNA abundance (green). (C) c-Jun sgRNA abundances in Brunello and Avana dropout screens. Log 2 c-Jun sgRNA abundances from GM12878 (red) and P3HR-1 (blue) cells 21 days post library selection were determined via deep sequencing and compared to input library sgRNA abundance (green). (D) JunD sgRNA abundances in Brunello and Avana dropout screens. Log 2 JunD sgRNA abundances from GM12878 (red) and P3HR-1 (blue) cells 21 days post library selection were determined via deep sequencing and compared to input library sgRNA abundance (green). (E) JunB dependency across CRISPR DepMap lymphoid cell lines ( DepMap.org ). Gene dependency scores were calculated by comparing JunB sgRNA abundance in individual lymphoid cell lines 21 days post lentivirus library transduction and selection in comparison to input library abundance. Each circle indicates an individual lymphoid cell line. Circle size represents Log JunB reads from RNA-seq analysis (DepMap, Broad, DepMap Public 25Q3 (2025)) Green circles versus clear circles indicate DepMap Burkitt versus non-Burkitt lymphoid cell lines, respectively. P-values were determined by one-sided Fisher’s exact test. * p<0.05, **p<0.005, ***p<0.0005

Article Snippet: Antibodies against the following proteins were used in this study: JunB (Cell Signaling Technology, #3753), c-Jun (Cell Signaling Biotechnology, #9165), JunD (Cell Signaling Biotechnology, #5000), β-Actin (Biolegend, #664802), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), BIM (Cell Signaling Biotechnology, #2933), CCND2 (Proteintech, 10934-1-AP), EBNA1 (a gift from Jaap Middeldorp), and EBNA3C A10 (hybridoma from Martin Rowe’s group[ ]).

Techniques: Selection, Sequencing, CRISPR, Transduction, Comparison, RNA Sequencing

Journal: bioRxiv

Article Title: CRISPR Screens Reveal Epstein-Barr Virus-activated JunB as a Key Lymphoblastoid B cell Dependency Factor that Represses Cyclin Dependent Kinase Inhibitor P18INK4c

doi: 10.64898/2026.03.31.715635

Figure Lengend Snippet: (A) Validation of JunB CRISPR-Cas9 knockout in MUTU I and GM15892 cells. Immunoblot was performed on WCL from Cas9+ MUTU I and GM15892 cells transduced with control or JunB sgRNA seven days post-selection using indicated antibodies. (B) Mean ± SD live cell numbers of Cas9+ MUTU I or GM11830 cells expressing control or JunB targeting single guide RNAs (sgRNA) from n=3 replicates. Cells transduced with lentiviruses expressing the indicated sgRNAs were puromycin selected. Cell numbers two days following puromycin selection (defined as day 0 of the graph) were set to 1. Live cell numbers were quantitated by CellTiter-Glo assay (C) Investigation of impact of JunB on other Jun AP-1 transcription factors. Immunoblot analysis of WCL from Cas9+ GM12878 cells transduced with control or JunB sgRNAs, ten days post selection, with indicated antibodies. Densitometry ratios of JunB and JunD to β-Actin is shown. (D) Investigation of combinatorial impact of JunB and JunD on LCL growth. (Left) Mean ± SD live cell numbers of Cas9+ GM12878 cells expressing control, JunD, JunB, or JunB+JunD targeting single guide RNAs (sgRNA) from n=3 replicates. Cells transduced with lentiviruses expressing control or JunD sgRNA were Zeocin selected two days post transduction. After one week, cells were then transduced with a separate control or JunB sgRNA. Cell numbers two days following puromycin selection (defined as day 0 of the graph) were set to 1. Live cell numbers were quantitated by CellTiter-Glo assay. (Right) Immunoblot validation of JunB and JunD knockouts. WCL of GM12878 cells transduced with both zeocin and puromycin sgRNAs was extracted two days following puromycin selection and immunoblot was performed using indicate antibodies. (E) PI cell cycle stainin of GM12878 cells transduced with JunB sgRNA. Representative FACS cell cycle plots from n=3 replicates of GM12878 cells analyzed ten days post selection. P-values were determined by one-sided Fisher’s exact test. * p<0.05, **p<0.005, ***p<0.0005

Article Snippet: Antibodies against the following proteins were used in this study: JunB (Cell Signaling Technology, #3753), c-Jun (Cell Signaling Biotechnology, #9165), JunD (Cell Signaling Biotechnology, #5000), β-Actin (Biolegend, #664802), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), BIM (Cell Signaling Biotechnology, #2933), CCND2 (Proteintech, 10934-1-AP), EBNA1 (a gift from Jaap Middeldorp), and EBNA3C A10 (hybridoma from Martin Rowe’s group[ ]).

Techniques: Biomarker Discovery, CRISPR, Knock-Out, Western Blot, Transduction, Control, Selection, Expressing, Glo Assay

Journal: bioRxiv

Article Title: CRISPR Screens Reveal Epstein-Barr Virus-activated JunB as a Key Lymphoblastoid B cell Dependency Factor that Represses Cyclin Dependent Kinase Inhibitor P18INK4c

doi: 10.64898/2026.03.31.715635

Figure Lengend Snippet: (A) CCND2 sgRNA abundances in Brunello P3HR-1 and GM12878 dropout screens. Log 2 CCND2 sgRNA abundances from GM12878 (red) and P3HR-1 (blue) cells 21 days post library selection were determined via deep sequencing and compared to input library sgRNA abundance (green). (B) CCND2 transcript levels during EBV-mediated B cell transformation. RNAseq was performed on primary B cells infected with B95-8 virus on indicated days post infection (dpi). Normalized reads for CCND2 are shown. (C) Immunoblot analysis of CCND2 levels in response to LMP1 expression. Daudi Burkitt cells harboring selectively inducible WT or transformation effector site (TES) mutant LMP1 cassettes were mock induced or treated with 250 ng / mL doxycycline for 24 hours. Cells were then harvested and lysates analyzed via immunoblot with indicated antibodies. (D) Effect of JunB knockout on CCND2 levels. Cas9+ P3HR-1, Jijoye, or GM12878 cells expressing control or JunB sgRNAs. Seven days post selection, WCL was extracted from cells and immunoblot performed using indicated antibodies. P-values were determined by one-sided Fisher’s exact test. * p<0.05, **p<0.005, ***p<0.0005

Article Snippet: Antibodies against the following proteins were used in this study: JunB (Cell Signaling Technology, #3753), c-Jun (Cell Signaling Biotechnology, #9165), JunD (Cell Signaling Biotechnology, #5000), β-Actin (Biolegend, #664802), LMP1 (Abcam, ab78113), LMP2A (Abcam, ab59028), EBNA2 PE2 (a gift from Fred Wang), DDX1 (Bethyl, A300-521A-M), Myc (Santa Cruz Biotechnology, SC-40), p100/p52 (EMD Millipore, 05-361), TRAF1 (Cell Signaling Biotechnology, #4715S), HA tag antibody (Cell Signaling Technology, # 3724), BIM (Cell Signaling Biotechnology, #2933), CCND2 (Proteintech, 10934-1-AP), EBNA1 (a gift from Jaap Middeldorp), and EBNA3C A10 (hybridoma from Martin Rowe’s group[ ]).

Techniques: Selection, Sequencing, Transformation Assay, RNA sequencing, Infection, Virus, Western Blot, Expressing, Mutagenesis, Knock-Out, Control