Journal: Endocrine Oncology

Article Title: Vitamin D receptor expression in germinal centre type diffuse large B-cell lymphoma cells is associated with vitamin D insensitivity

doi: 10.1530/EO-25-0057

Figure Lengend Snippet: Vitamin D receptor (VDR) protein is expressed in both GC- and ABC-type DLBCL cells. (A and B) VDR transcript expression (probe 840) and BCL6 transcript expression in cultured human DLBCL cell lines, determined by 2 −ΔΔCt real-time PCR analysis after normalisation to 18S , relative to expression in DB, n = 3, mean ± SD. (C and D) Western blot analysis of human DLBCL cell lines. GC- and ABC-DLBCL blots in C were developed in parallel, representative of at least two experiments. (E) Double immunohistochemistry detecting VDR (brown) and CD20 (blue) staining of OCI-Ly8 cells. (F) DepMap analysis of VDR and BCL6 transcript expression in 30 DLBCL cell lines subtyped as GC (black), ABC (grey) or unknown (white).

Article Snippet: Universal MasterMix II (LifeTechnologies) and the following TaqMan assays (all Applied Biosystems, UK) were used for quantitative amplification on a Chromo4 instrument (BioRad, UK): hVDR (Hs01045840_m1), hIRF4 (Hs00180031_m1), hIGF1 (Hs01547656_m1), hHPGD (Hs00960587_m1), hSLA (Hs00277129_m1), hBCL6 (Hs00153368_m1) and hIL6 (Hs00985639_m1).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining

Journal: The Journal of Immunology Author Choice

Article Title: Defining T Cell Subsets in Human Tonsils Using ChipCytometry

doi: 10.4049/jimmunol.2100063

Figure Lengend Snippet: Intracellular markers for ChipCytometry cell suspension staining

Article Snippet: 1 , BCL6 , REA373 , PE , Miltenyi Biotec.

Techniques: Suspension

Journal: The Journal of Immunology Author Choice

Article Title: Defining T Cell Subsets in Human Tonsils Using ChipCytometry

doi: 10.4049/jimmunol.2100063

Figure Lengend Snippet: Phenotyping of tonsil-derived, CD3-enriched cells by cell suspension ChipCytometry. Four human tonsils were processed to single-cell suspensions, positively enriched for CD3 + T cells, barcoded, loaded onto a microfluidic chip, fixed, and subsequently analyzed by ChipCytometry with a panel of 19 fluorochrome-labeled Abs. ( A ) Density plot shows t -SNE analysis of concatenated samples, including all CD3 + CD19 − cells. ( B ) Clusters that were identified via FlowSOM were projected onto the t -SNE coordinates and color coded as indicated. ( C ) A heatmap showing median marker intensities within each cell cluster was generated to characterize cell clusters and derive cell labels. The heatmap color represents median marker expression across all four aggregated samples. ( D ) T-SNE plots show the expression of 16 markers within the aggregated samples. ( E ) Top panel, Within CD4 + T cells, the subsets TFH, pre-TFH, and non-TFH cells were identified according to expression of CXCR5 and PD1. Center panel, BCL6 expression is shown within non-TFH (red), pre-TFH (blue), and TFH (orange) cell subsets in human tonsils. Bottom panel, T-SNE plot shows non-TFH (red), pre-TFH (blue), TFH (orange), and non-CD4 T cells (black). Plots in (E) are derived from one donor and are representative of four donors.

Article Snippet: 1 , BCL6 , REA373 , PE , Miltenyi Biotec.

Techniques: Derivative Assay, Suspension, Labeling, Marker, Generated, Expressing

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 9 Deficiency Protects against Effector T Cell-mediated Systemic Autoimmunity

doi: 10.1074/jbc.m111.233932

Figure Lengend Snippet: FIGURE 3. HDAC9 regulates genes expressed in follicular and extrafollicular T effector cells in MRL/lpr mice. A, shown is real time PCR analysis of ICOS mRNA in CD4 T cells from KO compared with WT mice. B, representative flow cytometric plots used for graph C for B220CD4CD44high ICOS T cells on splenocytes from KO are compared with WT mice. D, representative flow cytometric plots of CD4CD44highCD62LloPSGL-1lo T cells for graph E in splenocytes from KO are compared with WT mice. F, real time PCR analysis of mRNA expression of the indicated genes associated with follicular and extrafollicular T cells from KO are compared with WT mice. G, a real time PCR analysis of roquin mRNA in CD4 T cells from KO is compared with WT mice. H, shown is a Western blot analysis of roquin (from pooled samples of three mice from each group) that demonstrates increased expression in KO mice consistent with roquin mRNA expression; data are representative of two independent experiments. I, ChIP assays were performed in splenocytes from the KO and WT mice. Chromatin was immunoprecipitated with antibodies against total IgG, AcH3, Ac-H3K9, and AcK18. Primers flank the roquin gene, and precipitated DNA was analyzed by PCR. Non-immunoprecipitated samples served as an input control. A representative figure of two independent experiments from pooled samples of three mice in each group in each experiment is shown. J, real time PCR analysis demonstrates increased miRNA-101 in CD4 T cells from the KO compared with WT mice. miRNA-101 expression was normalized to control miRNA-U6. Real time PCR (K) and Western blot analysis (L) of BCL6 in splenocytes demonstrates decreased expression in KO compared with WT mice. Results represent the mean S.E. of six animals per group. *, p 0.05; **, p 0.01(Student’s t test).

Article Snippet: Protein Extraction and Western Blot Analysis—Protein extraction and Western blot analysis were performed using the following antibodies: Ac-H3, Ac-H3K9, Ac-H3K14, Ac-H3K18, Ac-H3K23 (Upstate Biotechnology), HDAC9 (BioVision); roquin (Novus); PPAR- (Santa Cruz Biotechnology); BCL6 (Novus) as described previously (20, 25).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunoprecipitation, Control