Journal: Journal of Cellular and Molecular Medicine

Article Title: NR1D1 ‐transactivated lncRNA NUTM2A‐AS1 promotes chemoresistance and immune evasion in neuroblastoma via inhibiting B7‐H3 degradation

doi: 10.1111/jcmm.18360

Figure Lengend Snippet: NUTM2A‐AS1 is associated with B7‐H3 and inhibits B7‐H3 ubiquitination level. (A,B) The graphs depict the protein scores of the 11 proteins identified through RNA pull‐down and LC–MS analysis. (C,D) The graphs illustrate the exponentially modified protein abundance index (emPAI) of the 11 proteins identified by RNA pull‐down assays and LC–MS. (E) B7‐H3 was specifically pulled down by biotin‐labelled sense NUTM2A‐AS1 (S) but not NUTM2A‐AS1 anti‐sense (AS) RNA in the indicated cells. (F) RNA immunoprecipitation (RIP) assays were conducted using anti‐B7‐H3 antibodies with extracts from neuroblastoma (NB) cells. Relative enrichment represents the RNA levels associated with B7‐H3 relative to an input control, comparing anti‐B7‐H3 antibody immunoprecipitation with IgG antibody. GAPDH mRNA served as the negative control. (G) B7‐H3 mRNA expression in NUTM2A‐AS1 knockdown cells was assessed by qRT‐PCR assay. (H,I) B7‐H3 protein expression in NUTM2A‐AS1 knockdown cells was examined by western blot assay. (J) NB cells with NUTM2A‐AS1 knockdown and control cells were treated with or without MG132 (5 μM) for 12 h, and cell lysates were analysed by western blotting. (K) NB cells with or without siRNAs specific for NUTM2A‐AS1 were treated with 20 μg/mL cycloheximide (CHX) or a vehicle for the indicated periods, and B7‐H3 levels were analysed by western blotting. (L) Western blot analysis of the ubiquitination of B7‐H3 in NUTM2A‐AS1 knockdown SK‐N‐SH cells. ***p < 0.001.

Article Snippet: Antibodies for western blot analysis are as follows: B7‐H3 (R&D Systems, #AF1027, 1:250) and GAPDH (Abcam, #EPR16891, 1:10000).

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Modification, Quantitative Proteomics, RNA Immunoprecipitation, Control, Immunoprecipitation, Negative Control, Expressing, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Generation of B7 ‐ H3 isoform regulated by ANXA2 / NSUN2 / YBX1 axis in human glioma

doi: 10.1111/jcmm.18575

Figure Lengend Snippet: Characterization of different isoforms of B7‐H3 expression. (A) Cartoon displays the transcript length and basic structure information of the different isomers of B7H3, as well as primers, antibodies and probes used in this study. The table at the bottom right gives the length of the different isoform PCR products, which can be used as a reference for all subsequent RT‐PCR results. (B) RT‐PCR show the PCR products of B7‐H3 isoform in five glioma cell lines used by P1 primers (top), P2 primers (middle) and β‐actin primers (bottom). (C) WB assay shows the protein bands of B7‐H3 isoform, ANXA2, NSUN2 and YBX1 in five glioma cell lines. The band locations of 4Ig and 2Ig and the absence of 3Ig are inferred by RT‐PCR. (D) WB assay shows the protein bands of B7‐H3, ANXA2, NSUN2 and YBX1 in 24 glioma tissues. The double bands represent 4Ig and 2Ig according to the protein size in (C). (E) Venn diagram shows the binding proteins on B7‐H3 pre‐mRNA via probe pull down and mass spectrum. YBX1, NSUN2 and ANXA2 are highlighted in different intersections. (F) Enrichment of ANXA2 on B7‐H3 in five glioma cells by RIP‐qPCR. The statistical significances compared between other four cells and U373MG via one‐way ANOVA are indicated by asterisk. **** p < 0.0001, *** p < 0.001 and ** p < 0.01.

Article Snippet: Primary antibodies against B7‐H3 (1: 2000, Cat. no. 58798, CST, USA), ANXA2 (1: 2000, Cat. no. 8235, CST), NSUN2 (1: 2500, Cat. no. 44056, CST), YBX1 (1: 2000, Cat. no. 9744, CST), β‐actin (1: 5000, Cat. no. 4967, CST) and Flag (1: 1000, Cat. no. 14793, CST) were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay

Journal: Journal of Hepatocellular Carcinoma

Article Title: Identification and Experimental Validation of the Prognostic Significance and Immunological Correlation of Glycosylation-Related Signature and ST6GALNAC4 in Hepatocellular Carcinoma

doi: 10.2147/JHC.S400472

Figure Lengend Snippet: Correlation analyses between the risk scores, GRGs, and the immune checkpoint genes in HCC. ( A ) Differential expressions of immune checkpoint genes in high- and low-risk groups of HCC. ( B ) Correlation analyses between risk scores and the multiple immune checkpoint gene expressions in HCC. ( C ) Correlation analyses between the expressions of GRGs and immune checkpoint genes in HCC. ( D ) Correlation dot plots of ST6GALNAC4 with multiple immune checkpoint genes in HCC from the TIMER database. ( E ) Correlation dot plots of B7-H3 with GRGs from the TIMER database.

Article Snippet: The antibodies for detection of GAPDH (5174), PD-L1 (13684), and B7-H3 (14058) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Journal of Hepatocellular Carcinoma

Article Title: Identification and Experimental Validation of the Prognostic Significance and Immunological Correlation of Glycosylation-Related Signature and ST6GALNAC4 in Hepatocellular Carcinoma

doi: 10.2147/JHC.S400472

Figure Lengend Snippet: Validation of ST6GALNAC4 expression in HCC tissues and its correlation with immune checkpoints. ( A ) Expression profile of ST6GALNAC4 in 47 HCC tissues compared to paracan-cerous tissues. ( B ) The mean expression level of ST6GALNAC4 in 47 paired HCC and paracan-cerous tissues (**p < 0.01). ( C ) The relative expression distribution of ST6GALNAC4, PD-L1, and B7-H3 in HCC tissues. ( D ) Correlation analyses of ST6GALNAC4 expression with PD-L1 and B7-H3 in HCC tissues. ( E ) Western blot analysis for ST6GALNAC4 protein expression in HCC and paracancerous tissues.

Article Snippet: The antibodies for detection of GAPDH (5174), PD-L1 (13684), and B7-H3 (14058) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Biomarker Discovery, Expressing, Western Blot

Journal: Journal of Hepatocellular Carcinoma

Article Title: Identification and Experimental Validation of the Prognostic Significance and Immunological Correlation of Glycosylation-Related Signature and ST6GALNAC4 in Hepatocellular Carcinoma

doi: 10.2147/JHC.S400472

Figure Lengend Snippet: Effect of ST6GALNAC4 knockdown on hepatoma cells. ( A ) Expression of PD-L1 and B7-H3 mRNA in hepatoma cells after ST6GALNAC4 knockdown. ( B and C ) Flow cytometric and Western blot analyses of PD-L1 and B7-H3 expression in hepatoma cells after ST6GALNAC4 knockdown. ( D ) CCK-8 analysis of the effect of ST6GALNAC4 knockdown on the proliferative capacity of hepatoma cells. ( E and F ) Effect of ST6GALNAC4 knockdown on the clone formation ability and cell cycle of hepatoma cells. ( G and H ) ST6GALNAC4 knockdown inhibited the migration and invasion ability of hepatoma cells. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies for detection of GAPDH (5174), PD-L1 (13684), and B7-H3 (14058) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Expressing, Western Blot, CCK-8 Assay, Migration

Journal: Cancer discovery

Article Title: Tuning the Antigen Density Requirement for CAR T Cell Activity

doi: 10.1158/2159-8290.CD-19-0945

Figure Lengend Snippet: (a) Schema of first generation CD19 CARs with either a CD8 or CD28 hinge-transmembrane region (CD19-CD8H/T-ζ and CD19-CD28H/T-ζ). (b) NALM6 clones expressing either 963 or 45,851 molecules of surface CD19 were cocultured at a 1:1 ratio with either CD19-CD28ζ, CD19–4-1BBζ, CD19-CD28H/T-ζ or CD19-CD8H/T-ζ CAR T cells and tumor cell killing was measured in an Incucyte assay. Representative of three experiments with different T cell donors. Statistical analysis performed with repeated measures ANOVA between CD19-CD28H/T-ζ and CD19-CD8H/T-ζ. (c) CD19-CD28ζ, CD19–4-1BBζ, CD19-CD28H/T-ζ, and CD19-CD8H/T-ζ CAR T cells were cocultured with NALM6 clones expressing various amounts of CD19 for 24 hours and secreted IL-2 was measured in the supernatant by ELISA. Representative of three experiments with different T cell donors. Statistical comparisons performed with the student’s t-test (two sided) between CD19-CD28H/T-ζ and CD19-CD8H/T-ζ. (d) Schema of a Her2 CAR containing a CD28 hinge-transmembrane region and 4–1BB costimulatory domain (Her2-CD28H/T-4–1BBζ). (e) One million 143b osteosarcoma cells were orthotopically implanted in the hind leg of NSG mice. After seven days, mice were treated with 10 million Her2–4-1BBζ CAR T cells, Her2-CD28H/T-4–1BBζ CAR T cells, or untransduced control T cells (MOCK). Leg measurements were obtained twice weekly with digital calibers. Measurements for individual mice are shown. Statistical analysis performed with repeated measures ANOVA. (f) Survival curves for mice treated as in (e). Statistical analysis performed with the log-rank test. (e-f) are representative of two experiments with different T cell donors (n=5 mice per group). (g) Schema of a B7-H3 CAR containing a CD28 hinge-transmembrane region and 4–1BB costimulatory domain (B7-H3-CD28H/T-4–1BBζ). (h) One million CHLA255 neuroblastoma cells were engrafted into NSG mice by tail vein injection in a metastatic neuroblastoma model. Six days later, mice were injected with 10 million B7-H3–4-1BBζ CAR T cells, B7-H3-CD28H/T-4–1BBζ CAR T cells, or untransduced control T cells (MOCK). Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Representative bioluminescent images are shown. (i) Quantified tumor flux values for individual mice treated as in (h). Statistical analysis performed with repeated measures ANOVA. (j) Survival curves for mice treated as in (h). Statistical analysis performed with the log-rank test. (h-j) are representative of two experiments with different T cell donors. For in vitro experiments, error bars represent SD and for in vivo experiments, error bars represent SEM. p < 0.05 was considered statistically significant, and p values are denoted with asterisks as follows: p > 0.05, not significant, NS; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: B7-H3 expression on tumor lines was assessed with PE conjugated mouse anti-human B7-H3 antibody (R&D, clone MAB1027) and Her2 with PE or APC conjugated anti-human CD340 antibody (Biolegend, clone 24D2).

Techniques: Clone Assay, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Software, In Vitro, In Vivo