Journal: The Journal of Pathology

Article Title: Spatial transcriptomics exploration of the primary neuroblastoma microenvironment in archived FFPE samples unveils novel paracrine interactions

doi: 10.1002/path.6457

Figure Lengend Snippet: Identification of a spatially defined NRTN – GRFA2/RET interaction and experimental validation in NB cells. (A) Chord plot showing the number of predicted outgoing signaling interactions from the AC‐like cluster. Number of interactions with NE1 and NE2‐CA and examples of common interactions given. See supplementary material, Table for detailed results. (B) Spatial gene expression plots showing NRTN , GFRA2 , and RET expression in sections, as indicated. (C) Bar plot indicating percentages of NRTN expressing primary NB tumors with high (>90th percentile), intermediate, and low (<10th percentile) AC‐like signatures, as indicated. p values calculated using chi‐square test. (D) Kaplan–Meier survival plots comparing overall survival between NB patients with high (>90th percentile), intermediate (10th–90th percentile), and low (<10th percentile) gene expression, as indicated. p values calculated using the log rank test. (E) Western blot showing expression of GFRA2 in four different NB cell lines, as indicated. Normalized densitometry values indicated below each blot. (F, G) Time‐dependent effect of NRTN (100 ng/ml; n = 3) and/or selpercatinib (500 n m ; n = 3) application on (F) relative wound density (RWD; measured using a scratch/wound migration assay), and (G) cell growth (measured on Incucyte S3 system) on different NB cell lines, as indicated. p values for indicated timepoints calculated using an unpaired, two‐sided Student's t ‐test.

Article Snippet: Samples were subjected to western blotting analyses using the following antibodies: PITPNM3 (1:1,000) (Thermo Fisher Scientific, Cat# PA5‐21903, RRID:AB_11154802 ), GFRA2 (1:1,000) (Proteintech, Cat# 21973‐1‐AP, RRID:AB_11124728 ), and beta‐actin (1:10,000) (Cell Signaling Technology, Danvers, MA, USA; Cat# 4970, RRID:AB_2223172 ).

Techniques: Biomarker Discovery, Gene Expression, Expressing, Western Blot, Migration

Journal: Epigenetics

Article Title: Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood

doi: 10.4161/epi.28153

Figure Lengend Snippet: Table 2. Top 10 list of the most significant CpG sites based on ordinary-least squares regression analysis modeling the association between M -value and log 10 arsenic, adjusted for infant sex and 6 imputed leukocytes cell mixture coefficients

Article Snippet: Specifically, log 10 arsenic was modestly associated with increased methylation at cg04597393, cg16321474 (Ref Gene, VDR ), cg01783894, cg03783410 (Ref Gene, SOX2OT ), cg06448705 (Ref Gene, TRPC7 ), cg15346830 (Ref Gene, COL11A2), cg15641060, and a decrease in methylation at cg00122779 (Ref Gene, TNXB ) and cg02095504 (Ref Gene, CIB4 ). table ft1 table-wrap mode="anchored" t5 caption a7 CpG Site Effect estimate (M-val / log 10 As) Unadjusted P value q-value Location Infinium Type Nearby SNPs* RefGene Name RefGene Group Chromosome RefGene Accession cg00498691 0.26 5.89 x 10 −8 0.021 Island II 19 cg04597393 0.34 4.54 x 10 −7 0.056 I Yes 8 cg16321474 0.23 6.99 x 10 −7 0.058 Island I Yes VDR TSS1500 12 NM_001017535 cg00122779 -0.66 4.25 x 10 −7 0.056 II TNXB Body 6 NM_019105 cg01783894 0.28 7.87 x 10 −7 0.058 S_Shore I 1 cg03783410 0.21 9.49 x 10 −7 0.058 N_Shore II SOX2OT Body 3 NR_004053 cg02095504 -0.24 1.16 x 10 −6 0.061 II CIB4 3′UTR 2 NM_001029881 cg06448705 0.25 1.34 x 10 −6 0.062 II TRPC7 5′UTR 5 NM_001167576 cg15346830 0.22 1.79 x 10 −6 0.065 II COL11A2 Body 6 NM_080679 cg15641060 0.21 1.96 x 10 −6 0.065 N_Shore I 11 Open in a separate window * CpGs with polymorphism in Infinium probe or target CpG. caption a8 Table 2.

Techniques:

Journal: Molecular Therapy Oncolytics

Article Title: Adoptive T cell immunotherapy for medullary thyroid carcinoma targeting GDNF family receptor alpha 4

doi: 10.1016/j.omto.2021.01.012

Figure Lengend Snippet: P4-10 CAR T cells specifically respond to GFRα4 protein in vitro (A) Schematic of CAR construct inserted downstream of an EF1a-derived promoter within a 3 rd -generation lentiviral vector plasmid. (B) Primary human T cells were activated using anti-CD3/anti-CD28-coated beads followed by transduction with lentiviral vectors encoding the indicated CAR genes or were left non-transduced (NTD). Seven days later, T cells were stained with biotinylated F(ab′) 2 -specifc goat anti-mouse or donkey anti-rabbit followed by streptavidin-AF647 and analysis by flow cytometry. (C) NFAT-GFP reporter Jurkat cells expressing no CAR (NTD), 19bbz, or the P4-10bbz CAR were cultured in wells that had been coated overnight with GFRα1, GFRα2, GFRα3, GFRα4, and OKT3 proteins. Following overnight culture, cells were analyzed by flow cytometry for GFP expression. A representative of 3 independent experiments is shown. (D and E) NFAT-GFP reporter Jurkat cells (D) or primary human T cells (E) expressing no CAR (NTD), or the P4-10bbz CAR were cultured in wells that had been coated overnight with OKT3 or GFRα4 (bound) or in media with soluble GFRα4 (soluble). Following overnight culture, cells were analyzed by flow cytometry for GFP expression (*p < 0.001 compared to media control, ANOVA, Dunnett’s multiple comparisons test). Representatives of 2 independent experiments each are shown. Error bars indicate 1 standard deviation of the mean.

Article Snippet: Expression of GFRα1, GFRα2, and GFRα3 was assessed using polyclonal rabbit anti-GFRα1, anti-GFRα2, and anti-GFRα3 (Sino Biologicals, Wayne, PA, USA) followed by AF647-conjugated goat anti-rabbit antibody (Thermo Fisher Scientific).

Techniques: In Vitro, Construct, Derivative Assay, Plasmid Preparation, Transduction, Staining, Flow Cytometry, Expressing, Cell Culture, Standard Deviation