Journal: eLife

Article Title: Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages

doi: 10.7554/eLife.69982

Figure Lengend Snippet: ( A, B ) Total IKKα, p38 MAPK, phospho-IKKα(Ser180)/IKKβ(Ser181), and p38 MAPK (Thr180/Tyr182) ( A ) and the ratio of phospho/total proteins ( B ) analyzed by flow cytometry in mTEC lo from WT and ΔCD4 mice. Data are representative of two independent experiments (n = 3–4 mice per group and experiment). ( C ) Scatter plot of gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) of mTEC lo from WT versus ΔCD4 mice. Genes with fold difference ≥2 and p-adj<0.05 were considered as upregulated or downregulated genes (red and blue dots, respectively). RNA-seq was performed on two independent biological replicates with mTEC lo derived from 3 to 5 mice. ( D ) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated (left panel) and the proportion of upregulated TRAs compared to those in the all genome (right panel). ND, not determined. ( E ) Numbers of induced Aire-dependent, Fezf2-dependent, Aire/Fezf2-dependent, and Aire/Fezf2-independent TRAs. ( F ) The expression of Aire-dependent ( Meig1, Nov ), Fezf2-dependent ( Fcer2a, Kcnj5 ), Aire/Fezf2-dependent ( Krt1, Reig1 ), and Aire/Fezf2-independent ( Crp, Rsph1 ) TRAs measured by qPCR in WT (n = 3–4) and ΔCD4 (n = 3–4) mTEC lo . ( G ) Expression fold change in HDAC3-induced transcriptional regulators and other transcription factors significantly upregulated in WT versus ΔCD4 mTEC lo . The color code represents gene expression level. ( H ) Heatmaps of genes encoding for cell adhesion molecules and cytokines that were significantly downregulated in mTEC lo from ΔCD4 mice. ( I ) Hierarchical clustering and heatmap of mean expression of these cell adhesion molecules and cytokines in mTEC subsets identified by scRNA-seq. Error bars show mean ± SEM, *p<0.05, **p<0.01 using two-tailed Mann–Whitney test for ( A ), ( B ) and ( F ) and chi-squared test for ( D ).

Article Snippet: Sequence-based reagent , Meig1-RV , Sigma-Aldrich , PCR primers , CAAGGTTTCAAGGTGGGTGT.

Techniques: Flow Cytometry, Expressing, RNA Sequencing Assay, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages

doi: 10.7554/eLife.69982

Figure Lengend Snippet: ( A ) The expression of Aire-dependent ( Meig1, Nov ), Fezf2-dependent ( Fcer2a, Kcnj5 ), Aire/Fezf2-dependent ( Krt1, Reig1 ), and Aire/Fezf2-independent ( Crp, Rsph1 ) tissue-restricted self-antigens (TRAs) was measured by qPCR in purified mTEC lo from WT (n = 3), ΔCD4 (n = 3), and MHCII -/- (n = 3) mice. ( B ) Itgb6, Cdh2, Il21, and Il5 mRNAs were measured by qPCR in WT (n = 4) and ΔCD4 (n = 4) mTEC lo .

Article Snippet: Sequence-based reagent , Meig1-RV , Sigma-Aldrich , PCR primers , CAAGGTTTCAAGGTGGGTGT.

Techniques: Expressing, Purification

Journal: eLife

Article Title: Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages

doi: 10.7554/eLife.69982

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Meig1-RV , Sigma-Aldrich , PCR primers , CAAGGTTTCAAGGTGGGTGT.

Techniques: Cell Isolation, Recombinant, Software, Real-time Polymerase Chain Reaction, Sequencing, Plasmid Preparation