Journal: Cell metabolism

Article Title: CD38-NAD + Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response

doi: 10.1016/j.cmet.2017.10.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mycoplasma testing was done in Mehrotra lab. N/A Experimental Models: Organisms/Strains C57BL/6 Jackson Laboratory Stock# 000664 C57BL/6-Tg(HLA-A2.1)1Enge/J Jackson Laboratory Stock# 003475 B6.129S7- Rag1 tm1Mom /J Jackson Laboratory Stock# 002216 B6.129P2- Cd38 tm1Lnd /J Jackson Laboratory Stock# 003727 B6;129- Sirt1 tm1Ygu /J Jackson Laboratory Stock# 008041 Tg(Cd4-cre)1Cwi/BfluJ Jackson Laboratory Stock# 017336 B6.SJL- Ptprc a Pepc b /BoyJ Jackson Laboratory Stock# 002014 B6.129S7- Ifng tm1Ts /J Jackson Laboratory Stock# 002287 B6.Cg- Rag1 tm1Mom Tyrp1 B-w Tg(Tcra,Tcrb)9Rest/J Jackson Laboratory Stock# 008684 B6.Cg- Thy1 a /Cy Tg(TcraTcrb)8Rest/J Jackson Laboratory Stock# 005023 C57BL/6-Tg(TRAMP)8247Ng/J Jackson Laboratory Stock# 003135 IFNγ Thy1.1 knockin Casey T. Weaver, University of Alabama at Birmingham (UAB) ( Harrington et al., 2008 ) Foxo1 fl/fl Lck cre Melanie Gubbels Bupp, Randolph-Macon College, VA ( Gubbels Bupp et al., 2009) TCR-I Jennifer Wu, MUSC ( Staveley-O’Carroll et al., 2003) Oligonucleotides Primers for qPCR, see Table S4 Recombinant DNA MSGV1-TRP-1 vector ( Kerkar et al., 2011 ) N/A LV-TCR/sr39TK-GFP vector ( Koya et al., 2010 ) N/A Software and Algorithms FlowJo 10.2 TreeStar, OR https://www.flowjo.com/solutions/flowjo/downloads/ Prism 5 GraphPad https://www.graphpad.com/scientiflc-software/prism/ Agilent Seahorse Wave 2.4 Agilent http://www.agilent.com/en-us/products/cell-analysis-(seahorse )/seahorse-wave-software CFX Manager 3.1 Biorad http://www.bio-rad.com/en-us/sku/soft-cfx-31-patch-cfx-manager-software-v3-1-upgrade GenomeStudio software Illumina https://www.illumina.com/techniques/microarrays/array-data-analysis-experimental-design/genomestudio.html PIMENTo ( Sásik et al., 2004 ) https://github.com/MUSC-CGM/PIMENTo ToppGene Suite ( Chen et al., 2009 ) https://toppgene.cchmc.org/ Venny This paper http://bioinfogp.cnb.csic.es/tools/venny/index.html Open in a separate window KEY RESOURCES TABLE Th1/17 cells with effector and stemness features exhibit durable tumor control High glutaminolysis of Th1/17 cell regulates its viability and anti-tumor response NAD + -Sirt1-Foxo1 axis is central to the anti-tumor phenotype of Th1/17 cells Targeting NADase CD38 on T cells increases NAD + levels and controls tumor growth

Techniques: Recombinant, Staining, Cell Isolation, Synthesized, Lysis, Extraction, Cell Based Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, cDNA Synthesis, SYBR Green Assay, Microarray, Knock-In, Plasmid Preparation, Software

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 4. A - NAC can reverse the transfer of NFATc3 from nucleus to cytoplasm caused by As4S4 combined with radiotherapy. RMS A-673 cells were treated with 2 Gy radiation, 1 μM As4S4, and combination therapy，respectively. The expressions of NFATc3 was detected by Western blot 24 h later. B - shC3-1 and shC3-2 were effectively knocked down, Western blot assay were used to evaluate expression levels of NFATc3, RAG1, and γ-H2AX. C - Western blot analysis of γ-H2AX expression changes in RMS A-673 cells after transfection with (shC3-1 + shRAG1-1), shC3-1, and shRAG1-1, respectively.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: Western Blot, Expressing, Transfection

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 3. As4S4 enhances radiosensitivity via activating NFATc3-RAG1 mediated DSB in RMS cells. All samples were run in triplicate. A - RMS cells were treated with radiotherapy, As4S4, and combination therapy, respectively. Western blot assay and qPCR were used to evaluate expression levels of NFATc3 and RAG1. B - RMS cells were treated with radiotherapy, As4S4, and combination therapy, respectively. Western blot assay and qPCR were used to evaluate expression levels of p53 and γ-H2AX. C - RMS cells were treated with radiotherapy, As4S4, and combination therapy, respectively. Immunofluorescence assays for the expression of γ-H2AX. Left, γ-H2AX (red); middle, DAPI (blue); right, γ-H2AX and DAPI merged.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 6. A, B - IHC images (NFATc3 and RAG1) in RMS tissues of in vivo xenograft models (40X). C, D - Histograms of NFATc3 and RAG1 in qPCR experiments.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: In Vivo

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 7. NFATc3 and RAG1 are independent prognostic factors for RMS patients. A, B - IHC images of NFATc3 and RAG1 in RMS tissues (40X). C - The Kaplan-Meier survival curves of the overall survival of two groups defined as low or high expression (NFATc3 and RAG1) in 59 RMS patients. D - Boxplots of expression (NFATc3 and RAG1) between tumor and adjacent normal tissues. E - Nomogram of the prediction model. F. ROC curves predicting 5-year survival of NFATc3-RAG1 based prediction model, TNM staging, and Risk level.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: Expressing

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 8. Schematic figure describing that As4S4 enhances radiosensitivity in RMS via activating NFATc3-RAG1 mediated DSB.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques:

Journal: Signal transduction and targeted therapy

Article Title: Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

doi: 10.1038/s41392-023-01544-y

Figure Lengend Snippet: Fig. 1 Base-edited monkeys were generated using the CBE4max system. a Schematic illustrating the process of generating base-edited monkeys. b Specific gRNAs designed to target RAG1 and IL2RG genes. The gRNA target sites are highlighted in red, while the edited bases are indicated in green. c The status of the embryos after CBE4max systemic injection. d T7E1 assay for base-edited embryos. e Following the microinjection of 152 embryos, 35 embryos in the developmental stage were transferred to surrogate female monkeys. Subsequently, five recipient monkeys were confirmed to be pregnant, resulting in the successful acquisition of six newborn monkeys. f The editing efficiency in the peripheral blood of the edited animals was assessed using Sanger sequencing-based EditR software. g The genotype of the base-edited monkeys (mutation) was determined. Red boxes indicate the presence of stop codons after the substitution, while the asterisk (*) denotes a substituted base. The gRNA sequencing map of the RAG1 gene is derived from monkey NO.1, while the gRNA sequencing map of the IL2RG gene is from monkey NO.3. h The frequencies of base substitution in deceased base-edited monkeys determined through targeted deep sequencing

Article Snippet: Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies.

Techniques: Generated, Injection, Microinjection, Sequencing, Software, Mutagenesis, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

doi: 10.1038/s41392-023-01544-y

Figure Lengend Snippet: Fig. 2 Phenotypic and histopathological changes in base-edited monkeys. a, b Comparison of body weights, body lengths, and head circumferences between mutant monkeys and age-matched WT monkeys while being subjected to the same feeding environment. c, d Analysis of thymus and spleen in base-edited monkeys. e H&E staining depicting the development of thymus and spleen in base-edited monkeys. Scale bars: 50 µm. f–k Evaluation of RAG1 and IL2RG protein expression in the thymus and spleen of mutant monkeys. Western blot analysis illustrating RAG1 and IL2RG protein expression levels and quantification in the thymus, spleen, liver, and lung of mutant and WT monkeys (f, g). Immunohistochemistry depicting the expression and quantification of RAG1 and IL2RG in the thymus and spleen of mutant and WT monkeys (h–k). Statistical analysis was performed using an unpaired two-tailed t-test to compare the two groups. P < 0.05 was considered statistically significant. Three replicates were performed for each analysis. Scale bars: 20 µm

Article Snippet: Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies.

Techniques: Comparison, Mutagenesis, Staining, Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Signal transduction and targeted therapy

Article Title: Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

doi: 10.1038/s41392-023-01544-y

Figure Lengend Snippet: Fig. 4 Off-target analysis in base-edited monkeys. a Whole-genome sequencing analysis was conducted to identify single-nucleotide variations (SNVs) and indels in three base-edited monkeys (NO.1, NO.2, and NO.3) as well as three wild-type monkeys (WT1, WT2, and WT3). The height of the bars represents the average number of mutations in each group, while black dots indicate the number of mutations in each sample. b The distribution of mutation types in base-edited and wild-type monkeys. The number within each cell indicates the proportion of a specific mutation type among all mutations. c The genomic feature regions of mutations in base-edited and wild-type monkeys. A large proportion of SNVs were detected in intronic and intergenic regions, and no difference was observed between base-edited and wild-type monkeys. d Evaluation of the potential for mutations in base-edited and wild-type monkeys to impact gene function. No difference was observed between base-edited and wild-type monkeys. e Distribution of indel fragment lengths. f, g Assessment of Off-target base editing frequency in the RAG1 (f) and IL2RG (g) genes in base-edited monkeys generated by the CBE4max system. Potential off-target sites (OT1–OT9) were detected by deep sequencing and predicted using Cas-OFFinder. Deep sequencing was employed to determine the frequency of substitutions at predicted target sites in the spleen (NO.1 and NO.3) and peripheral blood (NO.2) of three deceased base-edited animals

Article Snippet: Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies.

Techniques: Sequencing, Mutagenesis, Generated

Journal: Experimental & molecular medicine

Article Title: Extracellular vesicles derived from lung M2 macrophages enhance group 2 innate lymphoid cells function in allergic airway inflammation.

doi: 10.1038/s12276-025-01465-6

Figure Lengend Snippet: Fig. 1 GW4869 injected into asthmatic mice reduced the activation of ILC2s. a WT mice and Rag1−/−mice were sensitized via i.p. injection with 200 μg of papain on day 0 and 7, followed by i.n. administration of papain (100 μg) and GW4869 (1.5 mg/kg) on days 14–17. After the last treatment was completed, the mice were euthanized 24 h later, and samples were collected for analysis. b Flow cytometric analysis of lung ILC2s (CD45+Lin−CD90+ST2+); n = 4 mice. c The proliferation of ILC2s was indicated by Ki67 staining; n = 4 mice. d Flow cytometric analysis of IL-5 and IL-13 in ILC2s; n = 4 mice. e, f IL-5 (e) and IL-13 (f) concentrations (pg/ml) in BALF were determined by ELISA; n = 4 mice. g The frequencies of eosinophils (CD11c−Siglec F+) in BALF were analyzed by flow cytometry; n = 4 mice. h, i Representative H&E staining (h) and PAS staining (i) of lung tissue sections from the indicated groups; n = 4 mice. The data are representative of two or three independent experiments and are presented as mean ± s.e.m. P values were calculated using two-sided Student’s t-tests or one-way ANOVA, followed by a multiple comparison test. ns not significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: C57BL/6 female mice and Rag1−/− female mice, aged between 6 and 8 weeks and weighing 20–22 g, were sourced from GemPharmatech Co., Ltd. and Cyagen Biosciences Co., Ltd., respectively.

Techniques: Injection, Activation Assay, Staining, Enzyme-linked Immunosorbent Assay, Cytometry, Comparison

Journal: Journal of Clinical Investigation

Article Title: CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis

doi: 10.1172/jci63139

Figure Lengend Snippet: Figure 6). Thus, CDK4 deficiency leads to lymphomas that harbor rampant genomic alterations, and this is associated with dysregulation of at least some cancer-associated genes. A CDK4/RAG1/2 pathway controls genomic stability and tum- origenesis in Eμ-Myc B cells. To determine the mechanism by which CDK4 deficiency renders Eμ-Myc B cells susceptible to genomic alterations, we assessed factors known to con- trol genomic instability in lymphoid cells. Elevated levels of the lymphocyte-specific recombinase complex composed of RAG1 and RAG2, which are physically linked in the genome and are coordinately expressed (38, 39), are sufficient to drive aberrant translocations, recombination, and rear- rangements of chromosomes that promote lymphomagen- esis (40, 41). Notably, RAG1 and RAG2 mRNA and protein levels were selectively elevated in premalignant and neoplas- tic Eμ-Myc Cdk4–/– B cells (Figure 4, A and B). Given the established roles of the RAG1/RAG2 complex in driving chromosomal alterations, we tested whether enforced coexpression of RAG1 and RAG2 was sufficient to provoke genomic instability and accelerate lymphoma onset in a fetal liver–derived Eμ-Myc HSC transplant model (20) and whether it was sufficient to augment the tumorigenic potential of extant Eμ-Myc lymphoma. Notably, lethally irradiated congenic recipients transplanted with Eμ-Myc HSCs cotransduced with MSCV-RAG1– and MSCV-RAG2– expressing retroviruses had a greatly accelerated course of lymphoma onset (Figure 4, C and D). As predicted, we found that RAG1/RAG2-expressing tumors had markedly increased numbers of genomic alterations (translocations, genomic deletions, and cleavage events at bona fide and for- tuitous RSSs) that were associated with enhanced recombi- nation at Igh and Bcl11b loci compared with the later-onset lymphomas arising in recipients transplanted with Eμ-Myc HSCs that were transduced with control MSCV virus (Figure 4, E and F, and Supplemental Table 4, C and D). To further test whether RAG1 and RAG2 overexpression was sufficient to augment the tumorigenic potential of extant Eμ-Myc lymphoma, we cotransduced a randomly chosen tumor with MSCV-Rag1-IRES-Puro and MSCV- Rag2-IRES-Hygro viruses or with control MSCV-Puro virus. Syngeneic C57Bl/6 mice were then transplanted i.v. with 1 × 106 lymphoma cells via the tail vein, and the recipient mice were followed for their course of disease and overall survival. Notably, enforced RAG1 and RAG2 coexpression in extant Eμ-Myc Cdk4+/+ lymphoma induced more rapid disease in these recipients than in those transplanted with lymphoma transduced with MSCV-IRES control virus (Supplemental Figure 7). Again, the accelerated course of disease in recipients bearing RAG1/RAG2-expressing lym- phomas was associated with marked increases in genomic instability (translocations, deletions, etc.; data not shown). Thus, enforced expression of RAG1 and RAG2 is sufficient to accelerate the onset and augmented tumorigenic poten- tial of Myc-driven lymphoma, and this is associated with marked increases in genomic instability. CDK4 controls Rag1 and Rag2 transcription via FOXO1. To define the mechanism by which CDK4 loss controls RAG1 and RAG2 expression, we assessed known regulators of Rag1/Rag2 transcription. The FOXO family transcription factors FOXO1 and FOXO3a were candidates, as FOXO1

Article Snippet: Equal amounts of lysate protein (50 μg) were separated on 8% to 12% SDS-PAGE gels, transferred to PVDF membranes (Amersham), and then incubated with primary antibodies specific to CDK4 (SC-260; Santa Cruz Biotechnology Inc.), CDK2 (2546; Cell Signaling Technology), CDK6 (SC-177; Santa Cruz Biotechnology Inc.), p-FOXO1 (S329) (ab52857; Abcam), RAG1 (NPP1-74190; Novus Biological), RAG2 (ab133609, i.e., Epitmics 5485-1 for mouse cells; Abcam); RAG2 (ab959955 for human cells; Abcam), p62 (SC-28359; Santa Cruz Biotechnology Inc.), p27Kip1 (610242; Transduction Labs), p53 (Ab-7; Oncogene Research), p19Arf (ab80; Abcam), FOXO1 (2880; Cell Signaling Technology), p-FOXO1-S249 (441245G; Invitrogen), FOXO3a (2497; Cell Signaling Technology), p-318/321-FOXO3a (9465; Cell Signaling Technology), or β-actin (A00702; Sigma-Aldrich).

Techniques: Derivative Assay, Irradiation, Expressing, Transduction, Control, Virus, Over Expression

Journal: Journal of Clinical Investigation

Article Title: CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis

doi: 10.1172/jci63139

Figure Lengend Snippet: Figure 4 CDK4 controls RAG1 and RAG2 expression in Eμ-Myc B cells, and forced coexpression of RAG1 and RAG2 is sufficient to augment the development of Myc-driven lymphoma. (A) Rag1 and Rag2 mRNA levels in Eμ-Myc Cdk4–/– and Eμ-Myc Cdk4+/+ premalignant bone marrow B220+ and lym- phoma cells (n = 5 mean ± SD, P < 0.005, respectively). (B) RAG1 and RAG2 protein levels in Eμ-Myc Cdk4–/– and Eμ-Myc Cdk4+/+ premalignant B220+ and lymphoma cells (data shown are representative of analyses of five cohorts of Eμ-Myc Cdk4–/– and Eμ-Myc Cdk4+/+ B220+ B cells and lymphomas). (C) Overexpression of RAG1 and RAG2 in Eμ-Myc HSCs. HSCs from E13.5–E15.5 Eμ-Myc fetal livers were transduced with MSCV-IRES-Puro (MSCV) control virus or were cotrans- duced with MSCV-Rag1-IRES-Puro and MSCV-Rag2-IRES-Hygro (MSCV-RAG1/RAG2) retrovi- ruses. Lysates from these HSCs were analyzed by Western blotting. (D) Enforced coexpression of RAG1 and RAG2 accelerates lymphoma development. 3 × 106 HSCs (nontransduced, MSCV, or MSCV-Rag1/Rag2-HSCs; n = 15) were transplanted into lethally irradiated congenic recipients that were then followed daily for lymphoma onset (P < 0.001). (E) FISH analyses of lymphomas arising in recipient mice engrafted with Eμ-Myc HSCs transduced with MSCV control retrovirus or cotransduced with MSCV-RAG1/RAG2-expressing retroviruses. Metaphase cells from each cohort were assessed by FISH with 5′ (green) and 3′ (red) Igh probes. An intact Igh locus had colocalized red and green signals, while a broken, translocated locus had split red and green signals. Original magnification, x1,500. (F) Statistical analysis of Igh translocations in the two cohorts of lymphomas, Eμ-Myc-MSCV and Eμ-Myc-MSCV-RAG1/RAG2 (n = 6, P < 0.01).

Article Snippet: Equal amounts of lysate protein (50 μg) were separated on 8% to 12% SDS-PAGE gels, transferred to PVDF membranes (Amersham), and then incubated with primary antibodies specific to CDK4 (SC-260; Santa Cruz Biotechnology Inc.), CDK2 (2546; Cell Signaling Technology), CDK6 (SC-177; Santa Cruz Biotechnology Inc.), p-FOXO1 (S329) (ab52857; Abcam), RAG1 (NPP1-74190; Novus Biological), RAG2 (ab133609, i.e., Epitmics 5485-1 for mouse cells; Abcam); RAG2 (ab959955 for human cells; Abcam), p62 (SC-28359; Santa Cruz Biotechnology Inc.), p27Kip1 (610242; Transduction Labs), p53 (Ab-7; Oncogene Research), p19Arf (ab80; Abcam), FOXO1 (2880; Cell Signaling Technology), p-FOXO1-S249 (441245G; Invitrogen), FOXO3a (2497; Cell Signaling Technology), p-318/321-FOXO3a (9465; Cell Signaling Technology), or β-actin (A00702; Sigma-Aldrich).

Techniques: Expressing, Over Expression, Transduction, Control, Virus, Western Blot, Irradiation

Journal: Journal of Clinical Investigation

Article Title: CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis

doi: 10.1172/jci63139

Figure Lengend Snippet: Figure 5 CDK4-dependent control of the levels and phosphorylation of FOXO1 in Eµ-Myc B cells. (A) Levels of the indicated proteins were determined by immunoblotting lysates of Eμ-Myc Cdk4+/+ versus Eμ-Myc Cdk4–/– lymphomas. Right: ratio of p-FOXO1/total FOXO1 (n = 5 for each cohort). p-FOXO1-S326 levels were markedly reduced in Eµ-Myc Cdk4–/– lymphomas (P < 0.01). (B) CDK4 silencing affected the levels and phosphorylation of FOXO1 and RAG1 and RAG2 expression in Eµ-Myc Cdk4+/+ lymphomas. Bottom: ratio of p-FOXO1/total FOXO1. Note the marked reduction in p-FOXO1-S326 following knockdown of CDK4 in malignant Eµ-Myc Cdk4+/+ B cells (P < 0.05, n = 5). (C) Restoring CDK4 expression in Eμ-Myc Cdk4–/– lymphoma cells affected the levels and phosphorylation of FOXO1 and RAG1 and RAG2 expression. Bottom: ratio of p-FOXO1/total FOXO1. Note the marked increase in p-FOXO1-S326 following restoration of CDK4 in malignant Eμ-Myc Cdk4–/– B cells (P < 0.05, n = 5). (D) Increased levels of nuclear FOXO1 are a hallmark of Eμ-Myc Cdk4–/– lymphoma. Left panels: FOXO1; middle left panels: DAPI; middle right panels: light microscopy; right panels: merge. Quantification of FOXO1 localization is shown at right: C, cytoplasm; N, nucleus; C+N, cytoplasm and nucleus. The mean of triplicate experiments is shown (n = 100 Eμ-Myc Cdk4–/– and Eμ-Myc Cdk4+/+ lymphoma cells). Original magnification, ×600.

Article Snippet: Equal amounts of lysate protein (50 μg) were separated on 8% to 12% SDS-PAGE gels, transferred to PVDF membranes (Amersham), and then incubated with primary antibodies specific to CDK4 (SC-260; Santa Cruz Biotechnology Inc.), CDK2 (2546; Cell Signaling Technology), CDK6 (SC-177; Santa Cruz Biotechnology Inc.), p-FOXO1 (S329) (ab52857; Abcam), RAG1 (NPP1-74190; Novus Biological), RAG2 (ab133609, i.e., Epitmics 5485-1 for mouse cells; Abcam); RAG2 (ab959955 for human cells; Abcam), p62 (SC-28359; Santa Cruz Biotechnology Inc.), p27Kip1 (610242; Transduction Labs), p53 (Ab-7; Oncogene Research), p19Arf (ab80; Abcam), FOXO1 (2880; Cell Signaling Technology), p-FOXO1-S249 (441245G; Invitrogen), FOXO3a (2497; Cell Signaling Technology), p-318/321-FOXO3a (9465; Cell Signaling Technology), or β-actin (A00702; Sigma-Aldrich).

Techniques: Control, Phospho-proteomics, Western Blot, Expressing, Knockdown, Light Microscopy

Journal: Journal of Clinical Investigation

Article Title: CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis

doi: 10.1172/jci63139

Figure Lengend Snippet: Figure 8 CDK4 expression is suppressed, and FOXO1 and RAG1 expression is elevated in several subtypes of human B cell lymphomas. (A) 125 cases of randomly collected patient B cell lymphoma samples from The Ohio State University Department of Pathology and US Biomax Inc., along with 10 normal LNs, were stained with antibodies specific to CDK4 (top panels), p-FOXO1-S329 (second panels), FOXO1 (third panels), or RAG1 (bottom panels). Original magnification, ×400. (B) For statistical analysis, the samples were graded on a scale of staining scores from 1 (lowest) to 5 (maximum), based on signal intensity for CDK4, p-FOXO1-S329, FOXO1, or RAG1. Approximately 90% of B cell lymphoma samples (111 of 125) had low or nondetectable levels of CDK4 (stages 1 and 2), and nearly 70% (87 of 125) of B cell lymphoma samples had low levels of p-FOXO1-S329 (stages 1 and 2). Accordingly, approximately 70% of these tumors (86 of 125) had elevated levels of total FOXO1 (stages 4 and 5), and approximately 64% (80 of 125) had elevated levels of RAG1 (stages 4 and 5). Among the lymphomas with elevated levels of FOXO1, more than 70% (61 of 86) had elevated levels of RAG1.

Article Snippet: Equal amounts of lysate protein (50 μg) were separated on 8% to 12% SDS-PAGE gels, transferred to PVDF membranes (Amersham), and then incubated with primary antibodies specific to CDK4 (SC-260; Santa Cruz Biotechnology Inc.), CDK2 (2546; Cell Signaling Technology), CDK6 (SC-177; Santa Cruz Biotechnology Inc.), p-FOXO1 (S329) (ab52857; Abcam), RAG1 (NPP1-74190; Novus Biological), RAG2 (ab133609, i.e., Epitmics 5485-1 for mouse cells; Abcam); RAG2 (ab959955 for human cells; Abcam), p62 (SC-28359; Santa Cruz Biotechnology Inc.), p27Kip1 (610242; Transduction Labs), p53 (Ab-7; Oncogene Research), p19Arf (ab80; Abcam), FOXO1 (2880; Cell Signaling Technology), p-FOXO1-S249 (441245G; Invitrogen), FOXO3a (2497; Cell Signaling Technology), p-318/321-FOXO3a (9465; Cell Signaling Technology), or β-actin (A00702; Sigma-Aldrich).

Techniques: Expressing, Staining