Journal: Cancer cell

Article Title: Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells

doi: 10.1016/j.ccell.2019.01.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 4Ig-B7-H3-His , Novus Biologicals , CAT# NBP2-59914-250ug.

Techniques: Generated, Polymer, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Software, Imaging

Journal: Cellular & Molecular Biology Letters

Article Title: The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

doi: 10.1186/s11658-020-00233-w

Figure Lengend Snippet: Nutlin-3 stabilizes MDM2-CD276 protein-protein interactions in situ . a Proximity ligation assay of A375 cells treated with DMSO or 10 μM Nutlin-3 for 24 h. Green fluorescence spots suggest that both target proteins (MDM2-CD276) are within interacting range. The cells were counterstained with DAPI (blue). b Quantitation of CD276-MDM2 interaction after incubation with DMSO or Nutlin-3. The results represent the mean ± SD of technical triplicates. Statistical analysis was performed by one-way ANOVA with a Bonferroni post hoc test, *** p < 0.001. c Western blot of A375 cells treated with the indicated concentrations of Nutlin-3 (Nut-3). MDM2, PD-L1, CD276 and β-actin have apparent molecular weights of 90, 50, 60 and 42 kDa, respectively

Article Snippet: Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [ ], p53 (DO-1) [ ], β-actin and GAPDH (both Santa Cruz Biotechnology) were used.

Techniques: Protein-Protein interactions, In Situ, Proximity Ligation Assay, Fluorescence, Quantitation Assay, Incubation, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

doi: 10.1186/s11658-020-00233-w

Figure Lengend Snippet: Nutlin-3 induces CD276 and PD-L1 proteins on the surface of HCT116 cells. Representative flow cytometry profiles and quantitation of cell surface CD276 ( a , b) and PD-L1 ( c , d ) on HCT116 p53-wt and p53-null cells treated with DMSO or 10 μM Nutlin-3 for 24 h. CD276 and PD-L1 were measured using a FITC- and c APC-conjugated antibody, respectively. In a and c the grey areas correspond to an isotype antibody as a negative control. In b and d , results represent the mean ± SD of technical triplicates, each with 30,000 counted cells. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test, * p < 0.05, ***p < 0.001

Article Snippet: Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [ ], p53 (DO-1) [ ], β-actin and GAPDH (both Santa Cruz Biotechnology) were used.

Techniques: Flow Cytometry, Quantitation Assay, Negative Control

Journal: Cellular & Molecular Biology Letters

Article Title: The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

doi: 10.1186/s11658-020-00233-w

Figure Lengend Snippet: Nutlin-3 induces CD276 and PD-L1 on the surface of melanoma A375 cells. Representative flow cytometry profiles and quantitation of cell surface CD276 ( a , b) and PD-L1 ( c , d ) on A375 p53-wt and p53-null cells treated with DMSO or 10 μM Nutlin-3 for 24 h. CD276 and PD-L1 were measured using a FITC- and c APC-conjugated antibody, respectively. In a and c the grey areas correspond to an isotype antibody as a negative control. In b and d , results represent the mean ± SD of technical triplicates, each with 30,000 counted cells. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test, *p < 0.05, ***p < 0.001. e Measurement of PD-L1 basal level A375 p53-wt (wt) and in single cell- p53 gene edited clones (Supplementary Fig. ). The data are plotted as the mean fluorescent intensity (MFI) of PD-L1 for individual clones. f The effect of the ATM inhibitory molecule KU55993 and Nutlin-3 (Nut-3) on cell surface PD-L1. A375 p53-wt and A375 p53-null cells were incubated with KU55993 or Nutlin-3 at the indicated concentrations (μM) for 24 h. Western blot of lysates prepared from g A375 p53-wt and h A375 p53-null cells treated with indicated concentrations of Nutlin-3 (Nut-3) and KU55993. MDM2, p53, CD276, PD-L1 and β-actin have apparent molecular weights of 90, 50, 60, 50 and 42 kDa, respectively

Article Snippet: Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [ ], p53 (DO-1) [ ], β-actin and GAPDH (both Santa Cruz Biotechnology) were used.

Techniques: Flow Cytometry, Quantitation Assay, Negative Control, Clone Assay, Incubation, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

doi: 10.1186/s11658-020-00233-w

Figure Lengend Snippet: Cell surface PD-L1 as a function of ATG5 status. a A375 p53-wt and A375 p53-null cells were treated with 10 μM MG-132 for the indicated times. PD-L1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control antibody have apparent molecular weights on Western blots of 50 and 37 kDa, respectively. b A549-Atg5-wt cells (wt) were processed using gene editing (CRISPR-Cas9) to create mutant A549-Atg5-null cells (null). Atg5-GFP was introduced into A549-Atg5-null cells to create a partial Atg5 rescue phenotype (resc). Both PD-L1 and GAPDH were detected in these cells. Representative flow cytometric graphs of c A549-Atg5-wt, d A549-Atg5-null and e A549-Atg5-resc. Cell surface PD-L1 was measured using APC-conjugated antibody. Results represent the mean ± SD of technical triplicates, each with 30,000 counted cells. f Normalized mean fluorescent intensity (MFI) of PD-L1 derived from FACS on c - e . Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test, * p < 0.05, *** p < 0.001. g PD-L1 (upper row) and CD276 (lower row) were measured after treatment with 10 μM chloroquine for the indicated times by FACS using APC- and FITC-conjugated antibodies, respectively

Article Snippet: Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [ ], p53 (DO-1) [ ], β-actin and GAPDH (both Santa Cruz Biotechnology) were used.

Techniques: Control, Western Blot, CRISPR, Mutagenesis, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

doi: 10.1186/s11658-020-00233-w

Figure Lengend Snippet: Summary of the genetic and pharmacological events that distinguish regulation and function of CD276 or PD-L1 by the MDM2/p53 axis. The left and right panels summarize data highlighting the elevation of PD-L1 or CD276, respectively, after treatment with Nutlin-3

Article Snippet: Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [ ], p53 (DO-1) [ ], β-actin and GAPDH (both Santa Cruz Biotechnology) were used.

Techniques:

Journal: Cellular & Molecular Biology Letters

Article Title: The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

doi: 10.1186/s11658-020-00233-w

Figure Lengend Snippet: Effects of CD276 depletion on cell cycle parameters. HCT116 p53-wt were transfected with siRNA towards CD276 ( CD276si ) for 48 h. a Detection of CD276 and β-actin on Western blot at molecular weights of 60 and 42 kDa, respectively, NTC – non-targeting control. b Flow cytometry analysis of cell cycle parameters. Distribution of G1 and G2 phases in HCT116 cells with normal and depleted CD276 protein level ( CD276si ) and after treatment with 20 μM Nutlin-3 or untreated for 48 h. Results represent the mean ± SD of technical triplicates, each with 10,000 counted cells. c Representative histograms of cell cycle after staining with propidium iodide

Article Snippet: Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [ ], p53 (DO-1) [ ], β-actin and GAPDH (both Santa Cruz Biotechnology) were used.

Techniques: Transfection, Western Blot, Control, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: B7-H3 as a promising target for cytotoxicity T cell in human cancer therapy

doi: 10.18632/oncotarget.8784

Figure Lengend Snippet: Shaded histogram represents cells stained with anti-B7-H3 mAb and un-shaded histogram represents cells stained with the control mouse IgG1. Mean Fluorescent Intensity (MFI) values obtained with anti-B7-H3 mAb staining divided by the control isotype staining are indicated in the upper right of the histogram.

Article Snippet: Anti-B7-H3 mAb (R&D System, Minneapolis, MN, USA) was reacted with sulfo-SMCC and anti-CD3 (OKT3, eBioscience) was reacted with Traut's reagents as the method previously described [ , , ].

Techniques: Staining, Control

Journal: Oncotarget

Article Title: B7-H3 as a promising target for cytotoxicity T cell in human cancer therapy

doi: 10.18632/oncotarget.8784

Figure Lengend Snippet: A. The heteroconjugated product of equimolar concentrations of B7-H3Bi-Ab was quantified by Coomassie blue staining of SDS-gel. B. Flow cytometry based binding assay for B7-H3Bi-Ab was tested. ATC was stained by B7-H3BiAb (shaded histogram), or a combination of OKT3 with anti-B7-H3 (the black line), then FITC-anti-mouse-IgG1 was added to detect the B7-H3 moiety of B7-H3Bi-Ab (a), or an FITC-anti-mouse IgG2a to detect the anti-CD3 moiety of the B7-H3Bi-Ab (b). (c) Hela-luc cells were incubated with B7-H3Bi-Ab (shaded histogram) or a combination of OKT3 and anti-B7-H3 (the black line), then B7-H3Bi-Ab binding was confirmed by FITC-anti-mouse IgG2a to detect the anti-CD3 moiety of the B7-H3Bi-Ab. (d) LL/2-luc-M38 cells were incubated with B7-H3Bi-Ab (shaded histogram) or a combination of OKT3 and anti-B7-H3 (the black line), then B7-H3Bi-Ab binding was analyzed by combined FITC-anti-mouse IgG2a with an anti-mouse-IgG1-FITC. C. Titer of B7-H3Bi-Ab armed ATC was measured. ATC was armed with B7-H3Bi-Ab ranging from 5 to 500 ng/10 6 cells at E/T ratio of 10:1, and cytotoxic effects of B7-H3Bi-armed ATC on Hela-luc cells were tested in vitro. After 18 hour incubation with B7-H3Bi-armed ATC, the percentage of viability was calculated at each concentration.

Article Snippet: Anti-B7-H3 mAb (R&D System, Minneapolis, MN, USA) was reacted with sulfo-SMCC and anti-CD3 (OKT3, eBioscience) was reacted with Traut's reagents as the method previously described [ , , ].

Techniques: Staining, SDS-Gel, Flow Cytometry, Binding Assay, Incubation, In Vitro, Concentration Assay

Journal: Oncotarget

Article Title: B7-H3 as a promising target for cytotoxicity T cell in human cancer therapy

doi: 10.18632/oncotarget.8784

Figure Lengend Snippet: Target cells were incubated either with B7-H3Bi-armed ATC or a combination of OKT3 and anti-B7-H3 mAb with ATC (unarmed ATC), or ATC for 18 hours, and luciferase quantitative assays were performed to determine cytotoxicity against different target cells at indicated E/T ratio. A. Bioluminescence image signal in photons per second was converted into living cell number and the cytotoxicity assays were measured at the at different E/T ratio (5:1, 10:1, and 20:1). B. Bioluminescence image of MDA-MB231-luc cell was as a representative after incubation with B7-H3Bi-armed ATC or unarmed ATC, or ATC at the indicated E/T ratio. Shown is a representative experiment of at least three, and the data for (A) are mean ± SD of triplicate determination. ** P < 0.01, *** P < 0.001, B7-H3Bi-armed ATC was compared with unarmed ATC or ATCunder similar conditions.

Article Snippet: Anti-B7-H3 mAb (R&D System, Minneapolis, MN, USA) was reacted with sulfo-SMCC and anti-CD3 (OKT3, eBioscience) was reacted with Traut's reagents as the method previously described [ , , ].

Techniques: Incubation, Luciferase

Journal: Oncotarget

Article Title: B7-H3 as a promising target for cytotoxicity T cell in human cancer therapy

doi: 10.18632/oncotarget.8784

Figure Lengend Snippet: A. Expression of B7-H3 on human primary breast cancer (BC) and lung cancer (LC) cell culture. Gray histogram represents cells stained with anti-human B7-H3 mAb and black histogram represents cells stained with the isotope control mAb. B. (a) Lactate dehydrogenase (LDH) activity assay was performed by B7-H3Bi-armed ATC or unarmed ATC against freshly isolated tumor cell. Supernatants of co-cultures at E/T ratio of 10:1 were harvested at 18 hour and LDH activity assay was performed to determine cytotoxicity against freshly isolated tumor cell. (b) IFN-γ, (c) TNF-α and (d) IL-2 secretion by B7-H3Bi-armed ATC against freshly isolated tumor cell. Supernatants of co-cultures at E/T ratio of 10:1 were harvested at 18 hours and analyzed for cytokine production using specific ELISA Kit. C. Target tumor cells were incubated either with B7-H3Bi-armed ATC or unarmed ATC for 18 h at E/T ratio of 10:1, and real-time photograph was taken at x 200 magnification. The data of (B) are mean ± SD of triplicate determination. Shown is a representative experiment of at least three experiments. * P < 0.05, *** P < 0.001, B7-H3Bi-armed ATC was compared with unarmed ATC under similar conditions.

Article Snippet: Anti-B7-H3 mAb (R&D System, Minneapolis, MN, USA) was reacted with sulfo-SMCC and anti-CD3 (OKT3, eBioscience) was reacted with Traut's reagents as the method previously described [ , , ].

Techniques: Expressing, Cell Culture, Staining, Control, Activity Assay, Isolation, Enzyme-linked Immunosorbent Assay, Incubation