mouse ifn β kit  (InvivoGen)

Journal: Nature Communications

Article Title: BATF2 is a glutamine-responsive tumour suppressor required for type-I interferon-dependent anti-tumour immunity

doi: 10.1038/s41467-025-68027-2

Figure Lengend Snippet: a We confirmed the establishment of Batf2 -targeted deletion in mice (left panel). The mRNA levels of Batf2 in BMDM from indicated genotypes are shown (right panel, n = 3 biologically independent samples; data are presented as mean ± SEM; unpaired two-tailed t -test). b BMDM were transfected with 2.0 µg/mL ISD and 1.0 µg/mL poly(I:C) for 8 h, and total mRNA was extracted for bulk RNA sequencing. The transcriptomes were compared between wildtype and Batf2 −/− BMDM. Data represent biologically independent samples ( n = 2 per group). c GSEA shows the significantly altered pathways between wildtype and Batf2 −/− BMDM in response to STING stimulation. d – g BMDM from wildtype and Batf2 −/− mice were transfected with 3.0 µg/mL ISD ( d ), 1.0 µg/mL cGAMP ( e ), 1.0 µg/mL poly(I:C) ( f ) or 1.0 µg/mL 5′ppp-dsRNA ( g ) for 16 h. Total mRNA was extracted for qPCR. The fold changes of Ifnb1 , pan -Ifna and Cxcl10 are shown as indicated. Data represent biologically independent experiments ( n = 3). h , i Wild-type and Batf2 −/− BMDM were transfected with 3.0 µg/mL ISD ( h ) or 1.0 µg/mL cGAMP ( i ) for 24 h. The concentration of Ifn-β in the supernatant was measured by ELISA. Data represent biologically independent experiments ( n = 3). j Wild-type and Batf2 −/− BMDM were incubated with 10.0 µg/mL diABZI for 8 h. Ifn-β in the supernatant was measured by ELISA. Data represent biologically independent experiments ( n = 3). k , l Wild-type and Batf2 −/− BMDM were transfected with 1.0 µg/mL poly(I:C) ( k ) or 1.0 µg/mL 5′ppp-dsRNA ( l ) for 24 h. Ifn-β in the supernatant was quantified by ELISA. Data represent biologically independent experiments ( n = 3). m , n Wild-type and Batf2 −/− BMDM were transfected with 3.0 µg/mL ISD ( m ) or 1.0 µg/mL cGAMP ( n ). Cell lysates were collected at the indicated time points for immunoblot analysis. The experiment was performed in two biologically independent experiments. Comparisons in ( d – l ) were made using two-way ANOVA followed by multi-comparison tests and data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: The amount of Ifn-β and Tnf-α in the supernatant was measured by a mouse Ifn-β kit (luex-mifnbv2, Invivogen) and a Tnf-α ELISA kit (BMS6073, Invitrogen) according to the manufacturer’s instructions.

Techniques: Two Tailed Test, Transfection, RNA Sequencing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Comparison

Journal: Nature Communications

Article Title: BATF2 is a glutamine-responsive tumour suppressor required for type-I interferon-dependent anti-tumour immunity

doi: 10.1038/s41467-025-68027-2

Figure Lengend Snippet: a , b BMDM were cultured in media containing glutamine (0 g/L, 0.5 g/L or 5.0 g/L) for 24 h, followed by transfection with STING agonists. c , d BMDM were cultured in media containing 0.5 g/L ( c ) or 5 g/L ( d ) glutamine and transfected with cGAMP with or without of BPTES. Data are compared using one-way ANOVA. e , f Ifn-β in the supernatant from this figure ( a , b ) was quantified by ELISA. g BMDM were cultured in media containing varied levels of glutamine and then transfected with cGAMP. The protein lysates were subjected to immunoblotting. Data represent two biologically independent experiments. h BMDM were cultured in media containing varied levels of glutamine and transfected with ISD. ChIP analysis of the H3K27me3 mark in the promoter regions of Batf2 is compared using one-way ANOVA. i BMDM were cultured in media containing 0 g/L or 5.0 g/L glutamine for 24 h and transfected with ISD and OCR was compared. j Wild-type and Batf2 −/− BMDM were transfected with ISD and OCR was compared. k Female C57BL6/J mice on a control diet or glutamine-rich diet were implanted with NOOC1 cells subcutaneously. Mice were treated with PBS or c-di-AMP intratumoural injection ( n = 8). l , m The combination of CDA and JHU-083 was administered for 2 weeks ( n = 8). n UMAP rendering of scRNA-Seq of the TILs separated from 5k ( n = 8 mice per group), shown above is generated. o The relative cluster size change percent is shown. p The Batf2 expression level in each cluster is shown. q The expression levels of Batf2 were compared using a Wilcoxon test. r Bulk RNA-Seq was performed for 15 HNSCC specimens. A correlation analysis between BATF2-IFN-I target genes and glutamine metabolism is shown. Data in this figure ( a – h ) represent biologically independent experiments ( n = 3, mean ± SEM). Data in this figure ( i , j ) were compared by an unpaired two-tailed t -test and represent biologically independent samples ( n = 3, mean ± SEM). Comparisons in ( a , b and e , f ) were made using two-way ANOVA, followed by multi-comparison tests. The tumour growth in this figure ( k – m ) was compared using the generalized estimating equation (GEE) model.

Article Snippet: The amount of Ifn-β and Tnf-α in the supernatant was measured by a mouse Ifn-β kit (luex-mifnbv2, Invivogen) and a Tnf-α ELISA kit (BMS6073, Invitrogen) according to the manufacturer’s instructions.

Techniques: Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Injection, Generated, Expressing, RNA Sequencing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: BATF2 is a glutamine-responsive tumour suppressor required for type-I interferon-dependent anti-tumour immunity

doi: 10.1038/s41467-025-68027-2

Figure Lengend Snippet: a , Stable EV control and Batf2-expressing MOC2-E6/E7 cells were transfected with Sting plasmid for 16 h. The mRNA was quantified by qPCR ( n = 3). b , c EV control and Batf2-expressing MOC2-E6/E7 cells were transfected with Sting plasmid for 24 h. b The amount of Ifn-β in the supernatant was measured by ELISA ( n = 3). c protein lysates were harvested for immunoblotting against the indicated markers. d EV or Batf2-expressing MOC2-E6/E7 cells were implanted subcutaneously and monitored for tumour burden in the wild-type female C57BL6/J mice ( n = 5 each group). The tumour growth was compared using the GEE model. e EV or Batf2-expressing MOC2-E6/E7 cells were implanted in Ifnar1 −/− mice ( n = 9 in EV group; n = 10 in Batf2-MOC2-E6/E7 group). Tumour growth was compared using the GEE model. f–i One million the EV or Batf2-expressing MOC2-E6/E7 cells were implanted subcutaneously in wild-type mice. On days 7, 14 and 21 after implantation, tumour tissues were processed into single-cell suspensions. Flow cytometry was used to evaluate the presence of p-Tbk1 + , p-Irf3 + , CD8⁺ and TCRγδ⁺ cells in the TME ( n = 4 in the Day 7 Batf2 and the Day 14 EV groups; n = 5 for other groups). j UMAP rendering of single-cell immune profiling of the TILs separated from the control ( n = 5) and Batf2-expressing ( n = 5) tumours grown in wild-type C57BL6/J mice from this figure ( d ) is shown. The significantly altered clusters between the two groups are labeled in red. k The relative percentage of TILs is shown. l T-cell clusters (13 + 16) were separated, and the volcano plot shows the differentially expressed genes between groups. m A gene set enrichment analysis was performed to identify the most significantly altered pathways in T-cells from this figure ( d ). Comparisons in ( a , b ) were made using two-way ANOVA followed by multi-comparison tests. Comparisons in ( f – i ) were made using an unpaired two-way t -test. Data in ( a , b and d – i ) are presented as mean ± SEM. Source data are provided.

Article Snippet: The amount of Ifn-β and Tnf-α in the supernatant was measured by a mouse Ifn-β kit (luex-mifnbv2, Invivogen) and a Tnf-α ELISA kit (BMS6073, Invitrogen) according to the manufacturer’s instructions.

Techniques: Control, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Labeling, Comparison