pbabe puro c myc t58a  (Addgene inc)

Journal: The EMBO Journal

Article Title: HAND1 level controls the specification of multipotent cardiac and extraembryonic progenitors from human pluripotent stem cells

doi: 10.1038/s44318-025-00409-0

Figure Lengend Snippet: ( A ) Western blot of HAND1-AM-Tag (Active Motif) at day 3 and 4 of differentiation in different conditions compared to undifferentiated hESCs. ( B ) Motif enrichment analysis of HAND1 ChIP-seq peaks with control (low HAND1) and SB (high HAND1) sets merged. Significance is relative to the background control. ( C ) Doxycycline-inducible HAND1-BFP transgene expressed in HAND1-null cells for cell sorting by HAND1 expression (12-hour induction) and molecular analysis by ATAC- and RNA-seq (both performed on 3 biological replicates). ( C ’) Euler plots for the ATAC-seq analysis representing the number of differentially accessible chromatin regions in HAND1+ vs HAND1-negative cells and the subset with detected HAND1 binding. ( D ) Schematic illustration of the motifs enriched in chromatin with increased or decreased accessibility as a result of HAND1 expression. ( E ) Identification of HAND1[+] (activating) and HAND1[-] (repressing) regulons. ( F ) Chord plot showing significantly enriched gene ontology terms for HAND1 target genes. The red and blue text colouring indicates terms enriched in activated targets and repressed targets, respectively. ( G ) UMAP plots showing the activity of constant and HAND1-sensitive regulons in endoderm and mesoderm of wild-type and HAND1-null cells. The activity of the HAND1[+] and HAND1[-] regulons are shown with the domains of strong activity highlighted. ( H – J ) ATAC-seq in HAND1-null and HAND1+ (12 h induction) populations, and HAND1 binding at low-HAND1 (CTRL) and high-HAND1 (SB) levels at the ( H ) FOXF1 locus, ( I ) HOXB cluster and ( J ) CDX2 locus. ( K ) Gene regulatory network model of HAND1. * Direct early target of HAND1. The dashed line indicates a link at high HAND1 levels. CTRL control/vehicle-only, DEG differentially expressed genes, EMT epithelial-mesenchymal transition, GO gene ontology, WT wild-type. See also Appendix Fig. . .

Article Snippet: A TO-MYC system was introduced by transducing the hPSCs with two lentiviruses, one carrying a MYC T58A transgene under the control of a TRE-CMV promoter (Addgene ID 19775), and the second carrying the tetracycline transactivator (Addgene ID 19780).

Techniques: Western Blot, ChIP-sequencing, Control, FACS, Expressing, RNA Sequencing, Binding Assay, Activity Assay

Journal: The EMBO Journal

Article Title: HAND1 level controls the specification of multipotent cardiac and extraembryonic progenitors from human pluripotent stem cells

doi: 10.1038/s44318-025-00409-0

Figure Lengend Snippet: ( A ) Schematic illustration of HAND1 level with primitive streak-like patterning of hESCs into cardiac and extraembryonic mesoderm. ( B ) Live images (brightfield and fluorescence) showing HAND1-Tomato in EBs at day 5 of differentiation. ( C ) UMAP plots showing the target genes AUC score (regulon activity) for MYC and N-MYC with differentiation in wild-type cells. White indicates the score is below threshold. ( D ) Target genes AUC score for MYC, N-MYC, PGC-1α (PPARGC1A) and SRF through pseudotime of mesoderm differentiation. ( E ) Scaled target gene AUC scores in bulk populations by differentiation day and impact of induction of a dox-inducible MYC transgene at day 5–6. ( F ) Expansion of HAND1-high, HAND1-low, and HAND1-neg progenitors from SB, control and DMH1-treated conditions, respectively. HAND1-high progenitors were maintained in FGF8, BMP4, and CHIR; HAND1-low progenitors were maintained in FGF8, BMP4, and WNT3A, and HAND1-negative progenitors were maintained in FGF8-only (see Methods). Flow cytometric analysis of HAND1-Tom and NKX2-5-GFP after 6 days of expansion. ( G ) Differentiation of progenitors to epicardial and endothelial cells assessed by immunostaining for WT1 and CD31, respectively, and additionally by the flow cytometric analysis of CD31. ( H ) Differentiation of progenitors to cardiomyocytes assessed by immunostaining for cTroponin T. ( I ) Quantification of differentiated cell types by population. Data in ( I ) represent mean ± SEM, n = 3 independent biological experiments. Scale bars represent 150 μm. AUC area under the curve, EB embryoid body, Epi epicardial cells, Endo endothelial cells, CM cardiomyocyte, SEM standard error of the mean. See also Appendix Fig. . .

Article Snippet: A TO-MYC system was introduced by transducing the hPSCs with two lentiviruses, one carrying a MYC T58A transgene under the control of a TRE-CMV promoter (Addgene ID 19775), and the second carrying the tetracycline transactivator (Addgene ID 19780).

Techniques: Fluorescence, Activity Assay, Control, Immunostaining

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Primary mouse microglia can be immortalized through HRAS and CMYC transcriptional factors. ( a ) Timeline depicting the transduction of primary CD1 E17.5 microglial cells at DIV4, followed by doxycycline induction of CMYC T58A and HRAS G12V at DIV5. ( b ) Immunocytochemistry results showing IBA1 + (Green) non-transduced control microglia, indicating a high-purity microglial culture. ( c ) Live imaging of cell confluence using IncuCyte for microglial cells transduced with TET-O-CMYC T58A-HRAS G12V with doxycycline (DOX), compared to non-transduced primary microglia at DIV16-23. ( d ) FACS gating strategy for isolating CD11B + LY6C – cells for microglial single-cell colony expansion. ( e ) Relative expression levels of microglial genes C1qa and P2ry12 , as well as human oncogenes CMYC and HRAS, normalized to Gadph. This was assessed using qRT-PCR on CD11b + LY6C − microglial cells, comparing control and transduced TET-O-CMYC T58A-HRAS G12V with DOX microglial cells. Statistical significance was evaluated using an unpaired t-test ( N = 3 replicates), with bars representing mean ± SEM. ( f ) Normalized counts per million (norm CPM) of murine proliferation genes Myc , Hras , Mki67 , Mcm2 and Pcna in primary MG and 2E11 clonal line without doxycycline (2E11 CTRL) and with doxycycline (2E11 DOX) from RNA-sequencing of total mRNA.

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Transduction, Immunocytochemistry, Control, Imaging, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Comparison of commonly used microglia cell lines examining microglial markers and function in vitro. Table modified from Stansley, Post, and Hensley (2012).

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Comparison, In Vitro, Modification, Transformation Assay

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Experimental conditions tested for immortalization of microglia of human and mouse.

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: CD4Cre-driven constitutive MYC transcription synergizes with Tsc1 deletion in T cells to trigger a lethal phenotype in mice. Related to . (A) Survival curves for wild-type, CD4Cre Rosa26 MYC/+ , and CD4Cre Igs2 MYC(T58A)/+ mice. (B) Rows 1-3: Representative images of hematoxylin and eosin (H&E) staining of spleen, lymph node and liver in 4-week-old wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Original magnification, 21x. Scale bar 0.2mm. Row 4: Representative images of Giemsa stain of blood from 4-week-old wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Original magnification, 63x. Scale bar 20μm. (C) Quantification of total T cell numbers in spleen, lymph nodes, liver and blood from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. (D) Representative flow cytometric analysis of CD4 and CD8α expression (left panel) in splenic T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice at 4 weeks of age. Right panel: Frequencies of CD4 + , CD8 + , CD4 + CD8 + and CD4 - CD8 - splenic T cells (right panel) from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Enlarged dots represent statistics of expanded T cell populations. (E) Representative flow cytometric analysis of Ki-67 and Foxp3 expression (left panel) in splenic CD4 + T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice at 4 weeks of age. Frequencies of Foxp3 + Treg cells (middle panel) and Ki-67 + cells (right panel) among splenic CD4 + T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice at 4 weeks of age. (F) Representative flow cytometric analysis of CD4 and CD8α expression in the thymus from 4-week-old wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Data are representative of 4 independent experiments. (G) Representative flow cytometric analysis of CD4 and CD8α expression in the thymus from 7-week-old wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. Data are representative of 7 independent experiments. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [A: Log-rank (Mantel Cox) test; C, D, E: one-way ANOVA with Tukey’s multiple comparisons test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.]

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Staining, Giemsa Stain, Expressing

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: CD4Cre-driven constitutive MYC transcription synergizes with Tsc1 deletion in T cells to trigger a lethal phenotype in mice. Related to . (A) Survival curves for wild-type, CD4Cre Rosa26 MYC/+ , and CD4Cre Igs2 MYC(T58A)/+ mice. (B) Rows 1-3: Representative images of hematoxylin and eosin (H&E) staining of spleen, lymph node and liver in 4-week-old wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Original magnification, 21x. Scale bar 0.2mm. Row 4: Representative images of Giemsa stain of blood from 4-week-old wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Original magnification, 63x. Scale bar 20μm. (C) Quantification of total T cell numbers in spleen, lymph nodes, liver and blood from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. (D) Representative flow cytometric analysis of CD4 and CD8α expression (left panel) in splenic T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice at 4 weeks of age. Right panel: Frequencies of CD4 + , CD8 + , CD4 + CD8 + and CD4 - CD8 - splenic T cells (right panel) from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Enlarged dots represent statistics of expanded T cell populations. (E) Representative flow cytometric analysis of Ki-67 and Foxp3 expression (left panel) in splenic CD4 + T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice at 4 weeks of age. Frequencies of Foxp3 + Treg cells (middle panel) and Ki-67 + cells (right panel) among splenic CD4 + T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice at 4 weeks of age. (F) Representative flow cytometric analysis of CD4 and CD8α expression in the thymus from 4-week-old wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. Data are representative of 4 independent experiments. (G) Representative flow cytometric analysis of CD4 and CD8α expression in the thymus from 7-week-old wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. Data are representative of 7 independent experiments. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [A: Log-rank (Mantel Cox) test; C, D, E: one-way ANOVA with Tukey’s multiple comparisons test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.]

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Staining, Giemsa Stain, Expressing

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: Constitutive MYC transcription synergizes with Tsc1 deletion in T cells to result in lethality associated with a lymphoproliferative T cell phenotype. (A) Survival curves for littermate wild-type control, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice. (B) Survival curves for littermate wild-type control, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. (C) Rows 1-3: Representative images of hematoxylin and eosin (H&E) staining of spleen, lymph node, and liver in wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. Original magnification, 21x. Scale bar 0.2 mm. Row 4: Representative images of Giemsa stain of blood from wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. Original magnification, 63x. Scale bar 20 μm. (D) Quantification of total T cell numbers in spleen, lymph nodes, liver and blood from wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. (E) Frequencies by flow cytometry of CD4 + , CD8 + , CD4 + CD8 + , and CD4 - CD8 - T cells in spleen from wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. Enlarged dots represent statistics of expanded T cell populations. (F) Frequency of Foxp3 + Treg cells among CD4 + T cells in spleen from wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. (G) Frequency of Ki-67 + cells among CD4 + T cells in spleen from wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [A, B: Log-rank (Mantel Cox) test; D, E, F, G: one-way ANOVA with Tukey’s multiple comparisons test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.] See also .

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Control, Staining, Giemsa Stain, Flow Cytometry

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: Constitutive MYC transcription and Tsc1 deletion triggers PTCL with its gene expression signature marked patients with poor survival regardless of disease subtypes. (A) Pie charts showing the relative abundance of T cell clones in splenic CD4 + T cells isolated from wild-type and CD4Cre Tsc1 fl/fl mice and hCD2 + CD4 + T cells from CD4Cre Rosa26 MYC/+ , CD4Cre Rosa26 MYC/+ Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. T cell clone is inferred from transcripts mapping to the TCRβ CDR3 region in bulk RNA-sequencing. Plots shown are representative of analysis from two mice each for wild-type, CD4Cre Rosa26 MYC/+ , and CD4Cre Tsc1 fl/fl genotypes. Full analysis from three CD4Cre Rosa26 MYC/+ Tsc1 fl/fl and three dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice are also shown. For mice with over 50 unique TCRý CDR3 clones the top 50 are shown, while all clones are shown for mice with under 50 unique TCRβ CDR3 clones. (B) Heatmap of z-scored ssGSEA scores (averaged across 2-3 mice per condition) for select Hallmark signature gene sets. Gene sets included are those differentially enriched between wild-type and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice, but for heatmap visualization CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice are also shown. Genotypes are ordered horizontally by average CDR3 clonality, and gene sets are manually ordered vertically by functional annotation. (C-F) Representative flow cytometric analysis of MYC (C), phosphorylated S6 (D), and S6 (E) protein as well as FSC-A (F) staining (left panels) and MFI Quantification (right panels) in splenic CD4 + T cells from wild-type and dLckCre Tsc1 fl/fl mice and hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice at 8 weeks of age and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice at 13 weeks of age. (G) Scatter plot of ssGSEA scores in published PTCL patient microarray dataset for MYC Targets (V2) and mTORC1 Signaling Hallmark gene sets (left); MYC Targets (V2) and Mouse PTCL Signature (middle); and mTORC1 Signaling Signature and Mouse PTCL Signature (right). The Mouse PTCL Signature comprises the human orthologs of differentially expressed genes upregulated in CD4 + T cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl as opposed to wild-type mice. A linear fit and Pearson’s correlation are shown for each scatter plot, and points are colored by the reported PTCL subtype. (H) Kaplan-Meier curve showing overall survival of PTCL-NOS patients grouped by expression (ssGSEA score) above or below the median for the Mouse PTCL Signature (left), Hallmark MYC Targets V2 (middle), and mTORC1 Signaling (right). (I) Schematic illustrating how constitutive MYC transcription synergizes with Tsc1 deletion in a positive feedback circuit to drive peripheral T cell transformation. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [C, D, E, F: one-way ANOVA with Tukey’s multiple comparisons test; G: Pearson correlation based on Kolmogorov-Smirnov test; H: Log-rank (Mantel Cox) test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.] See also .

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Gene Expression, Clone Assay, Isolation, RNA Sequencing, Functional Assay, Staining, Microarray, Expressing, Transformation Assay

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: Clonally expanded T cells with constitutive MYC transcription and Tsc1 deletion display decreased effector functions. Related to . (A) Box plots of CDR3 clonality for wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. (B) Principal Component Analysis (PCA) plot of top two principal components computed from bulk RNA-sequencing of sorted CD4 + T cells from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. Percent of variance explained is shown in parentheses for each component. (C) Heatmap of z-scored ssGSEA scores (averaged across 2-3 mice per condition) for select Reactome signature gene sets. Gene sets included are those differentially enriched between wild-type and dLckCre Rosa26 MYC/+ Tsc1 fl/fl T cells, but for heatmap visualization CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl T cells are also shown. Genotypes are ordered horizontally by average CDR3 clonality, and gene sets are manually ordered vertically by functional annotation. (D) Representative flow cytometric analysis (left panel) and frequencies (right panel) of splenic CD4 + effector T cells expressing cytokines TNFα, IFNψ, IL-17A, or IL-4 from wild-type, CD4Cre Rosa26 MYC/+ , CD4Cre Tsc1 fl/fl , and CD4Cre Rosa26 MYC/+ Tsc1 fl/fl mice after 4 hours PMA/Ionomycin stimulation. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [A: one-way ANOVA with Tukey’s multiple comparisons test; D: unpaired t-test, two-tailed, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.]

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: RNA Sequencing, Functional Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: Large neutral amino acid transporter chaperone SLC3A2 is required to sustain high levels of MYC protein expression and mTORC1 signaling for T cell transformation. (A) Heatmap of z-scored expression of cell surface proteins positively differentially expressed in hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice as compared with CD4 + T cells from wild-type mice, ordered alphabetically. The two chains of the CD98 heterodimer encoded by Slc3a2 and Slc7a5 are highlighted in yellow. (B) Representative flow cytometric analysis (left panel) and MFI quantification (right panel) of CD98 protein expression in CD4 + T cells from wild-type and dLckCre Tsc1 fl/fl mice and hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice at 8 weeks of age as well as dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice at 13 weeks of age. (C) Survival curves for dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice compared to dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice. (D) Left panel: Representative flow cytometric analysis of splenic hCD2 + TCRβ + T cells from dLckCre Rosa26 MYC/+ , dLckCre Rosa26 MYC/+ Tsc1 fl/fl , dLckCre Rosa26 MYC/+ Slc3a2 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice at 13 weeks of age, as well as dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice at 26 weeks of age. Right panel: Frequencies of splenic hCD2 + TCRβ + T cells among CD45 + cells from dLckCre Rosa26 MYC/+ and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice at 13 weeks of age and hCD2 + TCRβ + CD98 - T cells from dLckCre Rosa26 MYC/+ Slc3a2 fl/fl at 13 weeks of age and dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice at both 13 weeks and 26 weeks of age. (E-H) Representative flow cytometric analysis of MYC (E), phosphorylated S6 (F), and S6 (G) protein as well as FSC-A (H) staining (left panels) and MFI quantification (right panels) in splenic CD4 + T cells from wild-type mice, hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice, and hCD2 + CD98 - CD4 + T cells from dLckCre Rosa26 MYC/+ Slc3a2 fl/fl and dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice at 13 weeks of age. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [B, D: one-way ANOVA with Tukey’s multiple comparisons test; C: Log-rank (Mantel Cox) test; E, F, G, H: unpaired t-test, two-tailed, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.] See also .

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Expressing, Transformation Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: SLC3A2 deficiency corrects the transferrable lethal phenotype of PTCL cells. Related to . (A) Representative flow cytometric analysis of CD98 protein staining (left panel) and MFI quantification (right panel) in splenic CD4 + T cells from wild-type mice, hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ , dLckCre Rosa26 MYC/+ Tsc1 fl/fl , dLckCre Rosa26 MYC/+ Slc3a2 fl/fl and dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice at 13 weeks of age. (B) Survival curves for sub-lethally irradiated recipient mice (CD45.1/CD45.2) after receiving donor (CD45.2) hCD2 + CD4 + CD25 - T cells from dLckCre Rosa26 MYC/+ and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice, or hCD2 + CD98 - CD4 + CD25 - T cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice. (C) Quantification of dLckCre Rosa26 MYC/+ , dLckCre Rosa26 MYC/+ Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl donor T cell frequencies among total splenic TCRβ + T cells determined by the endpoint for recipient mice receiving dLckCre Rosa26 MYC/+ Tsc1 fl/fl donor T cells. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [A, C: one-way ANOVA with Tukey’s multiple comparisons test; B: Log-rank (Mantel Cox) test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.]

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Staining, Irradiation

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: SLC3A2-deficient PTCL cells have defective mitochondrial membrane potential and increased ROS production. (A-C) Box plot showing transcriptional expression (ssGSEA score) of Reactome Respiratory Electron Transport signature (A), Reactome Respiratory Electron Transport ATP Synthesis by Chemiosmotic Coupling and Heat Production by Uncoupling Proteins Pathway Signature (B), and Reactome Mitochondrial Translation Signature (C) in CD4 + T cells from wild-type mice, hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice, and hCD2 + hCD98 - CD4 + T cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice at 8 weeks of age. (D-F) Representative flow cytometric analysis of CD98 versus MitoTracker Green (D), TMRM (E), and MitoSox Red (F) staining (left panels) and MFI quantification (right panels) in splenic CD4 + T cells from wild-type mice and hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ , dLckCre Rosa26 MYC/+ Tsc1 fl/fl , dLckCre Rosa26 MYC/+ Slc3a2 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl Slc3a2 fl/fl mice. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. (D-F: one-way ANOVA with Tukey’s multiple comparisons test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.) See also .

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Membrane, Expressing, Staining

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: Leucine deprivation disrupts the synergy between constitutive MYC transcription and Tsc1 deletion to interrupt PTCL progression. (A) Survival curves for wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl , and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice switched to an isocaloric low leucine diet (13% leucine of normal chow) starting from 8 weeks of age and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice kept on normal chow control diet. (B) Serum leucine levels measured by mass spectrometry in wild-type, dLckCre Rosa26 MYC/+ , dLckCre Tsc1 fl/fl and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice fed with control or low leucine diet for 4 weeks. (C) Representative flow cytometric analysis (left panel) and quantification (right panel) of splenic hCD2 + TCRβ + T cells from dLckCre Rosa26 MYC/+ and dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice fed with control or low leucine diet for at least 4 weeks. (D-J) Representative flow cytometric analysis of MYC (D), phosphorylated S6 (E), and S6 (F) protein as well as FSC-A (G), MitoTracker Green (H), TMRM (I), and MitoSox Red (J) staining (left panels) and MFI quantification (right panels) in splenic CD4 + T cells from wild-type mice and hCD2 + CD4 + T cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice fed with control or low leucine diet for at least 4 weeks. All bar graphs are shown as mean ± SEM, each dot representing one mouse. Data are pooled from multiple experiments. [A: Log-rank (Mantel Cox) test; B, D, E, F, G: unpaired t-test, two-tailed; C, H, I, J: one-way ANOVA with Tukey’s multiple comparisons test, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, “ns” = not significant.] See also .

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Control, Mass Spectrometry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

doi: 10.1101/2024.04.03.587917

Figure Lengend Snippet: Leucine deprivation improves survival in advanced PTCL in the MYC/Tsc1 model. (A) Survival curves for sub-lethally irradiated recipient (CD45.1/CD45.2) mice receiving donor (CD45.2) CD4 + CD25 - T cells from wild-type mice (N = 2 donors, n = 5 recipients), hCD2 + CD4 + CD25 - T cells from dLckCre Rosa26 MYC/+ (N = 2 donors, n = 5 recipients) and dLckCre Tsc1 fl/fl (N = 2 donors, n = 5 recipients) mice, and hCD2 + CD4 + CD25 - PTCL cells from dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice (N = 5 donors, n = 16 recipients) fed on an isocaloric low leucine diet (13% leucine of normal chow) as well as recipient mice fed on normal chow control diet receiving PTCL cells from paired donor dLckCre Rosa26 MYC/+ Tsc1 fl/fl mice (N = 5 donors, n = 15 recipients). Recipient mice were maintained on control diet or switched to low leucine diet starting from the day of cell transfer. (B) Survival curves for sub-lethally irradiated recipient (CD45.1/CD45.2) mice receiving donor (CD45.2) CD4 + CD25 - T cells from wild-type mice (N = 2 donors, n = 5 recipients), hCD2 + CD4 + CD25 - T cells from dLckCre Rosa26 MYC/+ (N = 2 donors, n = 5 recipients) and dLckCre Depdc5 fl/fl (N = 2 donors, n = 5 recipients) mice, and hCD2 + CD4 + CD25 - PTCL cells from dLckCre Rosa26 MYC/+ Depdc5 fl/fl mice (N = 1 donors, n = 4 recipients) as well as recipient mice fed on normal chow control diet receiving PTCL cells from paired donor dLckCre Rosa26 MYC/+ Depdc5 fl/fl mice (N = 1 donors, n = 3 recipients). Recipient mice were maintained on control diet or switched to low leucine diet starting from the day of cell transfer. (C) Top: Schematic illustrating MYC cooperation with the growth factor arm of mTORC1 signaling to drive T cell transformation. Oncogenic lesions such as PTEN deletion frequently target the PI3K/AKT signaling axis to inactivate the Rheb GTPase GAP TSC complex, while the MYC oncogene is frequently over-transcribed due to gene amplification or translocation. Among MYC transcriptional targets are Slc7a5 and Slc3a2 encoding the heterodimeric large neutral amino acid transporter CD98. CD98-mediated uptake of leucine as an onco-nutrient is sensed via the KICSTOR/GATOR1/GATOR2/Sestrin supercomplex to activate the Rag GTPase complex that recruits mTORC1 to the lysosome surface to be activated by Rheb, which in turn promotes MYC translation in a positive feedback loop. Such aberrant mTORC1 signaling and MYC overexpression synergistically form an onco-circuit to drive PTCL development featuring increased mitochondrial mass, hyperpolarized mitochondrial membrane potential, and reduced mitochondrial ROS accumulation. Bottom: schematics illustrating how the cooperative oncogenic pathway can be disrupted either by genetic ablation of the onco-circuit component Slc3a2 (left) or deprivation of the key onco-nutrient leucine (right) in association with decreased mitochondrial membrane potential and increased mitochondrial ROS. [A, B: Log-rank (Mantel Cox) test, **** = p<0.0001, “ns” = not significant.] See also .

Article Snippet: CD4Cre, Rosa26 MYC/+ , Igs2 MYC(T58A)/+ , Tsc1 fl/fl , dLckCre, CD45.1 and CD45.2 mice were purchased from Jackson Laboratory.

Techniques: Irradiation, Control, Transformation Assay, Amplification, Translocation Assay, Over Expression, Membrane