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

Journal: Experimental & Molecular Medicine

Article Title: Discovery of ETI41 and ETI60: novel selective endosomal Toll-like receptor inhibitors for the treatment of autoimmune diseases

doi: 10.1038/s12276-025-01526-w

Figure Lengend Snippet: a Chemical structures of the main scaffold, TAC (red), and the potent inhibitors, ETI41 and ETI60. b Cell survival curve according to ETI concentration (1.6–200 μM) was measured by MTT assay in murine RAW 264.7 cell line and water-soluble tetrazolium assay in a human Daudi cell line. c Inhibitory effects of ETI15, ETI41 and ETI60 (ranging from 3.9 nM to 10 μM) on TLR7 and TLR9 were assessed by quantifying TNF-α secretion in murine RAW 264.7 cells and human Daudi cells, respectively. d ETI41 and ETI60 inhibited TLR3, TLR7 and TLR8 in a concentration-dependent manner (31.2 nM to 10 μM), as indicated by the reduction in TNF-α secretion in mouse RAW 264.7 cells. e Specificities of ETI41 and ETI60 were confirmed by measuring TNF-α secretion in surface TLRs (TLR1, TLR2, TLR4, TLR5 and TLR6. The cells were activated with agonistic ligands: TLR1/2 (FSL-1, 100 ng/ml, 4 h), TLR2/6 (Pam3CSK4, 100 nM, 4 h), TLR3 (poly I:C, 2 μg/ml, 24 h), TLR4 (LPS, 10 or 100 ng/ml, 4 h), TLR5 (FLA-ST, 500 ng/ml, 4 h), TLR7 (ORN06/LyoVec, 2 μg/ml, 24 h; and IMQ, 1 μg/ml, 4 h), TLR8 (TL8-506, 2 μg/ml, 24 h) and TLR9 (ODN2395, 1 μM, 4 h) at various concentrations in RAW 264.7 cells, human Daudi cells and THP-1 cells. Data are from at least three independent experiments ( n = 3) and statistical differences between the induced case and other cases were analyzed and verified using a one-tailed Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The Daudi cell line (Korean Cell Line Bank) was cultured in RPMI 1640.

Techniques: Concentration Assay, MTT Assay, WST Assay, One-tailed Test