Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Reduced MDM2 protein in T47D cells causes reduced chromatin phosphoproteins 53BP1 and MDC1. ( A ) Experimental design workflow of the SILAC analysis (created in BioRender. Harmon, K. (2025) https://BioRender.com/oyd7ala ). Chromatin isolated from a mixture of T47D vector control cells (MDM2-competent) cultured in natural amino acid medium, and T47Dshmdm2 cells (MDM2-depleted) cultured in heavy isotope amino acid medium, was subjected to proteolysis followed by phospho-peptide purification and enrichment. Scatter plot represents the H/L ratio versus abundance of peptides identified by mass spectrometry, with those corresponding to TP53BP1 (magenta), TP53 (blue), MCM2 (green), and MDC1(brown) highlighted. ( B ) Chromatin (5 μg) isolated from T47D vector control, T47Dshmdm2, and T47Dshmdmx cells was subjected to SDS–PAGE/western blot analysis for 53BP1, MDC1, MCM4, lamin A/C, and mtp53. ( C and D ) IF of total 53BP1 (i), phospho-53BP1 ser25 (ii), or phospho- 53BP1 ser1778 (iii) within T47D vector control nuclei [(i) 528, (ii) 549, and (iii) 501], T47Dshmdm2 nuclei [(i) 549,(ii) 520, and (iii) 501], and T47Dshmdmx nuclei [(i) 623, (ii) 376, and (iii) 501]. Confocal images for six fields for each were acquired and the number of 53BP1 foci per nucleus from each cell population indicated above was determined. Representative data ( n = 3) with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Multiplex sample analysis, Isolation, Plasmid Preparation, Control, Cell Culture, Purification, Mass Spectrometry, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: 53BP1 in breast cancer cells interacts with both mtp53 and MDM2. ( A ) Relative abundance of mtp53, MDM2, MDMX, 53BP1, and MDC1 within whole cell extracts (20 μg) prepared from T47D (L194F) and MDA-MB-231 (R280K) cell lines determined by SDS–PAGE/western blot analysis. ( B–D ) Association of mtp53, MDM2, and 53BP1 in vivo measured using the PLA. PLA analyses of mtp53-53BP1 (panel B), MDM2-mtp53 (panel C), and MDM2-53BP1 (panel D) within T47D and MDA-MB-231 cells were performed as described in the “Materials and methods” section; primary antibodies are PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 3 for T47D, n = 2 for MDA-MB-231). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: Panel B vector (T47D = 276; MDA-MB-231 = 108), shmdmx (T47D = 239; MDA-MB-231 = 128), and shmdm2 (T47D = 310; MDA-MB-231 = 106); Panel C vector (T47D = 92; MDA-MB-231 = 104), shmdmx (T47D = 108; MDA-MB-231 = 131), and shmdm2 (T47D = 101; MDA-MB-231 = 136); Panel D vector (T47D = 761; MDA-MB-231 = 129), shmdmx (T47D = 529; MDA-MB-231 = 142), and shmdm2 (T47D = 964; MDA-MB-231 = 163). **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: SDS Page, Western Blot, In Vivo, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2–53BP1 interaction is promoted by the mtp53 C-terminus. ( A ) The C-terminus of mtp53 R273H within MDA-MB-468 was modified using CRISPR–Cas9 to create the cell line MDA-MB-468 R273Hfs347Δ360-393 (termed G6; mtp53 derivative R273HΔC). ( B ) Relative protein levels of 53BP1, MDM2, and mtp53 within MDA-MB-468 (25, 12.5, 6.25, and 3.125 μg) and G6 (25 μg) cell lines was examined by SDS–PAGE/western blot analysis. ( C ) Loss of mtp53 C-terminus disrupts mtp53 co-IP with MDM2. Extracts from MDA-MB-468 and G6 cell lines were incubated with either normal mouse IgG (negative control) or anti-MDM2 antibodies 4B2 (lanes 1–7) or SMP14 (lanes 8–14), and IP reactions were examined for MDM2 and mtp53 by western blot analysis. Lanes 1–7: 4B2 IP reactions from 800 μg of extract (input); lanes contain 10% of total IP and 2% of input. Lanes 8–14: SMP14 IP reactions from 1600 μg of extract; lanes contain 12.5% each IP and 0.5% of input. Lanes labeled 2× (lane 7 for the 4B2 IPs and lane 14 for the SMP14 IPs) contain twice the amount of the G6 extract MDM2 IP; a lighter exposure of mtp53 input is presented due to the vast excess of mtp53 compared to MDM2 within the MDA-MB-468 cell lines. ( D–F ) PLA analysis of mtp53–53BP1 (panel D), MDM2–mtp53 (panel E), and MDM2–53BP1 (panel F) in MDA-MB-468 and G6. PLA analysis of the indicated proteins was measured using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 2A9 antibodies. Confocal images for 3–5 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 2). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (D) R273H = 120; R273HΔC = 144; (E) R273H = 131; R273HΔC = 159; (F) R273H = 102; R273HΔC = 138. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, CRISPR, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Incubation, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2-mtp53 and MDM2–53BP1 interactions are Nutlin 3a sensitive. ( A ) Western blot analysis of whole cell extracts (10 μg) from MCF7 and T47D cell line populations treated for the indicated time with either vehicle or 10 μM Nutlin 3a (labeled N3a or “+” in graphs) for the indicated proteins. ( B ) Nutlin 3a does not inhibit T47D cell cycle progression. Twenty-four hour post-treatment with either vehicle or 10 μM Nutlin 3a, cells were labeled with EdU for 20 min and assayed for Cyclin A2 by immunofluorescence. Confocal images from at least three fields were acquired and the number of EdU + and Cyclin A + nuclei were quantified (tabulated in S4, panel C) in each population of vehicle-treated [vector = 289; shmdmx = 278; shmdm2 = 249] and Nutlin 3a-treated [vector = 227; shmdmx = 283; shmdm2 = 245] cells. The S/G2 fraction (total Cyclin A + nuclei) for vehicle-treated: vector = 33.6%, shmdmx = 37.8%, shmdm2 = 36.8%; Nutlin 3a-treated: vector = 38.3%, shmdmx = 27.0%, shmdm2 = 29.4%. ( C and D ) Nutlin 3a disrupts mtp53–MDM2 and 53BP1–MDM2 PLA foci in T47D. Twenty-four hour post-treatment with vehicle or 10 μM Nutlin 3a cell populations PLA interactions were measured between MDM2–mtp53 (panel C) and MDM2–53BP1 (panel D) using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2 primary antibodies. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus from each cell population was determined ( n = 2 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the materials and methods from the indicated number of cells in panel: (C) vehicle-treated [vector = 328; shmdmx = 298; shmdm2 = 249], Nutlin 3a-treated [vector = 323; shmdmx = 364; shmdm2 = 285] and (D) vehicle-treated [vector = 543; shmdmx = 548; shmdm2 = 455], Nutlin 3a-treated [vector = 610; shmdmx = 545; shmdm2 = 618]. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Western Blot, Labeling, Immunofluorescence, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: MDM2 promotes 53BP1–MDC1 complex formation. ( A ) Reduced 53BP1–MDC1 PLA foci in T47D lacking MDM2. EdU-labeled T47D vector and shmdm2 cells were assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Shown under representative images ( n = 3) is the number of EdU + nuclei identified within each cell population and graphed is the number of PLA foci per nucleus. For the PLA analysis representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: vector (total = 329, S-phase = 100, G1-G2 = 229); shmdm2 (total = 251, S-phase = 79, G1-G2 = 172). ( B ) Activation of the DDR in T47D cell lines by Etoposide but not Nutlin-3a. Whole cell extracts (20 μg) from T47D cell lines treated with either 50 μM Etoposide for 5 h (Etop), or 10 μM Nutlin 3a for 24 h (N3a) were analyzed for the indicated proteins by WB. ( C and D ) MDC1–53BP1 PLA foci are disrupted by both DDR activation and Nutlin 3a. MDC1–53BP1 PLA analyses were performed within each T47D cell line at the indicated time points post Etoposide treatment (panel C) or 24 h-post Nutlin 3a treatment (panel D) using PLA goat anti-53BP1 and PLA rabbit anti-MDC1. Confocal images for 3–6 fields for each were acquired and the number of PLA per nucleus from each cell population was determined ( n = 3 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (C) vehicle-treated (0 h Etop) [vector = 208; shmdmx = 198; shmdm2 = 247], 2 h Etoposide [vector = 205; shmdmx = 184; shmdm2 = 231], 5 h Etoposide [vector = 189; shmdmx = 224; shmdm2 = 176]; (D) vehicle-treated [vector = 198; shmdmx = 270; shmdm2 = 336] and Nutlin 3a-treated [vector = 380; shmdmx = 339; shmdm2 = 252]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Labeling, Plasmid Preparation, Activation Assay

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. ( A ) Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. 53BP1 was immunoprecipitated from T47D CES as described in the “Materials and methods” section subjected to western blot analysis for 53BP1, MDC1, MDM2, and p53. Lanes are as follows: (1) cytoplasmic extract, (2) CES IP input, (3) IP with IgG, and (4) IP for 53BP1. ( B ) Inhibition of ATM promotes MDM2 activity. Western blot analysis for the indicated proteins within extracts (10 μg) from T47D populations treated for 24 h with either vehicle,10 μM ALRN-6924 (MDM2/X dual inhibitor), 10 μM KU-55933 (ATMi; ATM inhibitor) or both. ( C ) The MDM2 inhibitor ALRN-6924 reduces whereas the ATM inhibitor increases MDC1–53BP1 PLA foci in T47D. Twenty-four hour post treatment with either vehicle,10 μM ALRN-6924, 10 μM KU-55933 (ATMi) or both T47D vector and shmdm2 cells were labeled with EdU for 20 min and then assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Confocal images for several fields were acquired and the number of PLA foci/nucleus from each cell population was determined. Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in: vehicle-treated [vector = 186; shmdm2 = 87], ATMi-treated [vector = 163; shmdm2 = 188], ALRN-6924-treated [vector = 196; shmdm2 = 176], ALRN-6924 + ATMi-treated [vector = 154; shmdm2 = 159]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Inhibition, Activity Assay, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Depletion of MDM2 increases poly(ADP-ribose) modification (PARylation) levels of chromatin bound proteins. Cytosolic ( A ) and chromatin ( B ) fractions were prepared from T47D cells with constitutive shmdm2, shmdmx, or mir30 shRNA-expressing vector cells treated with either vehicle (DMSO), or a combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h, or combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h and then replaced with fresh media for an additional 24 h. Ten micrograms of cytosolic or chromatin protein was loaded on a SDS–PAGE and protein levels were determined by western blot analysis using the indicated antibodies.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, shRNA, Expressing, Plasmid Preparation, SDS Page, Western Blot

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 1. OX40 expression on pDCs in the TME of HNSCC. (A) OX40 expression in the TME (measured by flow cytometry) of HNSCC patients on differ- ent immune cell subsets — pDCs (n = 89), cDCs (n = 53), CD8+ T cells (n = 16), CD4+ T cells (n = 17), CD4+ Th1 T cells (n = 12), and CD4+ Treg cells (n = 14). T cell subsets were gated from live CD45+CD3+ cells. Th1 cells were defined as CD4+Tbet+ T cells and Treg cells were defined as CD4+Foxp3+ cells. (B) Gating strategy for FACS analysis and sorting of OX40+ and OX40lo/– pDCs from patient specimens. After selecting for singlets and live cells, pDCs were gated from HLA-DRhiLineage– cells, followed by CD11c–CD123+ cells. pDCs were further confirmed by expression of CD303 (BDCA-2). OX40 expression on pDCs was determined using internal negative controls. (C) Immunofluorescence of pDCs in the TME demonstrating OX40 and CD123 coexpression. n = 4, with 4 patient repeats. Original magnification, ×63. Scale bar: 5 μm. Red, OX40; green, CD123; blue, DAPI. (D) OX40 expression on pDCs from different anatomic sites: PBMC (n = 17), dLN– (n = 50) or dLN+ (n = 59), and primary tumor (n = 53). (E) Correlation (Pearson, with a line of best fit) between OX40 and ICOSL expression on matched patient TME pDCs (n = 28). One-way ANOVA followed by Tukey’s post hoc test (A and D). **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (A, D, and E).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 3. OX40+ pDCs promote antigen-specific CD8+ T cell responses. (A) Illustration of the antigen-specific in vitro coculture model, in which autologous OX40+/OX40lo/– pDCs from the TME/non-TME of HNSCC patients (n = 9) were cocultured with autologous TAA peptide-loaded mDCs and CD8+ T cells for 5 to 6 days, at which point antigen-specific CD8+ T cell responses were measured, (B) including for proliferation (eFluor 450–low) and IFN-γ production as demonstrated in flow plots of a patient’s CD8+ T cells cocultured with OX40+ or OX40lo/– pDCs sorted from their tumors. CD8+ T cell positivity was also measured for (C) Tbet and (D) eFluor 450–low in these coculture experiments. (E) CD8+ T cell positivity for CD69 after coculture with TAA peptide–loaded mDCs without pDCs (control) or with OX40+ or OX40lo/– pDCs from the TME versus non-TME (dLN–) (n = 5). (F) Illustration depicting the Transwell coculture assay in which OX40+ or OX40lo/– pDCs in the top chamber were separated from autologous CD8+ T cells and peptide-loaded mDCs in the bottom chamber. (G) Percentage of proliferating (eFluor 450–low) and GzB+ CD8+ T cells in Transwell versus contact coculture (n = 3). Representative flow plots show GzB production by CD8+ T cells cocultured with E7-loaded mDCs and OX40+ or OX40lo/– pDCs in coculture contact or separated by Transwell. (H) Flow plots comparing antigen presentation capacities of autologous OX40+ and OX40lo/– pDCs with mDCs, based on cytolytic CD8+ T cell responses (no peptide controls for these plots are shown in Supplemental Figure 2B). Shown is GzB production by CD8+ T cells in the presence or absence of OX40+/OX40lo/– pDCs (top) and IL-12p70 production by mDC/pDC subsets (bottom). n = 2; 2 experimen- tal repeats. One-way ANOVA followed by Tukey’s post hoc test (C–E and G). Bar graph data are mean ± SEM; *P < 0.05. NS, not significant. Middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (C–E and G).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: In Vitro, Control, Co-culture Assay, Immunopeptidomics, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 4. The OX40-OX40L axis is utilized by pDCs in the TME. (A) OX40L expression measured by flow cytometry on OX40+ and OX40lo/– pDCs (n = 7). Correla- tion (Pearson, with line of best fit) of OX40 and OX40L expression intensities (per-cell normalized counts, total weighting) on pDCs measured on an HNSCC TME tissue section. n = 4; 4 patient repeats. (B) Immunofluorescence images from the TME showing a cell’s coexpression of OX40 (red) with CD123 (green), sitting adjacent to CD8-expressing (magenta) cells. Gallery view of Z-stacks (collected at 0.29-μm intervals). Original magnification, ×63. Scale bar: 5 μm. n = 4; 4 patient repeats. (C) Processed multispectral image (steps outlined in Supplemental Figure 2E for same example image) of the TME. Original magnification, ×40. Inset: a cluster CD123+ (green) cells, including one with OX40 expression (orange), next to a CD8+ (magenta) cell. (D) Representative image file written using Phenoptr to calculate touching pairs of phenotyped OX40L+CD68+ macrophages with phenotyped OX40+ pDCs. (E and F) Percentage of phenotyped OX40+/ OX40lo/– pDCs touching other OX40L+ cells, including pDCs, macrophages (Mφ), and “other cells” (Methods), in the TME and non-TME of patients (n = 3). (G) Ratios of CD8+ T cell counts within 30 μm of pDC subsets to total counts of CD123+ pDC subsets (OX40+/OX40lo/–) in the TME and non-TME of patients (n = 5). (H) Intercellular distances of phenotyped pDC subsets to the closest tumor margin (μm). Original magnification, ×40. n = 3; 3 patient repeats. One-way ANOVA followed by Tukey’s post hoc test (F) and unpaired (E), and paired (A, G, and H) t tests. *P < 0.05; **P < 0.01; NS, not significant. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 6. OX40+ pDCs correlate to survival in cancer patients and suppress tumor growth. (A) Prospective recurrence-free survival (log-rank, Mantel-Cox test) of HNSCC cohort (n = 80), stratified by median (45%) intratumoral pDC OX40 expression, as measured by flow cytometry. (B) Overall survival (log-rank, Mantel-Cox test) of HNSCC patients (n = 500) from the GDC data portal, stratified first by median pDC gene signature Z scores followed by stratification of mean TNFRSF4 (encodes OX40) mRNA levels. (C) Correlation (Pearson, with line of best fit) of TNFRSF4 log2 mRNA levels (among cases with pDChi gene signatures) with CD8+ T effector scores in HNSCC (n = 172). (D) OX40 expression on intratumoral pDCs from different murine tumor mod- els. n =4; 4 experimental replicates. (E) gp100-specific Pmel-1 CD8+ T cell IFN-γ production by proliferating (eFluor 450–low) CD8+ T cells, measured in the presence or absence of pDCs from the dLNs of B16-F10– and B16CCR7-bearing mice. n = 2; 2 experimental repeats. (F) gp100-specific proliferating (eFluor 450–low) Pmel-1 CD8+ T cells in the presence or absence of B16CCR7 pDCs prestimulated with Resiquimod and OX86. n = 2; 2 experimental repeats. (G) Effect of pDC depletion (anti-PDCA1) in B16-F10– and B16CCR7-bearing mice compared with controls (anti-polyclonal IgG). Data are pooled from at least 2 independent experiments with 3 to 5 mice per group. (H) Quantification (by flow cytometry) of conventional cDCs (CD11c+CD11b–) and CD8a+ cDCs from B16CCR7-bearing mice treated with anti-PDCA1 or anti-polyclonal IgG. Data are pooled from individual experiments and normalized to 5 × 105 live cells. One-way ANOVA followed by Tukey’s post hoc test (D), 2-way ANOVA with Sidak’s test for multiple comparisons (H), and unpaired Student’s t test (G). **P < 0.01; ***P < 0.001. Tumor burden data and bar graph data are mean ± SEM.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry

Journal: Journal of Neuroscience

Article Title: CD200 Ligand Receptor Interaction Modulates Microglial Activation In Vivo and In Vitro: A Role for IL-4

doi: 10.1523/jneurosci.1781-07.2007

Figure Lengend Snippet: Figure1. CD200isexpressedonneuronsandisreducedinchronicneuroinflammatorycon- ditions.A,DoubleimmunofluorescenceforCD200(i)andIII-tubulin(ii)andamergedimage (iii) in cultured neurons. B, Double immunofluorescence for MHCII (i) and CD200R (ii) and a merged image (iii) in mixed cultured glia treated with A. C, CD200 protein expression de- creases with age as shown by Western blot (*p 0.05; n 13). D, Fluorescent images of CD200 in the dentate gyrus of young (i) and aged (ii) animals. E, Age-related increase in the expression of MHCII mRNA (***p 0.001; n 13). F, Images of MHCII staining in the hip- pocampalCA1regionofyoung(i)andaged(ii)animals.Scalebars:A,B,20M;D,10M;F,50 M. Error bars indicate SEM.

Article Snippet: Cells and sections were treated overnight at 4°C as follows: mouse monoclonal CD200 antibody (1:200; Abcam, Cambridge, UK), mouse monoclonal MHCII antibody (1:100; Serotec), goat polyclonal CD200R antibody (1:100; Santa Cruz Biotechnology), and mouse monoclonal III-tubulin antibody (1:200; Chemicon).

Techniques: Cell Culture, Immunofluorescence, Expressing, Western Blot, Staining

Journal: Journal of Neuroscience

Article Title: CD200 Ligand Receptor Interaction Modulates Microglial Activation In Vivo and In Vitro: A Role for IL-4

doi: 10.1523/jneurosci.1781-07.2007

Figure Lengend Snippet: Figure 2. CD200 expression is reduced in acute neuroinflammatory conditions both in vivo and in vitro. A, A decreases CD200 protein expression in vivo as assessed by Western blot analysis(*p0.05;n12).B,FluorescentimagesofCD200inthedentategyrusofcontrol(i) and A-treated (ii) animals. C, A treatment increases MHCII mRNA expression (***p 0.001;n20).D,FluorescentimagesofMHCIIincontrol(i)andA-treated(ii)animals.E,A decreasesCD200proteinexpressioninvitroasdeterminedbyWesternblotanalysis(*p0.05; n 6). F, Fluorescent images of CD200 in the control (i) and A-treated (ii) neurons. G, A treatment increases MHCII mRNA expression in vitro (***p 0.001; n 4). H, Fluorescent images of microglia in the control (i) and A-treated (ii) cells. Scale bars, 20 M. Error bars indicate SEM.

Article Snippet: Cells and sections were treated overnight at 4°C as follows: mouse monoclonal CD200 antibody (1:200; Abcam, Cambridge, UK), mouse monoclonal MHCII antibody (1:100; Serotec), goat polyclonal CD200R antibody (1:100; Santa Cruz Biotechnology), and mouse monoclonal III-tubulin antibody (1:200; Chemicon).

Techniques: Expressing, In Vivo, In Vitro, Western Blot, Control

Journal: Journal of Neuroscience

Article Title: CD200 Ligand Receptor Interaction Modulates Microglial Activation In Vivo and In Vitro: A Role for IL-4

doi: 10.1523/jneurosci.1781-07.2007

Figure Lengend Snippet: Figure4. IL-4increasesneuronalCD200expression.A,IL-4increasesCD200proteinexpres- sion in cultured neurons by Western blot (***p 0.001). B, Fluorescent images of CD200 expression in cultured neurons in the absence (i) and presence (ii) of IL-4. C, Intracerebroven- tricular injection of IL-4 increases CD200 protein expression in hippocampus (*p 0.05; n 3).D,FluorescentimagesofincreasedCD200expressioninhippocampusofIL-4-treatedrats(ii) versuscontrol(i).E,CD200expressionissignificantlydecreasedinhippocampaltissueprepared from IL-4 / mice (*p 0.05; n 4). F, CD200 expression is significantly decreased in cultured neurons prepared from IL-4 / mice (ii) compared with neurons prepared from wild-type mice (i). G, MHCII mRNA expression is increased in cultured glial cells prepared from IL-4 / mice (*p 0.05; n 5). H, IL-1 concentration is increased in cultured glial cells prepared from IL-4 / mice (**p 0.01; n 10). Scale bars, 20 M. Error bars indicate SEM. WT, Wild type.

Article Snippet: Cells and sections were treated overnight at 4°C as follows: mouse monoclonal CD200 antibody (1:200; Abcam, Cambridge, UK), mouse monoclonal MHCII antibody (1:100; Serotec), goat polyclonal CD200R antibody (1:100; Santa Cruz Biotechnology), and mouse monoclonal III-tubulin antibody (1:200; Chemicon).

Techniques: Cell Culture, Western Blot, Expressing, Injection, Concentration Assay

Journal: Scientific Reports

Article Title: Elimination of microglia improves cognitive function following cranial irradiation

doi: 10.1038/srep31545

Figure Lengend Snippet: ( A ) Immunofluorescence staining show that PLX5622 treatment for 6 weeks reduces ~70% of CD68 + activated microglia from the control (0 Gy + PLX5622) and irradiated (9 Gy + PLX5622) brains. (a1,a2) Representative high-resolution (60×) z stacks show ramified microglial morphology in the irradiated hippocampal dentate hilus (DH) and granule cell layer (GCL) compared to 0 Gy mice that received control chow. ( B ) Quantification (Autoquant and Imaris) of CD68 + activated microglia indicated an 80–90% reduction in the control and irradiated brains receiving PLX5622 (0 Gy + PLX5622 and 9 Gy + PLX5622) at 2 week and 6 week time points. ( C ) Analysis of pro-inflammatory markers from whole brains derived from irradiated mice (0 and 9 Gy) treated with PLX5622 for 1 week at 4 week post-irradiation show radiation-induced elevation in gene expression that was reduced significantly by PLX5622 treatment. Data are presented as mean ± SEM ( N = 4 mice/group). P values are derived from ANOVA and Bonferroni’s multiple comparisons test. * P < 0.01; ** P < 0.001 compared with 0 Gy group and + P < 0.01; ++ P < 0.01compared with 9 Gy group. Scale bars: 200 μm ( A ) and 20 μm (a1,a2).

Article Snippet: For the immunofluorescence analysis of microglia, the following primary and secondary antibodies were used: rabbit anti-IBA-1 (1:500, Wako), rat anti-mouse CD68 (1:500, AbD Serotec), donkey anti-rabbit or anti-mouse conjugated with Alexa Fluor 488 or 594 (Life Technologies/Invitrogen) and DAPI nuclear counterstain (Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Control, Irradiation, Derivative Assay, Gene Expression

Journal: Scientific Reports

Article Title: Elimination of microglia improves cognitive function following cranial irradiation

doi: 10.1038/srep31545

Figure Lengend Snippet: ( A,B ) Immunofluorescence staining and laser scanning confocal microscopy for the IBA-1 + and CD68 + cells show that cranial irradiation lead to significant elevation in microglial number (40% and 25% respectively) in the pre-limbic (PrL) and infra-limbic (IL) cortices of the medial pre-frontal cortex (mPFC). (a1,b1) Representative high-resolution (60×) z stacks showed characteristic activated microglial morphology (IBA-1, a1 and CD68, b1) in the irradiated PrL compared to 0 Gy group. ( C,D ) Treatment with PLX5622 for 6 weeks eliminates 80–90% of IBA-1 + and CD68 + microglia from the control (0 Gy + PLX5622) and irradiated (9 Gy + PLX5622) mPFC. Data are presented as mean ± SEM ( N = 4 mice/group). P values are derived from ANOVA and Bonferroni’s multiple comparisons test. * P < 0.05; ** P < 0.001 compared with 0 Gy group and + P < 0.05; ++ P < 0.01compared with 9 Gy group. Scale bars: 200 μm ( A ), 100 μm ( B ) and 50 μm (a1,b1).

Article Snippet: For the immunofluorescence analysis of microglia, the following primary and secondary antibodies were used: rabbit anti-IBA-1 (1:500, Wako), rat anti-mouse CD68 (1:500, AbD Serotec), donkey anti-rabbit or anti-mouse conjugated with Alexa Fluor 488 or 594 (Life Technologies/Invitrogen) and DAPI nuclear counterstain (Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Irradiation, Control, Derivative Assay

Journal: Pharmaceutical Biology

Article Title: Methamphetamine leads to the alterations of microRNA profiles in the nucleus accumbens of rats

doi: 10.1080/13880209.2020.1803366

Figure Lengend Snippet: The effects of METH on neuron differentiation by immunofluorescence. The NSE positive cells decreased (A) while GFAP positive cells (B) increased in the striatum, hippocampus, and NAc after METH treatment.

Article Snippet: The primary antibody of NSE (neuron specific enolase, catalog No. AF5169) and GFAP (glial fibrillary acidic protein, catalog No. AF2594) was obtained from the R&D system (Minneapolis, MN, USA).

Techniques: Immunofluorescence

Journal: Nature Communications

Article Title: MAIT cells protect against pulmonary Legionella longbeachae infection

doi: 10.1038/s41467-018-05202-8

Figure Lengend Snippet: Human MAIT cells are activated by Legionella infection via MR1 in vitro. a Jurkat.MAIT and C1R.MR1 cells were co-incubated for 16 h with lysates of L. pneumophila (L. pn.) or L. longbeachae or 5-OP-RU, acetyl-6-formylpterin (Ac-6-FP) or PBS. Activation, detected by staining with anti-CD69, is enhanced by bacterial lysate or by the activating ligand 5-OP-RU, but not by acetyl-6-FP. Activation was blocked by anti-MR1 antibody (26.5) but not by isotype control (W6/32) 2 h prior to co-incubation. Experiment performed in triplicate wells on two separate occasions with similar results. Data show mean fluorescence intensity, MFI (±SEM). Statistical tests: one-way ANOVA and post hoc Dunnett’s comparing all columns with the first column (black). Unpaired t -test (blue), with *** P < 0.001; **** P < 0.0001. b , c THP1 cells (WT) or THP1 cells overexpressing MR1 (THP1.MR1+, purple) or deficient in expression of MR1 (THP1.MR1−, blue) were infected for 27 h with live or heat-killed (HK) L. longbeachae (MOI: 100) or 10 nM 5-OP-RU, then co-cultured for 16 h with sorted CD3 + Vα7.2 + CD161 + human peripheral blood MAIT cells, or MAIT-depleted conventional T cells. MR1-5-OP-RU-tetramer+ MAIT cell activation was measured by intracellular cytokine staining for b TNF or c IFN-γ. b , c Percentage cytokine-positive cells as mean (±SEM) data from three independent donors performed on two separate occasions are shown. Statistical tests: unpaired t- tests with Bonferroni corrections, each comparing against MOI 0 for the specific cell line. Statistics with * P < 0.05; ** P < 0.01. d Immunofluorescence micrographs showing CD3+TCRVα7.2+ MAIT cell (white arrow) within healthy human lung tissue (top panel) and 24 h post infection (bottom panels) ex vivo with L. longbeachae . Yellow arrow: intracellular L. longbechae bacilli. Red, TCRVα7.2; green, CD3; magenta, polyclonal rabbit anti -L. longbeachae ; blue, nuclei (Hoechst)

Article Snippet: Following rehydration, the unfixed cryopreserved 8 μm sections of human lung were blocked with serum-free block and stained with purified anti-Vα7.2 TCR mAb clone 3C10 (Biolegend), rat anti-human CD3 mAb (BioRad) and polyclonal rabbit anti- Legionella antibody.

Techniques: Infection, In Vitro, Incubation, Activation Assay, Staining, Control, Fluorescence, Expressing, Cell Culture, Immunofluorescence, Ex Vivo

Journal: PLoS ONE

Article Title: Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation

doi: 10.1371/journal.pone.0054722

Figure Lengend Snippet: List of primary antibodies used for immunofluorescence studies.

Article Snippet: CD11b/OX42 , mouse anti-rat CD11b , 1∶150 , Serotec.

Techniques: Immunofluorescence, Immunopeptidomics, Purification, Binding Assay