Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2-induced different cell viability in M17, PC12 and MN9D cells as measured by MTT assay (A & B) and trypan blue dye exclusion assay (C). Each bar from Figs. represents data obtained from 5 separate experiments (N = 5 of independent cell culture preparations, which is the same in the following legends). *p < 0.05, **p < 0.01, ***p < 0.001, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: MTT Assay, Exclusion Assay, Cell Culture, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on DDRs in M17 and MN9D cells as demonstrated by measurements of γH2AX (A) and p53 (B). Cells were exposed to 100–400 μM H2O2 for 3 h. Top panels: autoradiograph obtained by western blotting. Button panel: the quantitative analysis of band densities in western blotting. Each bar from Figs. represents data obtained from 5–7 separate experiments (N = 5–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Autoradiography, Western Blot, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2 treatment induces single-strand DNA breaks as determined by the Comet assay in M17 and MN9D cells. Cells were exposed to different concentrations of H2O2 for 3 h. The cells were processed for comet assays run under alkaline conditions to identify DNA SSBs. Tail moment (B), tail length (C) and % DNA in the tail (D) were analyzed to evaluate DNA damage. Each bar from Figs. represents data obtained from 5 separate experiments (N = 5). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Single Cell Gel Electrophoresis, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on ROS production in M17 and MN9D cells. Cells were exposed to different concentrations of for 3 h. ROS was measured by the DCFH2-DA assay. Each bar from Figs. represents data obtained from 6 separate experiments (N = 6). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Ctr1 (A) and OGG1 (B) protein levels in M17 and MN9D cells. Each bar represents data obtained from 5 separate experiments (N = 5) in (A), and 4 separate experiments (N = 4) in (B). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Cav1.2 (A) and Cav1.3 (B) protein levels in M17 and MN9D cells as determined by western blotting. Cells were exposed to different concentrations of H2O2 for 3 h. Each bar in Figs. represents data obtained from 6 to 7 separate experiments (N = 6–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Western Blot, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic vaccination for glioblastoma elicited by retargeted oncolytic herpes virus

doi: 10.1136/jitc-2025-012840

Figure Lengend Snippet: R-115 effects on the immune components. (A) Representative flow cytometry histograms of CFDA-SE proliferating splenocytes derived from LS_R-115 and scNAIVE co-cultured with mHGG-HER2, mHGG or control (H-Fib). (B) Box plot of proliferating splenocytes co-cultured with mHGG-HER2 or mHGG tumor cells. LS_R-115 n=4, NLS_R-115 n=15, CTR n=5, scNAIVE n=6. (C) Box plot of proliferating CD4, CD8 and CD19 lymphocytes co-cultured with mHGG-HER2 or mHGG tumor cells. LS_R-115 n=4, scNAIVE n=6. (D) Scatter plot with bars (median) of Granzyme B, TNF-α, and IFN-γ released by splenocytes co-cultured with mHGG-HER2 or mHGG. LS_R-115 n=4, NLS_R-115 n≥10, CTR n=6, scNAIVE n=6. Data are analyzed with one-way ANOVA followed by Tukey’s multiple comparison test or with two-sided t-test. *p<0.05, **p<0.01. ANOVA, analysis of variance; mHGG, Murine high-grade glioma; NLS_R-115, not-long survivors treated with R-115; ReHV, retargeted oncolytic herpes simplex virus.

Article Snippet: The binding of the primary anti-mouse CD4 antibody was revealed with a secondary antibody anti-rat CF 660R (1:400, 20390, Biotium).

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Control, Comparison, Virus

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: The influence of GVHD on T cell development in allogeneic chimeras. Irradiated euthymic (A and C) or thymectomized (B and D) A.BY (Thy1.2, Ly5.2) recipients received a graft containing 10 7 T cell–depleted B6.PL (Thy1.1, Ly5.2) bone marrow cells (as a source of hematopoietic progenitors) with or without mature T cells (0.4 or 1.6 × 10 6 ) harvested from the LNs of B6.SJL donors (Thy1.2, Ly5.1). Chimeras' spleen cells were analyzed by three-color staining on day 100 ± 5 posttransplant. The origin of recipient T cells was determined according to their Thy1/Ly5 phenotype. Results are presented as the mean ± SD (three to four mice per group). Note that the ordinate scale is different in A and B versus C and D. nd, not determined.

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Irradiation, Staining

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: Skewing of Vβ usage by CD4 + and CD8 + T cells in athymic GVHD + chimeras. Three-color staining was performed using Abs against CD4 or CD8, Thy1.1 or Thy1.2, and specific Vβ elements. Chimeras were studied on day 100 ± 5. Results are presented as the mean ± SD (three to four mice per group).

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: Expansion of T cells from GVHD + mice after adoptive transfer into normal hosts. Three-color staining was performed on spleen cell suspensions using Abs specific for CD4 or CD8, Ly5.1 or Ly5.2, and Thy1.1 or Thy1.2. (A) Numbers of CD4 + and CD8 + T cells of B6.PL origin (Thy1.1, Ly5.2) found in the spleen of day 100 nonthymectomized GVHD + recipients (negative controls). (B) Numbers of CD4 + and CD8 + T cells of B6.PL origin (Thy1.1, Ly5.2) found in the spleen of irradiated/thymectomized B6.SJL or A.BY secondary hosts, on day 30 after injection of 10 7 T cell–depleted C57BL/6 bone marrow cells + 2 × 10 6 T cells harvested from the spleen of day 70 GVHD + mice (test group). (C) Numbers of Thy1.2 + Ly5.2 + T cells found in the spleen of irradiated/thymectomized B6.SJL or A.BY hosts, on day 30 after injection of 10 7 T cell–depleted B6.PL bone marrow cells + 2 × 10 6 T cells harvested from the spleen of normal syngeneic donors (A.BY for A.BY secondary recipients, C57BL/6 for B6.SJL secondary recipients) (positive controls). GVHD + mice were prepared as in Fig. C (irradiation followed by transplantation of 10 7 B6.PL bone marrow cells + 0.4 × 10 6 B6.SJL LN T cells). Results are presented as the mean ± SD (three to four mice per group). * P < 0.05, ** P < 0.005 relative with numbers of CD4 + or CD8 + T cells in GVHD + mice (Student's t test). No significant difference was found between T cell numbers presented in B and C.

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Adoptive Transfer Assay, Staining, Irradiation, Injection, Transplantation Assay

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: Adoptive transfer of normal host (A.BY)-tolerant T cells into nonthymectomized GVHD + mice and thymectomized GVHD − recipients. (A) Number of T cells in the spleen of day 100 GVHD + mice. GVHD + mice were prepared as in Fig. C (irradiation followed by transplantation of 10 7 B6.PL bone marrow cells + 0.4 × 10 6 B6.SJL T cells). (B) Number of T cells in the spleen of day 100 GVHD + mice previously injected, on day 60, with 5 × 10 6 host-tolerant T cells of C57BL/6 (Thy1.2,Ly5.2) origin. (C) Number of T cells in the spleen of thymectomized A.BY mice 40 d after irradiation and injection of 10 7 T cell–depleted B6.SJL bone marrow cells + 5 × 10 6 host-tolerant B6.PL T cells. Host (A.BY)-tolerant T cells used in B and C experiments were obtained from the spleen of nonthymectomized A.BY hosts, 60 d after irradiation and injection of T cell–depleted C57BL/6 or B6.PL bone marrow cells. Results are presented as the mean ± SD (three to four mice per group).

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Adoptive Transfer Assay, Irradiation, Transplantation Assay, Injection

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Schematic showing the domain organization of the reference HER2-specific CAR constructs and modifications made to introduce programmed membrane protein (proMP) transmembrane domains (TMDs). Bold, boxed sequence indicates the human CD28 TMD in the reference CD28TM and no cys CARs and designed proMP sequences in the monomeric (proCAR-1), dimeric (proCAR-2), and trimeric (proCAR-3) receptors. ( b ) BW5147 murine thymoma cells stably expressing proCARs and a destabilized GFP NF-κB reporter were surface labeled with anti-Myc antibody and analyzed by flow cytometry to assess surface expression levels. ( c ) Live cells from ( b ) were coated with polyclonal anti-IgG to bind CARs through the scFv domain and immunoprecipitated using protein G beads. Products were separated by nonreducing SDS-PAGE and immunoblotted using anti-Myc antibody to visualize surface-expressed CAR proteins. Molecular weight of the unglycosylated CAR polypeptide is 55 kDa. ( d, e ) Cells from ( b ) were co-cultured with HER2+ SKBR3 human breast adenocarcinoma cells for the indicated times and analyzed by flow cytometry for upregulation of activation marker CD69 ( d ) and GFP expression from the NF-κB reporter ( e ). All activation levels are normalized to the 8 hr time point in cells expressing the CD28TM CAR (% CD28TM Max). Bars represent the mean ± SD, and dots show the individual data points for three independent experiments. ( f ) Maximum target killing percentage at 20:1 effector to target ratio from 4 hr 51 Cr release assay. Bars show mean ± SEM with each data point representing an individual experiment (n = 3). p-Values determined from paired t -tests. ( g ) Cytokine production by primary mouse HER2 proCAR T cells following 24 hr co-culture with MC57-HER2 target tumor cells. Bars show mean concentration ± SEM with each data point representing an individual experiment (n = 5). Significance was determined from one-way ANOVA with multiple comparisons. Cytokine production on antigen-negative parental MC57 cells shown separately in .

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Construct, Introduce, Sequencing, Stable Transfection, Expressing, Labeling, Flow Cytometry, Immunoprecipitation, SDS Page, Molecular Weight, Cell Culture, Activation Assay, Marker, Release Assay, Co-Culture Assay, Concentration Assay

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Model of the CD28TM interface generated by mutagenesis of the CD3ζ TMD (PDB: 2HAC). Polar residues of the CD28 dimerization motif (orange) with predicted hydrogen bonds depicted (dotted lines). ( b ) Surface expression and ( c ) SDS-PAGE and immunoblot analysis of HER2 CARs possessing WT CD28TM or CD28TM mutations depicted in ( a ) expressed in the BW5147 cell line. ( d ) Quantitation of target cell killing measured by chromium release assay and cytokine production by primary mouse CD8 + CAR T cells in response to the MC57-HER2 target cell line (n = 4). Experiments performed as in . p-Values determined by paired t -tests. ( e ) Representative immunofluorescent confocal images of CAR-CD28 co-clustering in primary mouse CAR T cells. CAR clustering was induced with anti-Myc primary followed by crosslinking with fluorescent secondary antibody (magenta). Cells were then labeled for CD28 (cyan). Images are Z-projections over 12 m, scale bar represents 3 m. ( f ) Quantitation of CAR-CD28 co-clustering, each dot representing the percentage of CAR clusters in one cell that co-localized with a CD28 cluster. Lines show mean CAR-CD28 co-clustering percentage/per cell ± SEM, n ≥ 30 cells. p-Values determined by unpaired t -tests.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Generated, Mutagenesis, Expressing, SDS Page, Western Blot, Quantitation Assay, Release Assay, Labeling

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Flow cytometry gating strategy to determine the transduction efficiency of primary murine CAR T cells. Lymphocytes selected via morphology, live cells selected as zombie aqua negative, T cells selected as CD3 + CD8 + and mCherry + cells defined as CAR T cells. c-Myc co-expression with mCherry indicates surface CAR expression. ( b ) Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM CARs and ProCARs 1–3, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Transduction, Expressing, Labeling, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM chimeric antigen receptors (CARs) and proCAR-4, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Labeling, Transduction, Expressing, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Fluorescence, Microscopy, Recombinant, Sequencing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Modification, Software

Journal: medRxiv

Article Title: Th2 infiltration is a better predictor of survival than tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC)

doi: 10.1101/2023.06.02.23289891

Figure Lengend Snippet: Corresponding H&E pathological slides (A) and FC analysis (B) of a highly infiltrated group C tumor, BT127, showing 70% of TILs infiltration (A) and 20% of Th2/CD45 +cells (B), and a poorly infiltrated group A one, BT82, showing 5% of TILs stromal infiltration (A), and 6% of Th2/CD45+ cells (B). TILs count consists of mononuclear cells count in stromal fibrous spans (pink). Th2 cells are defined as living T helper lymphocytes (CD4+) expressing neither CXCR3 (contrary to Th1 and Th17 lymphocytes) nor CCR6 (contrary to Th17 lymphocytes). C: Complete characterization of the immune microenvironment of all tumors by FC showing the relative abundance of the different subpopulations (Percentage values in Supplementary Table 3). TC: T lymphocyte cells; APC: Antigen-presenting cells.

Article Snippet: BC: Breast cancer; SC: Stem cells; DBCC: Differentiated breast cancer cells; CD4+: helper T lymphocytes; CD8+: cytotoxic T lymphocytes; BT; lab number .

Techniques: Expressing

Journal: medRxiv

Article Title: Th2 infiltration is a better predictor of survival than tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC)

doi: 10.1101/2023.06.02.23289891

Figure Lengend Snippet: Correlation of TILs group as defined by the pathologist with total leukocytes, including helper CD4+ and cytotoxic CD8+ T cells, antigen-presenting cells (APC) or dendritic cells, and tumor cells characterized by flow cytometry (FC). Helper T cells could either trigger a Th1, Th17, or Th2 response. Only populations with significant correlation are shown on the figure. p values were derived from a Holm and a Kruskal-Wallis tests (for the comparison of respectively 2 and 3 groups). ns: non-significant; * p≤ 0.05; ** p≤ 0.005; *** p≤ 0.001.

Article Snippet: BC: Breast cancer; SC: Stem cells; DBCC: Differentiated breast cancer cells; CD4+: helper T lymphocytes; CD8+: cytotoxic T lymphocytes; BT; lab number .

Techniques: Flow Cytometry, Derivative Assay, Comparison