Journal: Nature Communications

Article Title: Ferroptotic damage promotes pancreatic tumorigenesis through a TMEM173/STING-dependent DNA sensor pathway

doi: 10.1038/s41467-020-20154-8

Figure Lengend Snippet: a Survival of Pdx1-Cre;Kras G12D/+ ;Gpx4 −/− (KCG) or Pdx1-Cre;Kras G12D/+ ; Gpx4 −/− ;Tmem173 −/− (KCGT) mice with or without control IgG or anti-8-OHG antibody treatment ( n = 10 mice/group; log-rank [Mantel–Cox] test). b Pancreatic weight of the indicated mice (6 months; n = 5 mice/group; two-way ANOVA with Tukey’s multiple comparisons test). c Representative pancreas histology of the indicated mice. d Percentages of histological structures in the pancreas of the indicated mice ( n = 5 mice/group; two-way ANOVA with Tukey’s multiple comparisons test). e Representative images of immunofluorescence staining of macrophages (red) in pancreas in indicated mice at the age of 3 months. f Relative gene expression in the pancreas of the indicated mice ( n = 3 mice/group; two-way ANOVA with Tukey’s multiple comparisons test). g Percentage of abnormal telomeres by FISH analysis in ductal cells from KCG and KCGT mice at 3 months of age ( n = 5 mice/genotype). h mRNA expression of Tmem173 in indicated mice at 3 months of age with or without clophosome treatment ( n = 3 mice/group; two-way ANOVA with Tukey’s multiple comparisons test). Data in b , d , and f – h are presented as mean ± SD. Data are from two or three independent experiments.

Article Snippet: To study the effects of 8-OHG inhibition on pancreatic tumorigenesis, 4–6 weeks old indicated that mice were randomly allocated into groups and injected i.p. with mouse monoclonal anti-8-OHG antibody (10 mg/kg; #GTX41980, RRID:AB_10732443, GeneTex) and control IgG2B (10 mg/kg; #MAB004, RRID:AB_357346, R&D Systems) per week for 12 weeks.

Techniques: Control, Immunofluorescence, Staining, Gene Expression, Expressing

Journal: Cell Reports

Article Title: Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity

doi: 10.1016/j.celrep.2018.08.006

Figure Lengend Snippet:

Article Snippet: The human/mouse Anti-UCP1 antibody (MAB6158, monoclonal Mouse IgG 2B Clone # 536435, R&D Systems) was conjugated to Alexa647 (ThermoFisher Alexa Fluor 647 antibody labeling kit A20186) and incubated with adipocytes at 1:300 for 1 h. Mouse IgG2B Alexa Fluor 647-conjugated Isotype Control antibody (R&D systems) was used as a control.

Techniques: Control, Recombinant, Blocking Assay, Antibody Labeling, Software, Flow Cytometry

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Impact of T-DM1 treatment on metastasis free survival in a prostate cancer left ventricle injection xenograft model. (A) Experimental design for the in vivo experiment. GFP/luciferase-expressing PC3 cells were injected into male SCID mice by left ventricle intracardiac (i.c.) injection. Human IgG (15 mg/kg) or T-DM1 (15 mg/kg) were injected by intraperitoneal (i.p.) injection every 3 days until 12 days after tumor injection IgG: n = 12. T-DM1: n = 14. (B) Kaplan-Meier analysis of time to formation of metastases visible by bioluminescence imaging or death. (C) Representative bioluminescence images of control mice (human IgG injected) and T-DM1 treated mice 124 days after tumor injection. (D) Antibody dependent cellular cytotoxicity (ADCC) assays using flow cytometry. PC3 cells were labeled with DiD for subsequent identification, and were treated with T-DM1 before addition of splenocytes from SCID mice. PC3 cells were identified as DiD positive and non-viable cells were identified as positive for DAPI. (E) Dead PC3 cells (%) quantified from data in panel (D) Data represent mean ± S.D. (N=3).

Article Snippet: Monoclonal mouse IgG2B (R&D systems, Cat #: IC0041V) was used as isotype control.

Techniques: Injection, In Vivo, Luciferase, Expressing, Imaging, Control, Flow Cytometry, Labeling

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: TRAIL induces the assembly of apoptotic and non-apoptotic signaling complex(es). ( a ) Surface levels of Death Receptor 5 (anti-DR5-488), Death Receptor 4 (anti-DR4 PE), Decoy Receptor 1 (anti-DcR1 PE) and Decoy Receptor 2 (anti-DcR2-488) in BJELR cells. Surface levels of Death Receptor 4 in Hela cells and surface level of Decoy Receptor 1 in BJ cells were used as a positive control for DR4 and DcR1 labeling, respectively. Isotypic IgG1PE or Alexa 488 or IgG2B Alexa 488 labeling was used as a control for background fluorescence. ( b ) Western blots assessing the co-immunoprecipitation of indicated proteins (canonical DISC components) with Death Receptor 5 (immunoprecipitation target; DR5 IP), Death Receptor 4 (immunoprecipitation target; DR4 IP) or Decoy Receptor 2 (immunoprecipitation target; DcR2 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with TRAIL for 30 min (+). NF, not detected. ( c ) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with Death Receptor 5 (DR5 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with 1 μ g/ml TRAIL for 30 min (+). ( d ) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with caspase-8 (immunoprecipitation target; C8 IP) from whole-cell lysates obtained from naïve cells (−) or cells treated with TRAIL for 30 min (+). ( b – d ) Depicted images correspond to one representative experiment out of three independent biological replicates. Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Positive Control, Labeling, Control, Fluorescence, Western Blot, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Apoptotic and pro-survival TRAIL-induced signaling complex(es) are assembled at the plasma membrane. ( a ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 and TRAF2 with Death Receptor 5 (DR5 immunoprecipitation: DR5 IP). ( b and c ) Western blots assessing the co-immunoprecipitation of DR5, DR4, DcR2, FADD, cFlip and TRAF2 ( b ) as well as caspase-8 ( c ) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP). ( d ) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DR5, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 with TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP). ( e ) Western blots assessing the co-immunoprecipitation of DR5, RIPK1 and TRAF2 with FADD (FADD immunoprecipitation: FADD IP). ( a–e ) Immunoprecipitations were performed from the purified plasma membrane (plasma membrane) obtained from either naïve cells (−) or cells treated with TRAIL (30 min '+' unless indicated otherwise in c ). Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control. Protein levels of indicated proteins at the plasma membrane are depicted as 'inputs'. Same blots are depicted as inputs for ( d ) and ( e ) as TRAF2 and FADD immunoprecipitation experiments were performed in parallel using the same protein input. Depicted images correspond to one representative experiment out of at least two independent biological replicates. N-Cadherin (N-Cdh) is shown as a plasma membrane marker. Controls for plasma membrane purification are depicted as . ND, not detected

Article Snippet: For surface expression of TRAIL receptors, Mouse IgG1 (IC002G), Mouse IgG2B (IC0041G), DcR1 (FAB6302P), DcR2 (FAB633G) and DR5 (FAB6311G) are from R & D Systems; DR4 (804-297TD-T100) from Enzo Life Sciences (Switzerland); and DR4 (cat. # 854.852.010) and DR5 (cat.# 854.862.010) from Diaclone (France).

Techniques: Clinical Proteomics, Membrane, Western Blot, Immunoprecipitation, Purification, Control, Marker

Journal: Nature immunology

Article Title: Complement-mediated regulation of the IL-17A axis is a central genetic determinant of the severity of experimental allergic asthma.

doi: 10.1038/ni.1926

Figure Lengend Snippet: Figure 8 C5a-induced IL-10 regulates IL-23 production by engaging Jnk and AP-1. (a) IL-10 production by medium- or HDM- treated BMDCs (BALB/c) in the presence of medium alone (Ctrl) or recombinant C3a (rC3a) or C5a (rC5a). *P < 0.05 and **P < 0.001 (one-way ANOVA). (b) IL-23 production by medium- or HDM- treated BMDCs (BALB/c) with (rC5a) or without (Ctrl) recombinant C5a in the presence of IgG1, anti-IL-10 (α-IL-10) or recombinant IL-10 (rIL-10). (c,d) Production of IL-23 (c) and IL-10 (d) by BMDCs (BALB/c) pretreated for 1 h with dimethyl sulfoxide (DMSO), SP600125, AS602868 or rapamycin, then treated with medium or HDM (100 μg/ml) in the presence (rC5a) or absence (Ctrl) of recombinant C5a. (e) Immunoblot analysis of c-Jun phosphorylated at Ser63 (p-c-Jun (Ser63)) and total c-Jun in medium- or C5a-treated RAW264.7 cells. (f) AP-1 activity in RAW264.7 cells treated with medium or various combinations of HDM, recombinant C5a and SP600125 (SP). (g) AP-1 activity in RAW264.7 cells stimulated with medium or various combinations of HDM, recombinant C5a, IgG1, anti-IL-10 and recombinant IL-10. (h) Il23a promoter activity in RAW264.7 cells cotransfected with pcDNA3.1 vector alone (p-cDNA) or vector containing c-Jun cDNA (p-c-Jun) or c-Fos cDNA (p-c-Fos), then treated with medium or HDM. (i) Jun and Fos mRNA in medium- or HDM-treated A/J and C3H/HeJ BMDCs, presented relative to S14 expression. Results are representative of two to three independent experiments (mean and s.e.m. of four to eight individual samples).

Article Snippet: Alternatively, BALB/c mice were treated as described in Supplementary Figure 1a; some mice received 120 μg rat mAb to mouse IL-17A or IgG2a intraperitoneally as described above, and 35 μg anti-C5aR (20/70; MCA2457EL; AbD Serotec) or IgG2b (MAB0061; R&D Systems) intratracheally on days −1 and +13 (relative to day 0 described above).

Techniques: Recombinant, Western Blot, Activity Assay, Plasmid Preparation, Expressing

Journal: Nanomaterials

Article Title: Scavenger Receptor A1 Mediates the Uptake of Carboxylated and Pristine Multi-Walled Carbon Nanotubes Coated with Bovine Serum Albumin

doi: 10.3390/nano11020539

Figure Lengend Snippet: Immunofluorescence microscopy and flow cytometric analysis of WT and SR-A1 knockout RAW 264.7 cells. Immunofluorescence images of (A) control WT RAW 264.7 cells without mouse anti-SR-AI/MSR Alexa Fluor ® 488-conjugated antibody, (B) WT RAW 264.7 cells incubated with mouse anti SR-AI/MSR Alexa Fluor ® 488-conjugated antibody, and ( C , D ) two different clones (C4 and B11) of SR-A1 knockout RAW 264.7 cells incubated with anti-mouse SR-AI/MSR Alexa Fluor ® 488-conjugated antibody. Hoechst 33342 staining is shown in blue and Alexa Fluor ® 488 staining is shown in green. All images are normalized to the same intensity scale, and the scale bars represent 20 μm. (E) Flow cytometry analyses where the black line represents WT RAW 264.7 cells incubated with the rat IgG2B Alexa Fluor ® 488-conjugated monoclonal antibody as the isotype control, the red line represents WT RAW 264.7 cells incubated with mouse anti-SR-AI/MSR Alexa Fluor ® 488-conjugated antibody, and the green and purple lines represent two different clones (C4 and B11, respectively) of SR-A1 knockout RAW 264.7 cells incubated with mouse anti-SR-AI/MSR Alexa Fluor ® 488-conjugated antibody. The x-axis denotes fluorescence detected in the 518–548 nm spectral region, and the y-axis denotes the number of events for each analysis.

Article Snippet: The cells were stained with 5 μg mouse SR-AI/MSR Alexa Fluor ® 488-conjugated antibody (R&D Systems cat. No. FAB1797G) or a rat IgG2B Alexa Fluor ® 488-conjugated monoclonal antibody (R&D Systems cat. No. IC013G) as the isotype control for 30 min at 4 °C in the dark.

Techniques: Immunofluorescence, Microscopy, Knock-Out, Control, Incubation, Clone Assay, Staining, Flow Cytometry, Fluorescence