Journal: Fungal Systematics and Evolution

Article Title: New and Interesting Fungi. 6

doi: 10.3114/fuse.2023.11.09

Figure Lengend Snippet: Collection details and GenBank accession numbers of isolates treated in this study, and associated ex-type strains where available. Species for which additional sequences were generated during the course of this study are also listed here. Novel GenBank accession numbers are indicated in bold font.

Article Snippet: Closest hits using the rpb2 (first part) sequence had highest similarity to Ruptoseptoria unedonis (strain CBS 755.70, GenBank MF951659.1; Identities = 768/771 (99 %), no gaps), Chuppomyces handelii (strain CBS 113302, GenBank MF951475.1; Identities = 677/773 (88 %), no gaps), and Neopenidiella nectandrae (strain ATCC 200932, GenBank MF951546.1; Identities = 580/740 (78 %), 16 gaps (2 %)).

Techniques: Generated

Journal: Fungal Systematics and Evolution

Article Title: New and Interesting Fungi. 6

doi: 10.3114/fuse.2023.11.09

Figure Lengend Snippet: Ruptoseptoria unedonis (CPC 44069). Conidia. Scale bar = 10 μm.

Article Snippet: Closest hits using the rpb2 (first part) sequence had highest similarity to Ruptoseptoria unedonis (strain CBS 755.70, GenBank MF951659.1; Identities = 768/771 (99 %), no gaps), Chuppomyces handelii (strain CBS 113302, GenBank MF951475.1; Identities = 677/773 (88 %), no gaps), and Neopenidiella nectandrae (strain ATCC 200932, GenBank MF951546.1; Identities = 580/740 (78 %), 16 gaps (2 %)).

Techniques:

Journal: Cell reports

Article Title: Collagen 1-mediated CXCL1 secretion in tumor cells activates fibroblasts to promote radioresistance of esophageal cancer.

doi: 10.1016/j.celrep.2023.113270

Figure Lengend Snippet: Figure 7. Col1 treatment promotes the secretion of CXCL1 in ESCC cells via NF-kB pathway (A) TRRUST enrichment analysis of elevated cytokines in Col1-treated KYSE450 cells. (B) qRT-PCR analysis of CXCL1 in ESCC cells treated with Col1 and RELA knockdown. The expression level was normalized to GAPDH (n = 3 biological rep- licates). (C) Western blot analysis of NF-kB phosphorylation in ESCC cells added to Col1-coated and uncoated plates for 24 h. (D) Representative immunofluorescence images of the activation and translocation of NF-kB P65 by Col1 treatment. KYSE450 cells were added to Col1-coated and uncoated plates for 24 h and then immunostained with NF-kB P65 antibody, followed by Cy3-conjugated secondary antibody (red) and DAPI (blue). White arrows indicate NF-kB P65 translocation in cells. Scale bar, 100 mm. (E) Quantification of the percentage of NF-kB P65 activated and translocated KYSE450 cells in (D) (n = 6 biological replicates). (F) Western blot analysis shows that the Akt inhibitor MK-2206 abrogated the phosphorylation of P65 and the increase of CXCL1 promoted by Col1 treatment. For all panels, data are presented as mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined using two-tailed Student’s t test. Each assay for western blot had three biological repeats, and the quantification results are presented below each band. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Fetal Bovine Serum Cell Technologies Cat# 30070 DMEM Corning Cat# 10-013-CV Advanced DMEM/F12 Gibco Cat# 12634028 Puromycin Gibco Cat# A1113803 Lipofectamine 2000 Invitrogen Cat# 11668027 Collagenase I Gibco Cat# 17100017 Collagenase IV Gibco Cat# 17104019 Hyaluronidase Sigma-Aldrich Cat# H1115000 EDHB Sigma-Aldrich Cat# E24859 MK-2206 MCE Cat# HY-108232 SB-265610 MCE Cat# HY-50688 Human recombinant CXCL1 MCE Cat# HY-P70508 Rat tail collagen type 1 Solarbio Cat# C8062 Critical commercial assays RNA-Quick Purification Kit ES Science Cat# RN001 TB Green Premix Ex TaqTMII (Tli RNaseH Plus) TaKaRa Cat# RR820A Masson’s Trichrome Stain Kit Solarbio Cat# G1340 Meilunbio FGSuper Sensitive ECL Luminescence Reagent Meilunbio Cat# MA0186-1 PierceTM Rapid Gold BCA Thermo Fisher Scientific Cat# A53226 Human Cytokine Array GS440 Raybiotech Cat# GSH-CAA-440-1 CXCL1 ELISA Kit Proteintech Cat# KE00133 Opal 5-Color Manual IHC Kit PANOVUE Cat# 10144100100 NF-kB Activation Nuclear Translocation Assay Kit Beyotime Cat# SN368 Deposited data ESCC WES and RNA-seq data of 19 PDX donors This paper GSA-Human: HRA004329 Single-cell RNA-seq raw data of 6 PDXmice This paper GSA: https://ngdc.cncb.ac.cn/gsa/browse/ CRA010501 Experimental models: Cell lines KYSE450 Dr. Y. Shimada JCRB1430 KYSE510 Dr. Y. Shimada JCRB1436 NIH/3T3 ATCC CRL-1658 Human derived esophagus normal fibroblast (NF) This paper N/A PDX-derived primary tumor cells (PDC) This paper N/A Experimental models: Organisms/strains NSG mouse IDMO N/A Oligonucleotides shRNA targeting sequences, see Table S3 JTSBIO Co., Ltd N/A siRNA, see Table S3 JTSBIO Co., Ltd N/A qRT-PCR primers, see Table S4 Tsingke Biotechnology N/A Software and algorithms R 4.2.2 R Core Team https://www.R-project.org/ Cell Ranger 6.0.2 10x Genomics https://10xgenomics.com/ Seurat (version 4.3.0) Hao et al.46 https://github.com/satijalab/seurat (Continued on next page) 18 Cell Reports 42, 113270, October 31, 2023

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Phospho-proteomics, Activation Assay, Translocation Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Memory like NK cells display stem cell like properties after Zika virus infection

doi: 10.1371/journal.ppat.1009132

Figure Lengend Snippet: ( A ) Hallmark pathway gene sets analysis in FACS purified CD27 + memory like and non-memory CD27 - NK cells a month post ZIKV infection. RNA-seq data are from 3 biological replicates for each group. ( B ) ATAC-seq tracks for selected loci of Wnt/β-catenin pathway genes in CD27 + memory like (green) and non-memory CD27 - NK cells (blue). ( C-D ) GSEA enrichment plots ( C ) and heat maps ( D ) for Wnt/β-catenin pathway ( C left ) and canonical Wnt targets ( C right ) among CD27 + memory like and non-memory CD27 - NK gene sets. (E - G ) Immunofluorescence images of FACS purified CD27 + memory like and non-memory CD27 - NK cells, isolated a month post ZIKV infection, and stained for TCF-1 (yellow) and β-catenin (red) show co-localization of both molecules in CD27 + cells. Scale bar corresponds to 10 μm. Top-CD27 + NK cells, Bottom-CD27 - NK cells. Data are representative of 2 independent experiments. ( G ) Relative fluorescent intensity of TCF-1 in CD27 + memory like and non-memory CD27 - NK. Data are representative of 2 independent experiments (n = 4 per experiment). ( H ) Transcription factor motif enrichment analysis of ATAC-seq from CD27 + memory like NK cells. ( I ) % of target sequences with TCF-1 motifs in genomic regions of CD27 + memory like and non-memory CD27 - NK cells. Chromatin opening by ATAC-seq ( J ) and gene expression by RNA-seq ( K ) of selected TCF-1 target genes in CD27 + memory like NK cells. Fold change values for peaks are plotted in ( J ). Mean ± s.d. two-sided Student’s t-test, *** P ≤ 0.001.

Article Snippet: This was followed by antibody staining step and cells were incubated with TCF-1-PE (4:100) (S33-966, BD Biosciences, #564217) and β-catenin-APC (4:100) (REA480, Miltenyi Biotech, #130124453) antibodies in PBS with 0.5% BSA for 45 min. Then, cells were washed with PBS containing 0.5% BSA, pelleted and subjected to cytospin for 2 minutes and taken on the slides.

Techniques: Purification, Infection, RNA Sequencing, Immunofluorescence, Isolation, Staining, Gene Expression

Journal: Cell reports

Article Title: Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

doi: 10.1016/j.celrep.2021.109105

Figure Lengend Snippet: (A) A generalized schematic of the platform. Human CD14 + monocytes are isolated from blood by density gradient separation of PBMCs followed by magnetic negative selection. Either PBMCs or monocytes may be cryopreserved for later editing . Cells are then nucleofected with preformed CRISPR-Cas9 RNPs and immediately put into differentiating culture under MDM- or MDDC-generating conditions. After allowing for 6–7 days of differentiation and washout of the targeted gene product, cells can be subjected to a wide variety of functional, phenotypic, and genotypic studies to assess the knockout efficiency and function of the targeted gene product. (B) Guide sequence-dependent knockout of targeted genes leads to loss of gene products. CD14 + monocytes were nucleofected with RNPs containing 1 of 5 distinct guide sequences against the indicated gene or a scrambled non-targeting control, cultured under MDM-generating conditions, and then lysed for immunoblot analysis. Blots show targeted gene protein product and untargeted housekeeping gene product β-actin protein levels in cells from 2 blood donors. GNE1 and ATP6V1A ran at their expected sizes of 79 and 69 kDa, respectively. (C) Knockout was quantified by digital densitometry and normalized on a per-sample basis in relative fluorescence units (RFUs) to untargeted housekeeping control protein β-actin. See also .

Article Snippet: To assess the quality of the CD14+ negative selection, samples before and after CD14 enrichment were stained with antibodies against CD14-PE (1:25)(Miltenyi 130–110-519) and CD16-APC (1:25)(Miltenyi 130–106-705).

Techniques: Isolation, Selection, CRISPR, Functional Assay, Knock-Out, Sequencing, Control, Cell Culture, Western Blot, Fluorescence

Journal: Cell reports

Article Title: Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

doi: 10.1016/j.celrep.2021.109105

Figure Lengend Snippet: (A) Principal-component analysis of RNA sequencing (RNA-seq) from the indicated cell types. (B) Normalized transcript abundance ( Z score) for selected markers of MDM or MDDC differentiation . (C) Dendrogram of hierarchical clustering of the data in (B) by Euclidean distance. (D and E) Among cells subjected to CRISPR-Cas9 RNP nucleofection, cell surface protein levels of CD16, CD14, and CD206 were compared between the cells that bear the desired β2 m knockout (pink) and those that do not (teal) by flow cytometry after 7 days of MDM differentiation. (D) shows gating, while (E) shows the expression of the indicated markers. (F) Representative images of unperturbed (left) and RNP-nucleofected (right) MDMs infected with GFP-expressing M. tuberculosis (Mtb-GFP) show that CRISPR-Cas9-targeted cells remain competent to phagocytose living pathogens. Top, membrane staining with Cell Mask Far Red; CENTER, Mtb-GFP; bottom, composite. Scale bars represent 100 μm. See also .

Article Snippet: To assess the quality of the CD14+ negative selection, samples before and after CD14 enrichment were stained with antibodies against CD14-PE (1:25)(Miltenyi 130–110-519) and CD16-APC (1:25)(Miltenyi 130–106-705).

Techniques: RNA Sequencing, CRISPR, Knock-Out, Flow Cytometry, Expressing, Infection, Membrane, Staining

Journal: Cell reports

Article Title: Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

doi: 10.1016/j.celrep.2021.109105

Figure Lengend Snippet:

Article Snippet: To assess the quality of the CD14+ negative selection, samples before and after CD14 enrichment were stained with antibodies against CD14-PE (1:25)(Miltenyi 130–110-519) and CD16-APC (1:25)(Miltenyi 130–106-705).

Techniques: Blocking Assay, Recombinant, Virus, Purification, Staining, Isolation, Cell Viability Assay, Plasmid Preparation, Software, Cell Analysis, Modification, Membrane, Western Blot, Cell Culture, Sterility