Journal: Cellular and Molecular Immunology

Article Title: The RNA binding proteins TIA1 and TIAL1 promote Mcl1 mRNA translation to protect germinal center responses from apoptosis

doi: 10.1038/s41423-023-01063-4

Figure Lengend Snippet: GC responses are impaired in the absence of TIA1 and TIAL1. a Analysis by flow cytometry of GCs in the spleen of control and Tia1 fl/fl Tial1 fl/fl AID Cre mice 7 and 14 days post-immunization with NP-KLH in alum. Left panels, representative dot plots. Right panels, quantitation of the percentage and number of GC B cells. b Percentage and number of IgG1+ GC B cells in the spleen of control and Tia1 fl/fl Tial1 fl/fl AID Cre mice 7 days after immunization. c Time course analysis of total and IgG1+ GC B cells in draining LNs from control and Tia1 fl/fl Tial1 fl/fl AID Cre mice. d Distribution of GC B cells in the DZ and LZ analyzed by flow cytometry (CXCR4 + CD86 - DZ GC B cells and CXCR4 - CD86 + LZ GC B cells). e Visualization of GCs in the spleen of control and Tia1 fl/fl Tial1 fl/fl AID Cre mice by confocal microscopy. Bottom panels show the size and LZ/DZ ratio in each of the 19 GCs analyzed from 4 different mice analyzed in two independent experiments. f Representative pseudocolor dot plots showing GC cell viability by flow cytometry. Proapoptotic and apoptotic cells were labeled with FITC-VAD-FMK in combination with the Zombie NIR fixable viability dye. Data in ( a ), ( b ), ( c ) and ( d ) are from two to four independent experiments performed with at least n = 5 mice per group. Data in ( f ) are representative of one of the three independent experiments performed. Mann‒Whitney tests were performed for statistical analysis

Article Snippet: Cell viability was assessed by combining Zombie NIR Fixable Viability and CaspACETM FITC-VAD-FMK in Situ Marker from Promega (1 μM per 10 7 cells incubated at 37 °C in RPMI-1640 containing 10% FCS, antibiotics, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol for 30 minutes).

Techniques: Flow Cytometry, Control, Quantitation Assay, Confocal Microscopy, Labeling

Journal: Cellular and Molecular Immunology

Article Title: The RNA binding proteins TIA1 and TIAL1 promote Mcl1 mRNA translation to protect germinal center responses from apoptosis

doi: 10.1038/s41423-023-01063-4

Figure Lengend Snippet: TIA1 and TIAL1 control the expression of MCL1 and survival of GC B cells. a Viability of antigen-specific GC cells in control and Tia1 fl/fl Tial1 fl/fl AID Cre mice at Days 7 and 14 post-immunization with NP-KLH. Cell viability was assessed using FITC-VAD-FMK and Zombie NIR viability dye. Data are from two or three independent experiments depending on the time point with at least five mice used per genotype and experiment. b Analysis by FACS of MCL1 expression in naïve, GC and memory B cells. Data from one of the four independent experiments performed are shown (n = 7 mice). c Genome viewer of TIA1 and TIAL1 binding sites annotated to Mcl1 mRNA. Polyadenylation sites with a central AAUAAA sequence are marked in yellow. d Quantitation of MCL1 expression in naïve and GC B cells from control and Tia1 fl/fl Tial1 fl/fl AID Cre mice. Data from one of the four independent experiments performed with at least 5 mice per genotype. e Mcl1 mRNA abundance in DZ and LZ B cells from control and Tia1 fl/fl Tial1 fl/fl AID Cre mice. Data from the mRNAseq analyses are shown in Fig. . P-adjusted values are from differential expression analyses with DESeq2. f MCL1 expression in DZ and LZ B cells from the mice shown in ( d ). g Expression of MCL1 in IgG1-class switched GC B cells in mice from ( d ). Mann‒Whitney tests were performed in ( a ), ( d ), ( f ) and ( g ). Paired t tests were performed for statistical analysis in ( b )

Article Snippet: Cell viability was assessed by combining Zombie NIR Fixable Viability and CaspACETM FITC-VAD-FMK in Situ Marker from Promega (1 μM per 10 7 cells incubated at 37 °C in RPMI-1640 containing 10% FCS, antibiotics, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol for 30 minutes).

Techniques: Control, Expressing, Binding Assay, Sequencing, Quantitation Assay, Quantitative Proteomics