Journal: PLoS ONE

Article Title: TESTIN Induces Rapid Death and Suppresses Proliferation in Childhood B Acute Lymphoblastic Leukaemia Cells

doi: 10.1371/journal.pone.0151341

Figure Lengend Snippet: Sequenom MassARRAY methylation data of (A) normal, somatic tissues, and (B) lymphoblastoid cell lines (95% confidence intervals are shown). (C) TES expression levels by quantitative RT-PCR were calculated relative to B2M expression and normalised to relative expression levels in the normal peripheral blood leukocyte sample (PBL2) (error bars are standard deviations).

Article Snippet: Lymphoblastoid cell lines were cultured as recommended by the ATCC or the RIKEN BioResource Center.

Techniques: Methylation, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Single cell discovery of m 6 A RNA modifications in the hippocampus

doi: 10.1101/2023.12.06.570314

Figure Lengend Snippet: (A) Schematic diagram of m 6 A detection with DART-seq in cultured cells. The YTH protein domain binds to m 6 A. When bound to APOBEC1, the APOBEC1 protein converts C-to-U in the vicinity of m 6 A. This results in a C-to-T mutation in cDNA. C-to-T mutations detected by RNA sequencing are indicative of m 6 A RNA modifications. (B) Left: Immunofluorescence (IF) of HEK293T cells transfected with Apobec1-Yth-HA- Egfp ( Yth-HA-E ) or Egfp-Apobec1-Yth-HA (E-YTH-HA ). Scale bar, 20μm, representative images are shown. Right: Quantification of EGFP and HA overlap. PCC: Pearson’s Correlation Coefficient. (C) Number of C-to-T editing events identified in each bulk DART-seq HEK293T cell replicate for Yth-E and E-Yth plasmids. Editing events identified in at least 2 replicates were considered for downstream analyses. The data was obtained following Apobec1-Yth-Egfp or Egfp-Apobec1-Yth transfection and EGFP FACS sorting. n=3, Rep: separately cultured replicate. (D) Metagene analysis showing m 6 A site counts along transcripts for Yth-E and E-Yth DART-seq results. 9% of all m 6 A sites occur in the first 10% of the 3’ UTR following the TTS for YTH-E, and 11% for E-YTH, respectively. Shown percentage indicates number of m 6 A sites upstream, within and downstream of coding sequence (CDS). (E) Metagene analysis showing m 6 A density 500nt 5’ and 500nt 3’ from stop codon (0nt) for YTH-E and E-YTH.

Article Snippet: HEK293T cells were purchased from Beijing Xiehe Cell Bank and cultured at 37°C with 5% CO2 in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS and 1% penicillin/streptomycin.

Techniques: Cell Culture, Mutagenesis, RNA Sequencing, Immunofluorescence, Transfection, Sequencing

Journal: Immunology

Article Title: The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for V δ 2 + γδ T cells in the modulation of immune response to tumour?

doi: 10.1111/imm.13248

Figure Lengend Snippet: V δ 2 + γδ T cells release granulysin in response to tumour. (a) The expression of exhaustion markers PD‐1 and Lag‐3 on, and the secretion of granulysin by, V δ 2 + γδ T cells during the 9‐day expansion process. (b) The percentage of V δ 2 + γδ T cells to express early activation marker CD69 following 24, 48 or 72 hr of culture with Daudi cells, Raji cells or Raji cells pre‐treated for 24 hr with 5 μ m zoledronic acid (ZA), as determined by flow cytometry. (c) The concentration of interferon‐ γ (IFN‐ γ ) found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (d) The percentage of V δ 2 + γδ T cells to express degranulation marker CD107a following 24, 48 or 72 hr of culture with Daudi cells, Raji cells or Raji cells pre‐treated for 24 hr with 5 μ m ZA, as determined by flow cytometry. (e) The concentration of granulysin found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (f) The concentration of granzyme B found within supernatants taken from 24, 48 or 72 hr co‐culture of V δ 2 + γδ T cells with tumour cell lines, as determined by ELISA. (g) Percentage killing of tumour cells by V δ 2 + γδ T cells following 24, 48 and 72 hr of culture, as determined by flow cytometry. Data shown are from six independent experiments, using V δ 2 + γδ T cells from six individual donors, with error bars (SD). Differences between groups were assessed by two‐way analysis of variance comparing negative control (V δ 2 + γδ T cells alone) with all other groups. * P < 0·05. ** P < 0·01. *** P < 0·001. **** P < 0·0001.

Article Snippet: Daudi and Raji B‐cell lymphoma lines (European Collection of Authenticated Cell Cultures, Salisbury, UK) were used in experiments as γδ T‐cell‐susceptible and ‐resistant target cells, respectively.

Techniques: Expressing, Activation Assay, Marker, Flow Cytometry, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Negative Control