Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A ) FC was used to monitor binding of FITC-labeled 4-1BB magnetic beads to activator cells following up to four rounds of selection using streptavidin–4-1BB magnetic beads. ( B ) FC was used to monitor 4-1BBL expression in activator cells following up to four rounds of enrichment with streptavidin–4-1BB magnetic beads. ( C ) The gRNA enrichment following first, second, third, or fourth round of selection using streptavidin–4-1BB magnetic beads was monitored using −log 10 (score). For each library, the percentages indicate the total gRNA counts for the gene of interest divided by the total number of gRNA counts for all genes (×100). ( D ) Immunoblotting was used to detect myc in 293 cells transfected with siglec-4–myc or 4-1BB–myc. β-Actin (β-act) was included as a loading control. ( E ) FC was used to measure the level of siglec-4 or 4-1BBL on the surface of 293 cells following transfection with pCMV EV (control), pCMV–siglec-4 (siglec-4), or pCMV–4-1BBL (4-1BBL). ( F ) Binding of 4-1BB–HIS to 293 cells transfected with EV (negative control), pCMV–4-1BB (positive control), or pCMV–siglec-4. Data in (A), (B), (D), (E), and (F) were representative of three experiments.

Article Snippet: For h4-1BB protein binding assay, HEK293 cells were transfected with pCMV6 (Origene, cat. no. PS100001) or pCMV3 (SinoBiological, cat. no. CV011) EV controls, pCMV-human siglec-4 (Origene, cat. no. RC208754), or pCMV-h4-1BBL (SinoBiological, cat. no. HG15693-CM) plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s manual.

Techniques: Binding Assay, Labeling, Magnetic Beads, Selection, Expressing, Western Blot, Transfection, Negative Control, Positive Control

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A to C ) FC was used to monitor (A) 4-1BB expression and 4-1BBL–Fc or siglec-4–Fc binding to activated T cells, (B) binding of siglec-4–Fc to stimulated T cells transfected with nonspecific (NS) or 4-1BB knockdown (KD) siRNAs, or (C) binding of siglec-4–Fc to stimulated T cells in the presence of increasing amounts of soluble 4-1BB–HIS protein. MFI, mean fluorescence intensity. Statistical analysis: unpaired t test. ( D ) ELISA was used to measure IFN-γ produced by activated T cells mixed with 293 cells overexpressing EV, siglec-4, 4-1BBL, or siglec-4 plus 4-1BBL. Statistical analysis: unpaired t test; P values correspond to comparisons between groups with or without siglec-4. ( E ) Luciferase assays were used to measure the viability of eGFP-FFLuc–labeled 293 target cells 24 hours after mixing with anti–TEM8–CAR-T cells. TEM8 knockout control cells (293/T8KO) were included as a specificity control. E:T, effector:target cell ratio. Statistical analysis: unpaired t test; P values correspond to comparisons between 293 and 293–Siglec-4 at each E:T cell ratio. ( F to H ) Immunoblotting was used to assess (F) p-c-Jun and c-Jun levels in 293 cells or 293–4-1BB cells following transient transfection with full-length siglec-4–myc or 4-1BB–myc, (G) p-c-Jun and c-Jun levels in unstimulated (U) or stimulated (S) T cells derived from two independent donors, and (H) p-c-Jun and c-Jun levels in T cells cocultured for 1 hour at a ratio of 1:1 with 293 cell transfected with EV (E) or siglec-4 (Sig4). Note that Siglec-4 expression can mediate the down-regulation of c-Jun only if 4-1BB is also present. β-Actin was used as a loading control in (F), (G), and (H). All data or images in (A) to (H) were representative of at least three independent experiments. For (C) to (E), n > = 3 biologically independent samples per group. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: For h4-1BB protein binding assay, HEK293 cells were transfected with pCMV6 (Origene, cat. no. PS100001) or pCMV3 (SinoBiological, cat. no. CV011) EV controls, pCMV-human siglec-4 (Origene, cat. no. RC208754), or pCMV-h4-1BBL (SinoBiological, cat. no. HG15693-CM) plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s manual.

Techniques: Expressing, Binding Assay, Transfection, Fluorescence, Enzyme-linked Immunosorbent Assay, Produced, Luciferase, Labeling, Knock-Out, Western Blot, Derivative Assay

Journal: FEBS Open Bio

Article Title: Development and characterization of a novel human CD137 agonistic antibody with anti‐tumor activity and a good safety profile in non‐human primates

doi: 10.1002/2211-5463.13494

Figure Lengend Snippet: Binding, specificity, and blocking profiles of PE0116. (A) The binding activity of PE0116, Urelumab, and Utomilumab toward CHOK1‐hCD137 and CHOK1‐cynoCD137 cells was analyzed as concentration‐mean fluorescence intensity (MFI) curve. (B) Specificity of PE0116 to CD137 was conducted by ELISA after capturing 1 μg·mL −1 humanOX40, GITR, and CD137 as the antigen. All assays were performed in duplicate, and all error bars indicate the SD. (C) Cell‐based blocking assay was performed to evaluate PE0116, Urelumab, and Utomilumab using CHOK1‐hCD137 stable cell line and CD137L‐hFc‐Biotin with streptavidin‐Alexa Fluor 488 conjugate.

Article Snippet: Flow cytometry assay was performed to evaluate the ability of anti‐hCD137 to block the binding of CD137 and CD137L based on CHOK1 cell overexpressing hCD137 and CD137L‐hFc‐Biotin (15693‐H01H‐B; Sino Biological, Beijing, China) protein.

Techniques: Binding Assay, Blocking Assay, Activity Assay, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Stable Transfection

Journal: FEBS Open Bio

Article Title: Development and characterization of a novel human CD137 agonistic antibody with anti‐tumor activity and a good safety profile in non‐human primates

doi: 10.1002/2211-5463.13494

Figure Lengend Snippet: Characterization of PE0116 in vitro functionality. (A) Luminescence value changed by HEK293‐CD137‐NF‐κB reporter cells treated with serially diluted PE0116, Urelumab, or Utomilumab (cross‐linking or not cross‐linking, antibodies were cross‐linked by anti‐Fc F(ab') 2 fragment, the molar ratio was 1 : 1.5). (B) Levels of IFN‐γ released by activated CD3 + T cells from three donors after 72 h of incubation with serially diluted PE0116, Urelumab, or Utomilumab in the presence of OKT3‐scFv displayed on the CHOK1 cell surface (cross‐linking or not cross‐linking). The EC50 value of Utomilumab non‐crosslink was blank in (B) (upper right panel), representing that the dates could not be analyzed by curve fitting. **** P < 0.0001 (hIgG4 vs. PE0116 at 6.67 n m in donor#2 or at 0.67 n m in donor#3 with cross‐linking) and *** P < 0.001 (hIgG4 vs. PE0116 at 66.7 n m in donor#2 without cross‐linking), as determined by two‐way ANOVA. All assays were performed in duplicate, and all error bars indicate the SEM.

Article Snippet: Flow cytometry assay was performed to evaluate the ability of anti‐hCD137 to block the binding of CD137 and CD137L based on CHOK1 cell overexpressing hCD137 and CD137L‐hFc‐Biotin (15693‐H01H‐B; Sino Biological, Beijing, China) protein.

Techniques: In Vitro, Incubation

Journal: Cells

Article Title: N -Glycosylation Facilitates 4-1BB Membrane Localization by Avoiding Its Multimerization

doi: 10.3390/cells11010162

Figure Lengend Snippet: The impact of N -glycosylation on 4-1BB/4-1BBL interaction. ( A ) The relative level of bound 4-1BBL to PNGase F-treated and untreated 4-1BB protein ( n = 3). ( B ) Octet analysis calculating the binding affinity of glycosylated (black) and deglycosylated 4-1BB (red) to 4-1BBL along with the K D values. N.S., not significant (Two-tailed student’s t -test).

Article Snippet: Native and deglycosylated 4-1BB were loaded on a Ni-NTA-coated 96-well plate and incubated at room temperature for 30 min. After three washes with PBST (phosphate-buffered saline (PBS)/0.1% Tween-20), human Fc-tagged 4-1BBL (Sino Biological, expressed in HEK293 cells, diluted in PBST) or mouse anti-human 4-1BB antibody (4B4-1, 1:200, BioLegend # 309802, San Diego, CA, USA) was added and incubated for another 30 min, followed by three washes.

Techniques: Binding Assay, Two Tailed Test

Journal: Oncotarget

Article Title: Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis

doi: 10.18632/oncotarget.14592

Figure Lengend Snippet: ( A ) Schematic diagrams of plasmid vectors used for the generation of the aAPC cell line. The costimulatory molecule vector (top) was used for transfection of wild-type K562 cells to generate puromycin-resistant K562 cells expressing CD64, CD137L and CD86 (K562A). K562A cells were further modified by co-transfection with the ZFN vector and EpCAM DNA donor vector (bottom) for AAVS1 locus-specific gene insertion to generate puromycin- and neomycin-resistant aAPCs expressing EpCAM (K562A-EpCAM). ( B ) PCR genome typing to demonstrate the AAVS1 locus-specific gene insertion of the EpCAM gene, as indicated by the presence of one single 1.5-kb band. ( C ) Phenotype analysis of K562A-EpCAM cells. Flow cytometric analysis demonstrates the surface expression of CD64, CD86, CD137L, and EpCAM. In the panels for CD64, CD86, and CD137L, left curves: isotype controls; right curves: antibodies. In the panel for EpCAM, left curve: K562 parental cells; right curve: K562A-EpCAM cells.

Article Snippet: The following antibodies were used for staining of cell lines: CD64, CD86, CD137L, and EpCAM (clone: HEA-125; Miltenyi, Bergisch Gladbach, Germany).

Techniques: Plasmid Preparation, Transfection, Expressing, Modification, Cotransfection

Journal: Frontiers in Immunology

Article Title: CD137 Agonists Targeting CD137-Mediated Negative Regulation Show Enhanced Antitumor Efficacy in Lung Cancer

doi: 10.3389/fimmu.2022.771809

Figure Lengend Snippet: The percentage of CD137+ Tregs was increased in the blood of lung cancer patients. (A) Tregs and CD137+ Tregs were gated by flow cytometry. (B) The percentage of Tregs in lung cancer patients (n=29) and healthy controls (n=34). (C) The percentage of CD137+ Tregs in the same patients and healthy controls. Differences are indicated as P values. The error bars represent the SEMs.

Article Snippet: To obtain a potential local target in tumors using CD137, we prepared CD137×hEGFR (mouse CD137 and human EGFR)-bispecific antibodies with Wt-mAb and Mut-mAb formats using a eukaryotic expression system (GenScript Co. Ltd.; ).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: CD137 Agonists Targeting CD137-Mediated Negative Regulation Show Enhanced Antitumor Efficacy in Lung Cancer

doi: 10.3389/fimmu.2022.771809

Figure Lengend Snippet: Enrichment and characterization of CD137+ Tregs in the tumor microenvironment. (A) Gated CD137+ Tregs derived from human cancer tissues via FACS; middle panel, CD137+ Tregs FMO control. (B, C) Treg and CD137+ Treg infiltration in tumors (n=10, unpaired samples) compared with the blood of lung cancer patients. P values are shown. (D) PI3K-AKT pathway-related molecules were differentially expressed in the CD137+ and CD137- Treg populations according to KEGG pathway analysis. (E) Comparative heatmap of the expression of Treg-associated markers, including transcription factors, in CD137+ Tregs (orange) and CD137- Tregs (blue). (F) The expression of eTreg-related markers in CD137+ Tregs and CD137- Tregs. (G) The expression of activated T cell markers in CD137+ Tregs and CD137- Tregs. (H–J) Differential expression of EBi3, IL-10 and LY108 in CD137+ Tregs and CD137- Tregs. ***Q value < 0.001, **Q value < 0.01. (K) The TCR clone frequency in CD137+ Tregs and CD137- Tregs in three individual samples was compared after TCB sequencing. (L) , TCR clone diversity in the two groups, **P< 0.01. The error bars represent the SEMs.

Article Snippet: To obtain a potential local target in tumors using CD137, we prepared CD137×hEGFR (mouse CD137 and human EGFR)-bispecific antibodies with Wt-mAb and Mut-mAb formats using a eukaryotic expression system (GenScript Co. Ltd.; ).

Techniques: Derivative Assay, Control, Expressing, Quantitative Proteomics, Sequencing

Journal: Frontiers in Immunology

Article Title: CD137 Agonists Targeting CD137-Mediated Negative Regulation Show Enhanced Antitumor Efficacy in Lung Cancer

doi: 10.3389/fimmu.2022.771809

Figure Lengend Snippet: Tumor sample characteristics and CD137+ Treg infiltration.

Article Snippet: To obtain a potential local target in tumors using CD137, we prepared CD137×hEGFR (mouse CD137 and human EGFR)-bispecific antibodies with Wt-mAb and Mut-mAb formats using a eukaryotic expression system (GenScript Co. Ltd.; ).

Techniques:

Journal: Frontiers in Immunology

Article Title: CD137 Agonists Targeting CD137-Mediated Negative Regulation Show Enhanced Antitumor Efficacy in Lung Cancer

doi: 10.3389/fimmu.2022.771809

Figure Lengend Snippet: Density of Foxp3+ and CD137+FOXP3+ cells in the tumor microenvironment and its correlation with prognosis. (A) Immune cells in the tumor microenvironment of a TMA core (20×). Magnified view of the red box: CD8+ T cells (B) Foxp3+ cells (C) CD137+ cells (D) and double-positive cells. (E) CD137+CD8+ cells are indicated by the yellow arrows, and CD137+Foxp3+ cells are indicated by the red arrows. (F) Impact of CD137+Foxp3+ cell density on patient OS. Dichotomization was based on the median; patients in the low-density group are indicated by the red line, and patients in the high-density group are indicated by the blue line. Log-rank P values are shown for each graph. (G) Correlation between CD137+Foxp3+ cell density and OS in patients with a high number of infiltrating CD137+CD8+ cells in the tumor microenvironment. CD137+CD8+ cell dichotomization was also based on the median. (H, I) IF intensity of CD137+FoxP3+ cells and CD137+CD8+ cells. (J) The mean IF intensity weighting of CD137 in CD137+CD8+ cells and CD137+FoxP3+ cells in TMA cores from 82 lung cancer patients. Differences are indicated as P values (paired t test). The error bars represent the SEMs.

Article Snippet: To obtain a potential local target in tumors using CD137, we prepared CD137×hEGFR (mouse CD137 and human EGFR)-bispecific antibodies with Wt-mAb and Mut-mAb formats using a eukaryotic expression system (GenScript Co. Ltd.; ).

Techniques:

Journal: Frontiers in Immunology

Article Title: CD137 Agonists Targeting CD137-Mediated Negative Regulation Show Enhanced Antitumor Efficacy in Lung Cancer

doi: 10.3389/fimmu.2022.771809

Figure Lengend Snippet: Targeting CD137+ Tregs with Wt-mAb enhanced antitumor efficacy in vivo . (A) CT26 (colon carcinoma) cells and (B) LLC cells treated with PBS (control) or the anti-CD137 mAbs Mut-mAb (with Fc mutation) and Wt-mAb (without Fc mutation). Mice (5 per group) were subcutaneously transplanted with CT26 or LLC cells. Six to 7 days later, when the tumor reached 0.5-0.7 cm in the largest diameter, the mice were injected intratumorally with mAbs (5 μg per mouse) 3 times at 2-day intervals. Tumor growth and mouse survival were monitored after transplantation. (C) The percentages of Tregs and CD137+ Tregs in blood and tumors from mice in the CT26 cell transplantation group and the changes in these percentages before and after mAb treatment (D) . (E) CD8+ T and CD137+ CD8+ T cell percentages in blood and tumors were compared before and after treatment. (F–H) The changes in the frequencies of T cell subsets were analyzed in mice in the LLC cell transplantation group as described for the CT26 cell transplantation group in (C–E) . A representative result of repeated experiments. The error bars represent the SEMs. *P < 0.05, **P < 0.01.

Article Snippet: To obtain a potential local target in tumors using CD137, we prepared CD137×hEGFR (mouse CD137 and human EGFR)-bispecific antibodies with Wt-mAb and Mut-mAb formats using a eukaryotic expression system (GenScript Co. Ltd.; ).

Techniques: In Vivo, Control, Mutagenesis, Injection, Transplantation Assay

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Regulation of CD137L , CDC42BPG and FST by p53. ( a ) Outline of the screening process. The expression profiles of 23813 genes in calvarial bone were detected by RNA sequencing, and 69 genes were selected by the indicated criteria as p53-induced genes. A second screening revealed three novel direct p53 targets. Of the 3 genes, one gene ( CD137L ) was up-regulated specifically in bone among 24 tissues. ( b ) At 24 h after transfection of each siRNA, U2OS cells were treated with ADR (2 μg/ml for 2 h). At 36 h after treatment, qPCR was performed. siRNA against EGFP was used as a control. β-actin was used for the normalization of expression levels. Error bars represent SD (n = 2). ( c ) qPCR was performed for the same calvaria sample as the RNA sequencing. β-actin was used for normalization of the expression levels. Error bars represent SD (n = 3). **P < 0.001, Student’s t-test. ( d ) qPCR was performed 36 h after treatment with ADR (2 μg/ml for 2 h) in p53 +/+ or p53 −/− calvarial osteoblasts. β-actin was used for normalization of the expression levels. Error bars represent SD (n = 3). **P < 0.001, Student’s t-test.

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: Expressing, RNA Sequencing, Transfection, Control

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Pathway analysis for the p53-induced and p53-repressed genes.

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: Significance Assay

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Identification of CD137L , CDC42BPG and FST as p53 direct target genes. ( a – c ) Luciferase assay of the p53BS in human (left) or mouse (right) CD137L ( a ), CDC42BPG ( b ) or FST ( c ) using SaOS2 cells. Luciferase activity is indicated relative to the activity of the mock vector with SD (n = 3). *P < 0.05, *P < 0.001, Student’s t-test. ( d – f ) CHIP assay for CD137L ( d ), CDC42BPG (BS-C) ( e ) or FST ( f ) was performed using SaOS2 cells that were infected with Ad-LacZ (lane 1) or Ad-p53 (lane 2-4). DNA-protein complexes were immunoprecipitated with an anti-p53 antibody (lanes 1 and 2) followed by qPCR. Input chromatin represents a small portion (1%) of the sonicated chromatin collected prior to immunoprecipitation. Immunoprecipitates with an anti-IgG antibody (lane 3) or in the absence of an antibody (lane 4) were used as negative controls. Columns, mean; error bars, SD (n = 3).

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Infection, Immunoprecipitation, Sonication

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Identification of CD137L as a bone-specific p53 target gene. ( a ) Induction of Cd137l among 24 tissues. Samples were categorized into 4 groups: (K) p53 −/− mice without irradiation, (W) p53 +/+ mice without irradiation, (KX) p53 −/− mice with irradiation and (WX) p53 +/+ mice with irradiation (n = 3 per group). We calculated the median FPKM value of WX/maximum FPKM value of the median FPKM value in K, KX or W for 24 tissues. ( b ) At 24 h after transfection with each siRNA, U2OS cells were treated with ADR (2 μg/ml for 2 h). At 36 h after treatment, whole cell extracts were subjected to western blotting with an anti-CD137L, anti-p21, anti-p53, or anti-β-actin antibody. siRNA against EGFP was used as a control. β-actin was used for normalization of the expression levels. These images were cropped from full-length blots (Supplementary Fig. ). ( c ) Immunohistochemical staining of Cd137l in mouse p53 α or p53 −/− calvaria with or without radiation exposure. Representative images from three tissues in each group are shown. Scale Bars (left) = 50 μm, Scale Bars (right) = 20 μm. Black arrowhead shows osteoblast.

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: Irradiation, Transfection, Western Blot, Control, Expressing, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Growth suppressive effects of p53 or CD137L. ( a ) p53 +/+ calvarial osteoblasts or p53 −/− calvarial osteoblasts were cultured on plates. Cell proliferation was assessed at 48 h. Error bars represent SD (n = 3). *P < 0.05, Student’s t-test. ( b ) Immunohistochemical staining of Ki67 in mouse p53 +/+ or p53 −/− calvaria with or without radiation exposure. Representative images from three tissues in each group are shown. Scale Bars (left) = 50 μm, Scale Bars (right) = 20 μm. ( c ) A colony formation assay was performed. After ectopic expression of CD137L or mock, the number of SaOS2, U2OS or LM8 cells was determined. Whole cell extracts were subjected to western blotting with a human or mouse anti-CD137L antibody. Error bars represent SD (n = 3). **P < 0.001, Student’s t-test. β-actin (U2OS and SaOS2) and α-tubulin antibody (LM8) was used as loading control. ( d ) Cd137l (n = 3) or mock (n = 3) stable expression cell lines were inoculated into the left and right flanks of C3H mice; each group contains 2 mice. The tumour volume was calculated every 2 or 3 days. *P < 0.05, Wilcoxon rank-sum test.

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: Cell Culture, Immunohistochemical staining, Staining, Colony Assay, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: The role of CD137L reverse or direct signalling in osteosarcoma. ( a ) SaOS2 cells were cultured with 10 or 20 μg/ml CD137-Fc or mock-Fc. Cell proliferation was assessed at 48 h. Error bars represent SD (n = 3). *P < 0.05, **P < 0.001, Student’s t-test. ( b ) p53 +/+ calvarial osteoblasts were cultured with 40 μg/ml Cd137-Fc or mock-Fc protein. Cell proliferation was assessed at 48 h. Error bars represent SD (n = 3). *P < 0.05, Student’s t-test. ( c , d ) IL2 production ( c ) and proliferation ( d ) of CD4 + or CD8 + T cells were assessed 48 h after stimulation with Cd137l-Fc. **P < 0.001, Student’s t-test. ( e ) C3H/HeJ mice (each group, n = 3) were subcutaneously injected into the left flank with 0.1 ml of PBS containing 1 × 10 6 LM8 cells. After tumours reached 0.5 cm in diameter, each of the mice was treated with Cd137l-Fc fusion protein or control mock-Fc by intraperitoneal injection for 3 days. The tumour volume was calculated every 2 or 3 days. Arrow shows the days of injection. *P < 0.05, Wilcoxon rank-sum test.

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: Cell Culture, Injection, Control

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: A schema of p53 or CD137L function. ( a ) A schema of the p53-regulated pathways or genes and the tumour suppressive roles. ( b ) A schema of CD137L function through direct or reverse signalling.

Article Snippet: Human anti-CD137L monoclonal antibody (EPR1172Y) was purchased from GeneTex (Irvine, CA, USA).

Techniques: