Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet:

Article Snippet: For microarray analysis, RNA was extracted from THP1 or stimulated human CD4+ T cells using the RNeasy Mini kit (QIAGEN).

Techniques: Recombinant, Multiplex sample analysis, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Reverse Transcription, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, DNA Library Preparation, Library Quantification, Control, Sequencing, Methylation Sequencing, Amplification, Software

Journal: BMC Medical Genomics

Article Title: Identification of protein-coding and non-coding RNA expression profiles in CD34 + and in stromal cells in refractory anemia with ringed sideroblasts

doi: 10.1186/1755-8794-3-30

Figure Lengend Snippet: Validation of gene expression data of CD34 + cells by real-time RT-PCR . Comparison of gene expression levels by real-time RT-PCR (black bars) and microarray experiments (gray bars) for eight select genes (names indicated at bottom) in CD34 + cells of MDS-RARS patients and healthy individuals. Positive and negative fold change values indicate the up- or down-regulation of expression in MDS-RARS patients in relation to the expression in healthy individuals, respectively.

Article Snippet: Data were normalized among the samples by quantile [ ] using Spotfire DecisionSite ® for Microarray Analysis (TIBCO Software Inc, Somerville, MA, USA).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Comparison, Microarray, Expressing

Journal: BMC Medical Genomics

Article Title: Identification of protein-coding and non-coding RNA expression profiles in CD34 + and in stromal cells in refractory anemia with ringed sideroblasts

doi: 10.1186/1755-8794-3-30

Figure Lengend Snippet: Validation of gene expression data of stromal cells by real-time RT-PCR . Comparison of gene expression obtained from real-time RT-PCR (black bars) and microarray experiments (gray bars) for four select genes (names indicated at bottom) in stromal cells of MDS-RARS patients and of healthy individuals. The positive and negative fold change values indicate up- or down-regulation of expression in MDS-RARS patients in relation to the expression in healthy individuals, respectively.

Article Snippet: Data were normalized among the samples by quantile [ ] using Spotfire DecisionSite ® for Microarray Analysis (TIBCO Software Inc, Somerville, MA, USA).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Comparison, Microarray, Expressing

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Identification of KSR1-regulated phosphoproteome in breast cancer cells. ( A ) Experimental schematic outline of SILAC experiment. ( B ) Scatter plot comparison of phosphosite ratios quantified from control vs KSR1-overexpressed MCF7 cells. ( C ) Gene ontology (GO) Classification of the KSR1-regulated phosphoproteome in MCF7 cells according to molecular functions, biological processes and cellular compartmentalisation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Multiplex sample analysis, Comparison, Phospho-proteomics, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 transcriptional activity in the presence or absence of etoposide by luciferase assays. ( A ) MCF7 cells were transiently co-transfected with either pCMV6 (vector) or pCMV6-KSR1 plasmids in the presence of four individual p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1, p53-CYCLIN G1 and p53-IGFBP3) following dimethylsulphoxide (DMSO) or etoposide (40 μ M ) treatment for 3 h. ( B ) MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 48 h, followed by transfection of three p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1 and p53-CYCLIN G1) for additional 24 h. DMSO or etoposide (40 μ M ) were subsequently added as described above. Firefly luciferase activity was measured (renilla luciferase activity was used to normalise transfection efficiency). The normalised luciferase activity of empty vector is set as 1. Results shown are the average of at least three independent experiments and error bars represent s.d. Student's t -test was performed using SPSS 16.0 statistical software (SPSS Inc.). (* P <0.05, ** P <0.01).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Construct, Expressing, Control, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 mRNA, total protein and neddylation levels and on p53 subcellular localisation. ( A ) Effects on p53 mRNA and total protein levels after KSR1 overexpression. MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, relative mRNA levels of TP53 and p53 total protein were measured by RT-qPCR and western blotting, respectively. Gene expression level from cells transfected with pCMV6 was set as 1. Results shown are the average of at least three independent experiments. Similarly, in MCF7 stably overexpressing KSR1 cells, p53 total protein was evaluated by western blot. Blots shown are representatives of at least three independent experiments. ( B ) Immunofluorescence staining of p53 cells after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. p53 was detected with an anti-p53 antibody while the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). Representative pictures of three independent experiments are shown. Subcellular fractionation assays were performed after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. Tubulin and histone deacetylase 1 (HDAC1) expression served as positive normalising control for cytoplasmic and nuclear proteins respectively. Blots shown are representatives of at least three independent experiments. ( C ) Neddylation assay on p53 after KSR1 overexpression. MCF7 cells were co-transfected with HA-NEDD8 and pCMV6 or pCMV6-KSR1 plasmids as indicated. p53 was immunoprecipitated using a p53-specific antibody (DO-1) and the neddylated-p53 was detected by immunoblotting using anti-NEDD8 and anti-p53-specific antibodies. Blots shown are representatives of at least three independent experiments. Abbreviations: IgG= immunoglobulin G; IP= immunoprecipitation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Gene Expression, Stable Transfection, Immunofluorescence, Staining, Fractionation, Histone Deacetylase Assay, Expressing, Control, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Mechanisms of KSR1-regulated p53 transcriptional activity. ( A ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 overexpression followed by etoposide treatment. MCF7 cells were transiently transfected with pCMV6 (vector) or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with various concentrations of etoposide (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( B ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 silencing followed by a titration of etoposide treatment. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 72 h followed by etoposide treatment (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( C ) Effect of KSR1 on p53 acetylation is through DBC1. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 in concordance with siCT or siDBC1 for 72 h followed by etoposide treatment (40 μ M , 3 h). Acetylated p53, DBC1 and KSR1 protein levels were assessed by immunoblotting with specific antibodies as indicated. ( D ) Effect of KSR1 on DBC1 phosphorylation is dependent on its intact kinase domain. MCF7 cells were transiently transfected with vector, wild-type KSR1 or mutant KSR1 (R502M) plasmids for 24 h followed by etoposide treatment (40 μ M , 3 h). DBC1 phosphorylation was measured by immunoblotting with specific antibody. ( E ) Interaction of DBC1 and SIRT1 after KSR1 overexpression with etoposide treatment by immunoprecipitation (IP). MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with etoposide (40 μ M , 3 h). The interactions between SIRT1 and DBC1 were detected by IP of SIRT1 or DBC1 followed by immunoblotting with DBC1 and SIRT1 antibodies respectively. Blots shown are representatives of at least three independent experiments. Quantification of blots was analysed by ImageJ software (NIH, Bethesda, MD, USA). ( F ) Schematic model illustrating the role of KSR1 on p53 transcriptional activity in breast cancer cells with (i) basal or (ii) up-regulated levels of KSR1. Abbreviation: IgG= immunoglobulin G.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, Western Blot, Titration, Control, Mutagenesis, Immunoprecipitation, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 silencing on breast cancer cell proliferation in vitro . SRB assays of MCF7, ZR75-1, SKBR3 and MDA231 cells after transfection with 20 n M of either siKSR1 or ‘non-targeting' siRNA (control siRNA) or vehicle (Hiperfect) for 6 days. Error bars represent s.d. of three experiements each in quintuplicates (* P <0.05, compared with control siRNA at day 6; Student's t- test).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: In Vitro, Transfection, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: KSR1 expression is altered in breast cancer tissues. Oncomine analysis was performed to examine KSR1 expression in breast normal and cancer tissues using online TCGA microarray data ( www.oncomine.org ).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Expressing, Microarray

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

Figure Lengend Snippet: T cell-depleted tumors and maturation trajectories of human DC vaccines (A) CIBERSORT deconvolution across TCGA cancer types. Population abundances were row normalized (C1, n = 1,313; C2, n = 1,210, C3, n = 688; C4, n = 222, C5, n = 2; C6, n = 111). (B) Overall survival of cancer patients’ transcriptome profiled before ICBs treatment (anti-PD-1/CTLA4/PD-L1 ICBs, or combinations thereof) sub-grouped in T cell-depleted C4/C5 tumors (n = 667) and immunogenic C2/C3/C6 tumors (n = 474). Statistics: log rank test. (C) GISTIC 2.0 analysis with indicated 12 genes. Statistical significance: false discovery rate (FDR) < 0.05 (random permutations to background score distribution, BH adjusted). Bladder cancer, n = 136; breast cancer, n = 880; colorectal adenocarcinomas, n = 585; glioblastoma multiforme, n = 580; head and neck cancer, n = 310; kidney cancer, n = 497; acute myeloid leukemia, n = 200; lung adenocarcinoma, n = 357; lung squamous cell carcinoma, n = 344; ovarian cancer, n = 563; endometrial cancer, n = 496. (D–J) Single-cell trajectory reconstruction exploration and mapping (STREAM) DC vaccine trajectory of 93 DC vaccines from 18 prostate adenocarcinoma patients vaccinated with five to eight vaccines. (D) Overview of STREAM DC vaccine trajectory. (E and F) Pseudo-time inferred from DC vaccines’ transcriptome based on variable genes. Principal graph initiated with epg_alpha = 0.01, epg_mu = 0.2, epg_lambda = 0.03, and epg_n_nodes = 5. Dots depict individual DC vaccines and dot color represents (E) patient number or (F) DC vaccine batch/cycle (chi-squared test of independence of variables). (G and H) Signature scores overlaid on the graph as streamplots. Type I IFN/ISG-response signature (G) or mature regulatory DC signature (H) were used as color intensity. (I and J) Patient outcomes were overlaid on the graph as streamplots. PSA doubling time at week 48 (I) and intensity of IFNγ production of peripheral blood mononuclear cell after antigen restimulation (J) were used as color intensity. Here, “n” represents different patients (biological replicates). See also Figure S1 .

Article Snippet: For DC vaccine creation, bone marrow derived DCs were stimulated with dying cancer cells in a 1:1 ratio or with TC1 antigens (i.e., Human Papillomavirus (HPV) e6/e7 epitopes: VYDFAFRDL/DKKQRFHNI, RAHYNIVTF/LCVQSTHVD)), with or without 2.5 ng/mL interferon beta (IFNβ) (R&D systems #8234-MB-010) for 48h.

Techniques: Vaccines

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

Figure Lengend Snippet: Optimization of DCvax-IT for T cell-depleted tumors (A) Metagene expression for indicated signatures in different subcutaneous tumors (from GEO: GSE85509 ). (B) Flow cytometry analysis of CD45 + fraction from subcutaneous MC38/TC1 tumors on day 23 after injection (percentage of CD8 + of CD3 + cells, n = 6; two-tailed Student’s t test). (C) Tumor volume of TC1-tumor-bearing mice treated with anti-PD-1/CTLA4 on day 9/16 after injection (n = 6; area under curve; one-way ANOVA, Kruskal-Wallis test). (D) Survival of WT, Ripk3 −/− , and Mlkl −/− TC1 cells 24/48 h after treatment (three or four repeats). (E) Cell death of WT and Mlkl −/− TC1 cells 48 h after treatment. p values depict comparison WT vs. Mlkl −/− TC1 cells (n = 3; two-way ANOVA, Sidak’s multiple comparisons test). (F) Schematic overview of the vaccine formulation process. (G and H) Functional analysis of DCs untreated or stimulated with LPS, IFNβ, or with untreated or dying TC1s (with/without IFNβ). (G) Flow cytometry of DC maturation (MHCII + CD86 + frequency of CD11c + ). p values depict comparison vs. UT DCs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (H) IFN-signature expression (qPCR). p values depict comparison vs. UT DCs (n = 3; one sample t test). (I) Flow cytometry of frequency of PD-L1 + PD-L2 + CD200 + of CD11c + cells (moDCs alone/cocultured with untreated/dying WT/ Mlkl −/− TC1 cells). p values depict comparison vs. UT moDCs (n = 4, LPS/IFNβ n = 3; one-way ANOVA, Fischer least significant difference [LSD]). (J) Flow cytometry of frequency of CD11b + F4/80 + in moDCs (alone/cocultured with untreated/dying WT/ Mlkl −/− TC1 cells) or bone-marrow-derived macrophages (BMDMs). p values depict comparison vs. BMDMs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (K) Cytokine secretion via cytokine array. From all values, the background was subtracted. Normalization was done using moDCs + untreated cancer cells (n = 3). Here, “n” represents biological replicates and error bars represent SEM. See also Figures S2 and .

Article Snippet: For DC vaccine creation, bone marrow derived DCs were stimulated with dying cancer cells in a 1:1 ratio or with TC1 antigens (i.e., Human Papillomavirus (HPV) e6/e7 epitopes: VYDFAFRDL/DKKQRFHNI, RAHYNIVTF/LCVQSTHVD)), with or without 2.5 ng/mL interferon beta (IFNβ) (R&D systems #8234-MB-010) for 48h.

Techniques: Expressing, Flow Cytometry, Injection, Two Tailed Test, Comparison, Formulation, Functional Assay, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

Figure Lengend Snippet:

Article Snippet: For DC vaccine creation, bone marrow derived DCs were stimulated with dying cancer cells in a 1:1 ratio or with TC1 antigens (i.e., Human Papillomavirus (HPV) e6/e7 epitopes: VYDFAFRDL/DKKQRFHNI, RAHYNIVTF/LCVQSTHVD)), with or without 2.5 ng/mL interferon beta (IFNβ) (R&D systems #8234-MB-010) for 48h.

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, Western Blot, Staining, Stripping, Liposomes, CRISPR, MTS Assay, ATP Assay, Reverse Transcription, Cell Isolation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Selection, Drug discovery, Vaccines, Single-cell Analysis, RNA Sequencing, Mutagenesis, Microarray, Purification, Software