vectastain abc hrp kit (peroxidase, rabbit igg )  (Vector Laboratories)

Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A ) Outline of metabolic inhibitors for screening. ( B ) Human hTERT-immortalized foreskin fibroblast (BJ-5ta, left) and mouse fibroblast (L929, right) cells were treated with inhibitors from A followed by 0.5 μg/mL HT-DNA transfection for 5 hours. Cells were harvested for qPCR analysis of human IFNB or mouse Cxcl10 . ( C ) BJ-5ta cells were treated with 1 mM AOA for 1 hour followed by HT-DNA (0.5 μg/mL) transfection for the indicated time. Cells were harvested for qPCR analysis of IFN response gene expression. ( D and E ) L929 ( D ) and MC38 ( E ) were treated with 0.5 mM AOA for 1 hour followed by DMXAA (50 μM) or HT-DNA (0.5 μg/mL) stimulation for the indicated time. Cells were harvested for qPCR analysis of IFN response gene expression. ( F – H ) Western blot detected phosphorylated (p-) p-IRF3 and p-STAT1 levels in BJ-5ta, L929, and MC38 cells treated as in C – E . ( I ) Representative immunofluorescence images of IRF3 in BJ-5ta cells treated with AOA 1 hour followed by HT-DNA (0.5 μg/mL) transfection or not. Scale bars, 5 μm. ( J ) Schematic of the main targets of AOA. ( K ) Western blot detected p-IRF3, p-STAT1, GOT2, and GPT2 levels in BJ-5ta cells treated as indicated. ( L ) The relative IFNB and CXCL10 mRNA expression in the control BJ-5ta cells versus GOT2 and GPT2 siRNA-silenced BJ-5ta cells after indicated treatment. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by unpaired t test ( C – E , I , and L ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: Transfection, Gene Expression, Western Blot, Immunofluorescence, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A ) Heatmap of metabolite changes in mock- and AOA-treated L929 cells followed with HT-DNA transfection or without HT-DNA transfection. The fold-change of metabolites abundances was normalized to the mock group. Each square represents the mean of 3 replicates ( n = 3 independent cultures). ( B ) L929 cells were treated with 0.5 mM AOA for 1 hour followed by DMXAA (50 μM) stimulation for 6 hours in the absence or presence of 20 mM aspartate or asparagine, and then cells were harvested for qPCR analysis of IFN response gene expression. ( C ) Heatmap from RNA-seq revealing the expression of ISGs in L929 cells with indicated treatment compared with mock group [ z -score–normalized log 2 (fold per million reads) values, n = 3 independent cultures]. ( D ) GSEA of interferon-beta response gene. ( E ) Western blot detected p-IRF3 and p-STAT1 levels in L929 cells treated as indicated. ( F ) Representative immunofluorescence images of IRF3 in BJ-5ta cells treated with AOA 1 hour followed by HT-DNA (0.5 μg/mL) transfection in the absence or presence of 20 mM aspartate. Scale bars, 5 μm. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by 1-way ANOVA followed by Tukey’s test ( B ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: Transfection, Gene Expression, RNA Sequencing, Expressing, Western Blot, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

doi: 10.1172/JCI199716

Figure Lengend Snippet: ( A ) Schematic showing that aspartate-mediated pyrimidine nucleotide synthesis is essential for intracellular DNA homeostasis. ( B ) Heatmap from RNA-seq revealing the expression of DNA repair enzymes in L929 cells with indicated treatment. The fold-change of expression was normalized to the mock group ( n = 3 independent cultures). ( C ) Genome browser screenshots of END-seq on mitochondria from L929 cells with indicated treatment. The expanded view of the indicated region shows more detailed genomic features. ( D ) The proportion of END-seq reads in mitochondrial and nuclear chromosomal regions. ( E ) Representative immunofluorescence images of mitochondrial (TOM20, red) or dsDNA (green) in BJ-5ta cells treated with AOA in the absence or presence of 20 mM aspartate for 6 hours. Scale bars, 10 μm. Lower panel: quantification of cytosolic dsDNA in BJ-5ta cells with indicated treatment ( n = 39–51 fields per group from 3 biological replicates). ( F ) Representative immunofluorescence images of BJ-5ta cells treated with ddC (100 μM) for 72 hours, followed by 6 hours of AOA treatment in the absence or presence of 20 mM aspartate. ( G ) 2D–structured illumination microscopy imaging of mtDNA externalization under the indicated conditions. ( H ) Western blot detected α-tubulin (cytosol), TOM20 (OMM), PDH (Matrix), COX IV (IMM), and histone H3 (nuclei) to validate protocol from in L929 cells treated as indicated. wcl, whole cell lysate; pel, pellet; cyt, cytosolic fraction. ( I ) L929 cells were treated with AOA in the absence or presence of 20 mM aspartate followed by HT-DNA transfection, and then cells were harvested for qPCR analysis of mtDNA or nDNA levels in cytosolic fractions. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by 1-way ANOVA followed by Tukey’s test ( E and I ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. DSBs, double-strand breaks.

Article Snippet: Six- to 8-week-old C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and age-matched Ifnar1 -KO C57BL/6J mice were obtained from Cyagen Biosciences (Suzhou) Inc. BJ-5ta and L929 cells were obtained from ATCC, while B16-F10 and MC38 cells were purchased from the National Collection of Authenticated Cell Cultures.

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Microscopy, Imaging, Western Blot, Transfection