Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet: Humanin-induced chemoresistance requires ATR signaling (A) hGBM1 cells were stimulated with HN or vehicle (Ctrl.), underwent transcriptomics, and differentially expressed genes (DEGs) were analyzed by bioinformatics. (B) Experiments described in (A) were repeated with hGBM-1, 2, and 3 cells providing 12 consistent DEGs, of which several components assembled in a network. (C) HUS1 was associated with outcome in human GBMs. (D) In a myeloid-free brain sample, hGBMs have a basal level of HUS1 expression, which is upregulated by interaction with hiPSC microglia in a GP130-dependent manner. (E and F) Contribution of the ATR pathway to humanin-induced GBM expansion (E) and chemoresistance (F) was demonstrated with the ATR inhibitor AZ20. (G) Western blots showing expression levels of HUS1, ATR and beta-actin (loading control) and a readout for of ATR activation (pT1989) in hGBM1 cells treated with bovine serum albumin (control), TMZ, HN, or AZ20. (H) In summary, AZ20 does not cooperate with TMZ per se, but blocks HN-induced TMZ resistance. The number of biological replicates is indicated (dots in graphs indicate data from individual experiments); all error bars are presented as mean ± SDM. Statistical significance is shown as FDR in (A), one-way ANOVA (D, E), or two-way ANOVA (F): ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Expressing, Western Blot, Control, Activation Assay

Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet: Humanin-induced chemoresistance can be blocked therapeutically (A) Tumor size of orthotopic HN-WT or HN-C8a tumors was compared in mice receiving TMZ or vehicle (in animals with established tumor growth, 5x per week for 2 weeks; pre-defined endpoint was at 3 weeks). (B) Orthotopic hGBM1 was infused with HN (100 nM) or vehicle (artificial cerebrospinal fluid, aCSF) and i.p. injected with bazedoxifene-A (5 injections of BZA per week; 40 mg/kg; for 2 weeks) or vehicle; brains were labeled for HUS1; HUS1 expression was quantified. (C) Mice with established, orthotopic HN-WT tumors received TMZ (50 mg/kg) and were cotreated with vehicle or BZA (as in B); after 3 weeks, tumor size was quantified (dashed line: average data from untreated WT GBMs). (D) Mice with HN-WT GBMs received TMZ and were cotreated with vehicle or BZA (as in C); GBM samples were immunostained for active caspase-3 and immunolabeling was quantified (dashed line: average data from untreated WT GBMs). (E) Intratumoral vascularization and vessel diameter were compared in HN-WT or HN-C8a tumors receiving TMZ. (F) The HN-WT GBM mouse model was i.p. injected with TMZ and cotreated either with BZA or vehicle and the extent of intratumoral vascularization was compared. The number of biological replicates is indicated (dots in graphs indicate data from individual mice); all error bars are presented as mean ± SDM. Statistical significance is shown by one-way ANOVA (A, E), two-way ANOVA (B–D), or t test (F): ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; NS, not significant. Scale bars in (B, C) indicate 1 mm; scales in (D) represent 500 (overview) or 10 μm (magnified).

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Injection, Labeling, Expressing, Immunolabeling

Journal: Cell Reports Medicine

Article Title: Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling

doi: 10.1016/j.xcrm.2024.101658

Figure Lengend Snippet:

Article Snippet: HUS1 shRNA lentiviral and non-target control constructs , BioCat , Cat#: TLHSU1400-3364-pZIP-hCMV-ZsGreen-GVO-TRI.

Techniques: Plasmid Preparation, Recombinant, Transfection, Fluorescence, Staining, Reverse Transcription, Expressing, Liposomes, Mutagenesis, shRNA, Control, Construct, Software, Imaging, Functional Assay, Dissection, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: Loss of E2F4 leads to defects in mouse ES cell growth. a Representative brightfield images (scale bar, 400 µm) and alkaline phosphatase (AP) staining (wells from a 6-well plate are shown) of wild-type (WT) and E2F4KO (KO) colonies one week after plating single cells ( n > 10 assays per genotype). b Quantification of the size of AP + colonies (unpaired t -test; n = 3 biological replicates per clone). c Comparison of the area of AP staining (undifferentiated cells) versus Giemsa staining (all cells) in an independent set of colonies ( n = 3 biological replicates per clone) (no significant differences). d Size of WT and E2F4KO individual cells as estimated by forward scatter in flow cytometric analysis (unpaired t -test; n = 2 biological replicates per clone). e Total number of cells per 10 cm dish one week after plating at low density (unpaired t -test; n = 3 biological replicates per clone). Data shown as the mean and standard error of the mean

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Staining

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: E2F4 mutant mouse ES cells are defective for cell cycle and cell survival. a Quantification of the percentage of cells in G1, S, and G2/M phases based on BrdU/PI FACS analysis (unpaired t -test; n = 2 biological replicates with 2 wild-type (WT) and 4 E2F4KO (KO) clones), performed after 4 days of low density plating. b Schematic of synchronizing mESCs in G0 using the Myc inhibitor (Myci) 10085-F4 (left), and quantification of the percentage of cells in G0/G1 in WT, E2F4KO (KO), and RB family triple knockout (TKO) populations at 24 and 48 h of treatment (unpaired t -test; n = 2–3 biological replicates with 2 clones of each genotype). c Quantification of the percentage of arrested and cycling cells post-release from Myci. Cell cycle structure was measured with BrdU/PI staining 6, 8, 10, and 12 h after withdrawal of Myci from the media (unpaired t-test was performed with all individual data points from n = 3–4 biological replicates with 1–2 clones of each genotype). d Quantification of the percentage of AnnexinVneg/PIneg cells (live cells) in WT and E2F4KO populations 4 days after low density plating (unpaired t -test; n = 3 biological replicates per clone). Data shown as the mean and standard error of the mean

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Mutagenesis, Clone Assay, Triple Knockout, Staining

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: Loss of E2F4 leads to genome-wide changes in gene expression. a Volcano plot of changes in gene expression upon E2F4 loss in mouse ES cells; y-axis represents -log10 transformation of p -value and x -axis represents log2 transformation of fold change. Red circles represent significantly changed genes ( q -value < 0.05) while orange circles represent significantly changed genes with a fold change > 0.5 (log2). b Overlap between E2F4 targets in mouse ES cells and genes differentially expressed in E2F4KO (KO)cells with respect to wild-type. c GO terms for biological processes enriched in genes downregulated in E2F4KO cells ( q -value < 0.05, fold change > 0.5 (log2)). GO terms were filtered for redundancy through REVIGO and the top 20 most significant are shown. d RT-qPCR validation of differentially expressed genes. Expression of downregulated genes in WT (dark green) and E2F4KO cells (light green); and upregulated genes in WT (pink) and E2F4KO cells (light pink), was normalized to Gapdh expression and then to expression levels in WT cells (unpaired t -test was performed with all individual data points from n = 2–4 biological replicates with 2 WT and 2 E2F4KO clones). Data shown as the mean and standard error of the mean

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Genome Wide, Expressing, Transformation Assay, Quantitative RT-PCR, Clone Assay

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: The transactivation and DNA binding domains of E2F4 are required for its function in mouse ES cells. a Schematic of the design of E2F4 mutant constructs. All constructs were fused C-terminal to a GFP tag. Wild-type (WT) E2F4 contains a DNA binding domain (DBD), a dual nuclear export signal (NES), a DP dimerization domain, a transactivation domain, and an RB family/pocket proteins binding domain (PPBD). GFP-DBD contains three point mutations that make contacts with the E2F consensus binding motif and the DP dimerization partners. GFP-T360 is a truncation mutant that lacks the last 50 amino acid residues, inactivating the transactivation domain. b Quantification of endogenous and exogenous E2F4 expression by immunoassay (from fluorescence units) in WT (gray) and E2F4KO (KO, green) cells ( n = 2 biological replicates with 1 WT and 1 E2F4KO clone). c Quantification of colony size by AP staining (unpaired t-test was performed with all individual data points from n = 2 biological replicates with 2 WT and 2 E2F4KO clones in each replicate) and d Total number of cells per well in 6-well plates one week after plating at low density (unpaired t-test was performed with all individual data points from n = 4 biological replicates with 2 WT and 2 E2F4KO clones in each replicate). e RT-qPCR analysis of E2F4 targets and canonical cell cycle genes. Expression of genes in WT and E2F4KO cells expressing each of the constructs, was normalized to Gapdh expression and then to expression levels in WT cells expressing GFP (unpaired t -test was performed with all individual data points from n = 2 biological replicates with 2 WT and 2 E2F4KO clones in each replicate). Data shown as the mean and standard error of the mean

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Binding Assay, Mutagenesis, Construct, Expressing, Fluorescence, Staining, Clone Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: Loss of E2F4 leads to defects in the growth of RB family TKO mouse ES cells. a Representative brightfield images (scale bar, 400 µm) and alkaline phosphatase (AP) staining (wells from a 6-well plate are shown) of RB family triple knockout (TKO) and RB family knockout, E2F4KO (QKO) colonies one week after plating single cells (n > 10 assays per genotype). b Quantification of the size of AP + colonies (unpaired t -test; n = 2 biological replicates per clone). c Total number of cells per 10 cm dish one week after plating at low density (unpaired t -test; n = 2 biological replicates per clone). d Quantification of the percentage of cells in G1, S, and G2/M phases based on BrdU/PI FACS analysis (unpaired t -test; n = 2–3 biological replicates with 2 TKO and 2 QKO clones), performed 4 days after low density plating. e Quantification of the percentage of AnnexinVneg/PIneg cells (live cells) in TKO and QKO populations 4 days after low density plating (unpaired t -test; n = 2 biological replicates per clone). Error bars represent ± s.e.m. Data shown as the mean and standard error of the mean

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Staining, Triple Knockout, Knock-Out, Clone Assay

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: E2F4 can access chromatin and regulate target genes in an RB family-independent manner. a Quantification of cytoplasmic and nuclear E2F4 expression by immunoassay in cycling and quiescent mouse embryonic fibroblasts (MEFs), WT and TKO mouse ES cells, and TKO MEFs ( n = 2–3 biological replicates per cell type). The percentage of nuclear E2F4 is shown. See Supplementary Fig. for fractionation controls. b Quantification of E2F4 binding to target genes and an Actin negative control in WT, TKO, and E2F4KO mouse ES cells, assayed by ChIP-qPCR (unpaired t -test was performed with all individual data points from n = 2–3 biological replicates of 2 WT, 2 TKO, and 1 E2F4KO clone(s)). Binding was normalized to 10% input and then to binding of an IgG control. c Overlap of genes that are differentially expressed in QKO versus TKO mouse ES cells, and genes that are differentially expressed in E2F4KO (KO) versus wild-type (WT) mouse ES cells ( q -value < 0.05 with a fold change > 0.5 (log2)). The sets of upregulated (1095) and downregulated (641) genes between E2F4KO/WT and QKO/TKO mouse ES cells are highly similar ( p -value close to zero). d RT-qPCR validation of differentially expressed genes. Expression of downregulated genes in TKO (dark green) and QKO mouse ES cells (light green); and upregulated genes in TKO (pink) and QKO mouse ES cells (light pink), was normalized to Gapdh expression and then to expression levels in TKO cells (unpaired t -test was performed with all individual data points from n = 2–4 biological replicates with 2 TKO and 2 QKO clones). Data shown as the mean and standard error of the mean

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Expressing, Fractionation, Binding Assay, Negative Control, Quantitative RT-PCR, Clone Assay

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: Transcriptional activation by E2F4 in mouse ES cells is mediated by chromatin modifiers. a Schematic representation of the affinity purification-mass spectrometry approach to identify the E2F4 interactome. b Silver stain of eluted fractions from RPE cells and mouse ES cells . Slices of gel were subjected to mass spectrometry, and the indicated protein bands were identified by analyzing the proteins enriched in each slice. c GO terms for cellular components enriched in the list of mouse ES cell-specific candidate interactors. d Validation of interactions between GFP-E2F4 and DP-1, HCFC1, YEATS2, LIN54, and LIN9, by co-immunoprecipitation followed by immunoassay, in mouse ES cells and human RPE cells. Transcriptional activators (HCFC1 and YEATS2) preferentially bind to E2F4 in mouse ES cells while members of the DREAM repressor complex (LIN54 and LIN9) bind preferentially to E2F4 in RPE cells. Molecular weights (kDa) are indicated on the left side (one experiment shown of at least two experiments)

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: Activation Assay, Affinity Purification, Mass Spectrometry, Silver Staining, Immunoprecipitation

Journal: Nature Communications

Article Title: E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family

doi: 10.1038/s41467-019-10901-x

Figure Lengend Snippet: ChIP-seq analysis of the role of E2F4 in gene activation in mouse ES cells. a Heatmap of enrichment scores of H3K9ac and H3K4me3 ChIP-seq signal across transcription start sites (TSS, −1/ + 1 kb) in 2 biological replicates of 2 TKO and 2 QKO clones (biological replicates are next to each other). b Average enrichment of H3K9ac and H3K4me3 signal around TSS (-1/ + 1 kb) in TKO and QKO clones. c Volcano plot showing genome-wide comparison of detected peaks in TKO and QKO cells for H3K9ac and H3K4me3. d , e Average enrichment of H3K9ac ( d ) and H3K4me3 ( e ) signal around TSS (−1/+1 kb) comparing all downregulated genes in QKO cells compared to TKO cells, genes that are downregulated and bound by E2F4, and a random set of downregulated genes. f Enrichr analysis of CHEA/ENCODE motifs and KEGG pathways significantly ( p < 0.05) enriched in genes with downregulated and upregulated H3K9ac and H3K4me3 signal upon E2F4 loss. Top motifs/pathways (by combined score) are shown

Article Snippet: Vectors expressing 3xFLAG-tagged E2F4 (FLAG-WT), the two mutant versions of E2F4 harboring point mutations in the HCF-1 and RB binding domains (FLAG-HCFC1m and FLAG-RBm), and truncated E2F4 (FLAG-T360) were ordered from VectorBuilder.

Techniques: ChIP-sequencing, Activation Assay, Clone Assay, Genome Wide

Journal: bioRxiv

Article Title: Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity

doi: 10.1101/2024.07.24.604840

Figure Lengend Snippet: A) Six (6) primary GBM samples were subjected to FACS isolation of DAPI-PDGFRβ+ cells. These cells were then transduced with a lentiviral library containing plasmids which contain unique CellTag DNA barcodes and GFP. After transduction, cells were transplanted onto week 8-12 human cortical organoids. Following a proliferation period of 12-18 days, the tumor cells were dissociated from the organoid and sorted based on GFP. GFP+ cells are harvested for scRNA-seq. The resultant data include paired CellTag DNA barcode information with transcriptomic profiles, allowing the construction of clonal relationships, illustrating neuronal and vascular clonal dynamics. B) Live-image of 3D cortical organoid with transplanted PDGFRβ+ CellTagged tumor cells in culture (4X magnification, scale bar = 500 μM). The inset depicts the cellular morphology of transplanted cells (20X magnification, scale bar = 100 μM). UMAPs depict the detection of CellTag associated genes GFP.CDS and CellTag.UTR, indicating that tumor cells were uniformly labeled by lentiviral transduction. Histogram displaying the distribution of clone sizes. Total clones: 1465, minimum size: 2, mean size: 3.28, maximum size: 28. C) Cells were captured for scRNA-seq and subject to standard processing and QC (Methods). Cell identity was annotated via projection onto the GBM meta-atlas . The bar graph depicts the likelihood of each cell type to be a member of a clone based on CellTag barcode analysis. Proportion of each cell type with a clone member was scaled to the proportion of cells in the entire dataset that are clone members. D) Representative clones are depicted on the UMAP. Each dot represents one cell, and cells of the same color belong to the same clone. Clone members in NVP share clonal identity with cells of various cell types in the annotated UMAP, including vascular, neuronal, and other NVP cells. E) UMAP highlighting cells that are members of a clone, colored by tumor. Clone members were distributed across cell types, with increased in the cycling cells cluster. F) UMAP highlighting clones (n=90) with at least one NVP member, colored by tumor. NVP cells harbored clone partners in a diverse array of cell types, including other NVP cells, cycling cells, neurons, and vascular cells. G) In parallel to PDGFRβ+ FACS sort and CellTag, in a subset of our tumors, we conducted a parallel infection, organoid transplantation, and scRNA-seq experiment. UMAP depiction of parent tumor scRNA-seq data is shown on the left. The right UMAP shows the PDGFRβ+ CellTagged cells overlayed on the UMAP space of the parent tumor, depicting that the majority of cell types could be derived from the sorted fraction. Notably, not all cell types are represented in the overlay. The bar graph depicts the number of unique cell types present in each tumor in either the parent sample or PDGFRβ+ CellTagged sample.

Article Snippet: The Vectorbuilder lentiviral library displayed a complexity of 7,989 barcodes, while the Addgene lentiviral library had a complexity of 19.974 barcodes.

Techniques: Isolation, Transduction, Labeling, Clone Assay, Infection, Transplantation Assay, Derivative Assay