Journal: Neoplasia (New York, N.Y.)

Article Title: Liver fluke granulin promotes extracellular vesicle-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

doi: 10.1016/j.neo.2020.02.004

Figure Lengend Snippet: Programmed CRISPR/Cas9 mutation of the human progranulin gene. Panel A, linear map of a pre-designed lentiviral CRISPR/Cas9 vector containing fused codon-optimized puromycin resistance marker (dark gray bar)-Cas9 (yellow bar) and green fluorescent protein (green bar) driven by the mammalian elongation factor alpha-1 promoter (dark blue arrow); guide RNA (gRNA) targeting human granulin exon 2 (red bar) is expressed from a single vector. The human U6 promoter (blue arrow) drove the gRNA. The vector backbone includes the 5′- and 3′-long terminal repeats (LTR) of the HIV-1 provirus (light gray blocks). Panel B, schematic representation of the partial human granulin gene precursor, huPGRN on chromosome 17: NC_000017.11 regions 44,345,086–44,353,106 (8021 bp) and protein structure. Nucleotide sequence in exon 2 encodes the N-terminus and part of the granulin/epithelin module (GEM) of progranulin; indicating locations of gRNA (4417–4438 nt; red colored-letter) predicted double-stranded break (DSB) (red arrow). Panels C–E, on target INDEL mutations in ΔPGRN-H69 analysis by NGS libraries and CRISPResso bioinformatic platform. Frequency distribution of position-dependent insertions (red bars) (C) and deletion (magenta) (D) the major INDELs; 6 and 2 bp deletions at the programmed CRISPR/Cas9 cleavage site. Other minor mutations including 1 bp insertion (red square) or base substitution (bold) were observed further (>10 bp) predicted cleavage site (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Total RNA and evRNA either from H69 or ΔhuPGRN-H69 cells were isolated using RNAzol (Molecular Research Center, Inc.) or total exosome RNA isolation kit (ThermoFisher) following the manufacturer’s instructions.

Techniques: CRISPR, Mutagenesis, Plasmid Preparation, Marker, Sequencing

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

Journal: Developmental cell

Article Title: ESRP1 mutations cause hearing loss due to defects in alternative splicing that disrupt cochlear development

doi: 10.1016/j.devcel.2017.09.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Total RNA (200ng) was used for poly A selected RNA-seq library preparation using the NEBNext® Ultra ™ Directional RNA Library Prep Kit from Illumina® (mRNA) (New England Biolabs) (products: NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490S) and NEBNext® Ultra ™ Directional RNA Library Prep Kit for Illumina® (E7420S).

Techniques: Transduction, Recombinant, Isolation, Derivative Assay, Modification, Sequencing, CRISPR, Software

Journal: Genes

Article Title: PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

doi: 10.3390/genes11091029

Figure Lengend Snippet: Sequence distribution of most efficient mutations identified with the ICE software around the IPK gene target site in Zea mays . Edited sequences were obtained after CRISPR-Cas9 RNP transfection to maize protoplasts. Forty-five μg of Cas9 preassembled with 15 μg of each gRNA were used in the protoplast transformation. Different exposure times of the RNP complex with the protoplast are presented. Cut sites are represented by black vertical dotted lines, insertions are represented by ‘N’ and deletions by black horizontal dotted lines.

Article Snippet: Cas9 (100 nM), gRNA, and 1 × NEBuffer 3 (New England Biolabs Inc, NEB) were incubated for 10 min at 25 °C to form the RNP complex.

Techniques: Sequencing, Software, CRISPR, Transfection, Transformation Assay

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 modulates pancreatic tumor growth. A) Immortalized human pancreatic duct cells (hTERT-HPNE and HPDE6c7), human and murine PDA cell lines, and human patient derived-organoids (PDOs) demonstrate nSMase activity. Values shown are technical replicates of one lysate per cell line. B) SMPD3 expression was analyzed in an existing human pancreas organoid RNA-Seq dataset and is depicted in normal (n=11), primary (n=35), and metastatic (n=9) human organoids. Log of normalized counts of SMPD3 expression are plotted. C) Immunocytochemistry demonstrates nSMase2 expression in two pancreatic metastatic PDOs. Scale bar is 100uM. D) Nanoparticle tracking analysis of CD63 + sEVs upon GW4869 and vehicle treatment is depicted. E) IVIS imaging results at Day 7, 14, and 19 are graphed. Eight animals were injected with each cell line. F) Images depict bioluminescence signal during IVIS imaging at day 19. G) Pancreas weight/body weight at day 19 is graphed. Average values for the KPC scrambled shRNA are 0.04540 ± 0.001635 (n=8) and KPC Smpd3 shRNA 1 are 0.02817 ± 0.0008858 (n=7). H) Rag 1 KO mice injected orthotopically with KPC Smpd3 shRNA1 and KPC Smpd3 shRNA2 cell lines lived significantly longer than Rag1 KO mice injected orthotopically with KPC scrambled shRNA cells ( P =0.0013 using log-rank test and P =0.0001 using log-rank test, respectively). The median survival of Rag 1 KO mice injected orthotopically with KPC scrambled shRNA cells (n=12) is 19 days, KPC Smpd3 shRNA 1 cells (n=6) is 31.50 days, and KPC Smpd3 shRNA 2 cells (n=6) is 36.50 days.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Derivative Assay, Activity Assay, Expressing, RNA Sequencing, Immunocytochemistry, Imaging, Injection, shRNA

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: S m pd3 regulates proliferation of PDA cells in vivo . A) SMPD3 is expressed in a panel of human pancreatic duct cells (n=2 cell lines), human PDA cells (n=12 cell lines), and mouse PDA cells (n=3 cell lines). B) qPCR demonstrates a reduction in Smpd3 expression in KPC Smpd3 shRNA 1 LucFlag (n=3) and KPC Smpd3 shRNA 2 LucFlag (n=3) cell lines when compared to the KPC scrambled shRNA LucFlag (n=3) control cell line. C) Mean ± standard deviation for tumor volume of cell lines injected subcutaneously are graphed. Results of an unpaired t test comparing KPC scrambled shRNA and KPC Smpd3 shRNA 2 cell lines at day 17 are depicted on the graph. For all cell lines, N=6 animals. D) Mean ± standard error of the mean for tumor weight at Day 17 of cell lines injected subcutaneously are graphed. For all cell lines, N=6 animals. E) Immunofluorescence images and quantification of epithelial (CK19 + , Vimentin - , Dapi + ), proliferative (Ki67 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag line (n=4) are 39.33% ± 2.046%, KPC Smpd3 shRNA 1 LucFlag line (n=5) are 28.88% ± 4.096%, and KPC Smpd3 shRNA 2 LucFlag line (n=5) are 15.83% ± 1.960. Scale bar is 50uM. F) Immunofluorescence images and quantification of epithelial (E-cadherin + , Vimentin - , Dapi + ), apoptotic (Cleaved caspase 3 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.299% ± 0.2075%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.082% ± 0.2126%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=5) are 0.9832% ± 0.1307. Scale bar is 50uM. G) CTG assay or colony formation assay results for the indicated cell lines are depicted. H) Reduction of Smpd3 in epithelial pancreatic cancer cells modestly reduces fibrosis. IHC demonstrates loss of nSMase2 in epithelial pancreatic cancer cells in tumors generated using the KPC Smpd3 shRNA 1 LucFlag and KPC Smpd3 shRNA 2 LucFlag cell lines. One scale bar (1000uM) on the picrosirius red image is representative for all picrosirius red and corresponding brightfield images. One scale bar (200uM) on the nSMase2 IHC image is representative for all nSMase2 IHC images. I) Quantification of polarized light using the picrosirius red stained images is depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.734% ± 0.3760%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.319% ± 0.1328%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=4) are 0.9188% ± 0.1124.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: In Vivo, Expressing, shRNA, Control, Standard Deviation, Injection, Immunofluorescence, Generated, CTG Assay, Colony Assay, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 expression is upregulated in PanIN. A) Schematic details the construct design for the Smpd3 floxed mouse. B-C) For examination of nSMase2 expression in the indicated normal murine pancreatic cell types, nSMase2 IHC sections from 3 C57BL/6J mice were scored. Preneoplasia and neoplasia lesions were scored from KPC mice (n=10 animals scored for acinar-to-ductal metaplasia (ADM), n=12 animals scored for PanIN, n=10 animals scored for primary tumor, and n=4 animals scored for metastasis). Scale bars for IHC images are 40 uM. nSMase2 expression was scored on a scale of 0-3 and is depicted in (C). The scoring system used is 0=absent, 1=low, 2=medium, 3=high. Values depicted are representative for individual animals. D) Subcellular localization of nSMase2 was scored using the same slides and animals used for expression scoring in . Results from Chi-squared tests using a 2 x 3 table are shown. E-F) H&E and IHC for nSMase2 images are displayed for the indicated genotypes.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 ablation reduces formation of neoplasia and prolongs survival of KPC mice. A) KPC; Smpd3 f/f mice lived significantly longer than KPC; Smpd3 wt/wt mice ( P =0.0039 using log-rank (Mantel-Cox) test). The median survival of KPC; Smpd3 f/f mice (n=41) is 23.71 weeks, KPC; Smpd3 f/wt mice is 21.57 weeks (n=45), and KPC; Smpd3 wt/wt mice (n=47) is 20.00 weeks. B) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt mice (n=30), KPC; Smpd3 f/wt mice (n=27), and KPC; Smpd3 f/f mice (n=24). (C) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt (n=29), KPC; Smpd3 f/wt (n=25), and KPC; Smpd3 f/f (n=22) mice. Using Fisher’s exact tests, no significant differences were observed in the number or site of macrometastatic lesions present at end-stage in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , or KPC; Smpd3 f/f mice. If macrometastasis was present, only one site was observed per animal in these cohorts. D-E) Quantification of alcian blue staining demonstrates significantly reduced PanIN lesions in KPC; Smpd3 f/f mice (n=5) when compared to KPC; Smpd3 wt/wt mice (n=9) ( P =0.0154). H&Es used to score edema are depicted. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PanIN-bearing KPC; Smpd3 f/f mice. F) Edema scoring for KPC; Smpd3 wt/wt (n=9), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=7) mice is depicted. The scoring system used is 1=low, 2=medium, and 3=high. G-H) The percentage of pancreatic area occupied by PDA is significantly higher in KPC; Smpd3 wt/wt mice (74.16 ± 8.705) when compared to KPC; Smpd3 f/f mice (30.51 ± 17.72) ( P =0.0497) at 19-21 weeks of age. Average percent area is graphed in H (n=4 KPC; Smpd3 f/f mice, n=4 KPC; Smpd3 f/wt mice, n=5 KPC; Smpd3 wt/wt mice). Dotted lines show the PDA area in representative images. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PDA-bearing KPC; Smpd3 f/f mice at 19-21 weeks of age.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of the KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. A-F) Western blot depicts nSMase2 expression levels in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. G) Pictures of cell lines taken immediately before RNA isolation for RNA sequencing are shown. Scale bars are 100uM.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: RNA sequencing demonstrates a role for Smpd3 in modulation of cellular pathways and regulation of PDA subtype. A) Average luminescence values from 3-5 independent experiments per cell line are graphed. Average values for primary PDA cell lines generated from KPC; Smpd3 wt/wt mice (n=17 cell lines) are 98,614 ± 5,684, KPC; Smpd3 f/wt mice (n=14 cell lines) are 106,061 ± 6,699, and KPC; Smpd3 f/f mice (n=15 cell lines) are 88,460 ± 6,672. B) Average number of colonies from at least 3 independent colony formation assays per cell line are depicted. Average values from KPC; Smpd3 wt/wt mice (n=14 cell lines) are 26.02 ± 3.652, KPC; Smpd3 f/wt mice (n=14 cell lines) are 24.16 ± 2.519, and KPC; Smpd3 f/f mice (n=13 cell lines) are 21.34 ± 4.872. C) Heat maps depict expression of significantly deregulated molecules in Tumor Microenvironment Pathway and Hepatic Fibrosis Signaling Pathway in all three PPT cell lines. Molecules shown in heat maps include those molecules within the Ingenuity pathway analysis (IPA) pathway that were significantly deregulated in any comparison between PPT groups. D) IPA results show a bubble category chart of significantly deregulated pathways when comparing KPC; Smpd3 f/f PPT and KPC; Smpd3 wt/wt PPT cell lines. A positive z-score represents upregulation, and a negative z-score indicates downregulation of a pathway in KPC; Smpd3 f/f PPT when compared to KPC; Smpd3 wt/wt PPT cell lines. A gray circle depicts significant overrepresentation of a pathway, the direction of which cannot yet be determined. Select pathways are annotated. E) Picrosirius red stained images are shown for KPC; Smpd3 wt/wt (n=6), KPC; Smpd3 f/wt (n=5), and KPC; Smpd3 f/f (n=6) mice at 10-11 weeks of age. The small piece of intestine in the lower left corner of the KPC; Smpd3 f/wt image was excluded from the quantification. Scale bar is 1000uM. Inset shows brightfield image. F) Transcriptional subtyping of end-stage pancreatic tumor cell lines generated from KPC; Smpd3 wt/wt (PPT and MPT), KPC; Smpd3 f/wt (PPT), KPC; Smpd3 f/f (PPT and MPT) as well as KPC Mock OE and KPC Smpd3 OE lines is depicted. Genes used to identify subtypes according to the Moffitt classification are shown. G) Pie charts depict the number of cell lines with the classical and basal-like Moffitt subtypes. When comparing KPC; Smpd3 wt/wt PPT vs KPC; Smpd3 f/f PPT using Moffitt classification and Chi square test, p-value=0.0339.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Generated, Expressing, Comparison, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of RNA sequencing data from murine polyclonal pancreatic cancer cell lines. A) Heat map showing Integrin Signaling in all three PPT cell lines. B) Estimated counts for select genes identified from Sleuth comparison of polyclonal cell lines from end-stage PPTs of KPC; Smpd3 wt/wt (n=8), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=10) mice are graphed. Each box plot represents the distribution of estimated counts quantified by kallisto with 100 bootstrap samples. For each gene, the most significant protein-coding isoforms are shown. C) The top twenty pathways from IPA comparing KPC; Smpd3 f/f MPT vs KPC; Smpd3 wt/wt MPT are shown. D) A heat map including significantly differentially regulated molecules in the Pentose Phosphate Pathway are shown for KPC; Smpd3 wt/wt MPT and KPC; Smpd3 f/f MPT groups. E) The six differentially regulated pathways when comparing KPC Smpd3 OE vs KPC Mock OE are shown. F) Log of transcript per million (TPM) values for Smpd3 gene are graphed for KPC Mock OE and KPC Smpd3 OE sequenced cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: K P C ; Smpd3 f/f pancreata display significantly fewer activated stellate cells and fibroblasts when compared to both KPC; Smpd3 f/wt and KPC; Smpd3 wt/wt pancreata. A) Immunofluorescence images depict co-labeling with alpha SMA, Gfap, Desmin, and Dapi in the pancreas of 10–11-week-old mice. For KPC; Smpd3 wt/wt , n=6 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=5 animals. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel C. Scale bar is 20uM. B) Immunofluorescence images depict co-labeling with alpha SMA, Fap, Vimentin, and Dapi in the pancreas of 10–11-week-old mice. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel D. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, KPC; Smpd3 f/f , n=6 animals. Scale bar is 50uM. C) Quantification of alpha SMA, Gfap, Desmin, and Dapi quadruple positive cells is depicted. D) Quantification of alpha SMA, Fap, Vimentin, and Dapi quadruple positive cells is depicted. E) Pie charts depict Moffitt classification PDA subtypes for KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of lipidomics data from murine polyclonal pancreatic cancer cell lines. A) Heat map shows IPA Ceramide Signaling Pathway in all three PPT cell lines. B) Heat map shows differentially expressed lipids in comparisons amongst our murine polyclonal pancreatic cancer cell lines. Groups compared are listed as A-B where depicted upregulation or downregulation of a lipid would be in A when compared to B. C) Principal component analysis of our polyclonal murine pancreatic cancer cell lines based on lipid composition is depicted. D) Box plots showing expression of selected ceramide species in our KPC; Smpd3 wt/wt PPT, KPC; Smpd3 f/wt PPT, KPC; Smpd3 f/f PPT, KPC; Smpd3 wt/wt MPT, and KPC; Smpd3 f/f MPT cell lines. E) Box plots showing expression of selected phosphatidylcholine species in our KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of exosomes isolated from KPC; Smpd3 wt/wt PPT and KPC; Smpd3 f/f PPT cell lines. A) CD63eGFP particles per cell are graphed for KPC; Smpd3 wt/wt CD63eGFP cell lines (n=6) and KPC; Smpd3 f/f CD63eGFP cell lines (n=6). B) Fractions were subjected to WB for sEV markers to determine the fractions that exosomes are contained within using the C-DGUC exosome isolation protocol. Expected band sizes are listed for each marker. C) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt uninjected mice (n=30), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=15), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=15), KPC; Smpd3 f/f uninjected mice (n=24), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=9), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . D) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt uninjected mice (n=29), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=14), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=12), KPC; Smpd3 f/f uninjected mice (n=22), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=8), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . E) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 wt/wt are 0.04396 ± 0.006112 (n=28), KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.05477 ± 0.007678 (n=15), and KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 f/f are 0.06809 ± 0.009409 (n=16). F) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 f/f are 0.05085 ± 0.005912, KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.06149 ± 0.01434, and KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 f/f are 0.04654 ± 0.008279. G) Pancreas images at dissection for the indicated groups are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Isolation, Marker, Injection, Dissection

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: PDA cell exosomes generated through nSMase2 accelerate PDA progression. A) Schematic of exosome injection study. B) Probability of survival for the indicated groups is graphed. Log-rank test was performed between the indicated groups. C-D) Volcano plot (C) and heat map (D) depict differentially expressed exosomal miRNAs isolated from KPC; Smpd3 f/f and KPC; Smpd3 wt/wt polyclonal PDA PPT cell lines. E) Exosomal protein abundance results for KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines are depicted. The x-axis shows the log2 fold change for KPC; Smpd3 f/f over KPC; Smpd3 wt/wt and the y-axis shows −log10(q-value). Significantly upregulated proteins (q-value < 0.05) are shown in green and significantly downregulated proteins are shown in red. F) Heatmap of top 40 differentially abundant exosomal proteins comparing KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt , displaying log2 normalized expression. Red indicates lower abundance of a given protein in each sample and green indicates higher abundance. Rows and columns are arranged based on hierarchical clustering dendrograms (not displayed). The color bar at the top indicates the genotype. G) IPA results depict significantly differentially regulated pathways when comparing exosomal proteins from KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Injection, Isolation, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: sEVs generated independently of nSMase2 promote a proinflammatory macrophage phenotype. A) Heat map depicts scaled marker expression in KPC; Smpd3 wt/wt and KPC Smpd3 f/f pancreata using our 17-marker myeloid spectral flow cytometry panel. B) t-SNE of 200,683 cells stained with our spectral flow panel depict 12 clusters identified for KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata through FlowSOM clustering. C) Heat map of scaled marker expression and number of cells for clusters shown in B is displayed. D) Bar graphs depict percent cellular subpopulations from KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata for the indicated markers after gating in FlowJo. E) Immunofluorescence images depict co-labeling with F4/80, iNOS, and Dapi at 10-11 weeks of age. Scale bar is 20uM. F) Quantification of intralobular F4/80 + and iNOS + copositive cells in KPC; Smpd3 wt/wt (n=5 animals), KPC; Smpd3 f/wt (n=6 animals), and KPC; Smpd3 f/f (n=5 animals) pancreata at 10-11 weeks of age is depicted. G) Schematic of experiment to assess how nSMase2-mediated exosome biogenesis affects polarization of macrophages. H-J) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Marker, Expressing, Flow Cytometry, Staining, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Functional effects of PDA cell sEVs on macrophages. A) Immunofluorescence images depict co-labeling with F4/80, Ly6G, and Dapi at 10-11 weeks of age. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=6 animals. Scale bar is 20uM. B) Quantification of interlobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. C) Quantification of intralobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. D-F) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Functional Assay, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: SMPD3 is an independent prognostic factor for pancreatic cancer patient survival. A) Log2 of the normalized counts are plotted for normal human pancreatic SMPD3 expression (n=39 samples) and primary PDA SMPD3 expression (n=39 samples). B) Log2 of the normalized counts are plotted for PPT SMPD3 expression (n=231 samples) and MPT SMPD3 expression (n=158 samples) in PDA patients. C) Kaplan-Meier survival curve of the whole cohort (N = 94) with the patients dichotomized into SMPD3 high and low expression based on the median mRNA expression value. SMPD3 high group had significantly better survival (median survival of 26.5 vs 15.0 months; P = 0.0364). A (0) value indicates a censored event and a (1) value indicates a death event. D) Kaplan-Meier survival curve of the subset of the whole cohort of patients who underwent adjuvant chemotherapy (> 95% single agent gemcitabine) after surgical resection (N = 58). SMPD3 expression was significantly associated with better survival (median survival of 34.3 vs 15.9 months; P = 0.0289) indicating SMPD3 is a biomarker of adjuvant gemcitabine response. E) Kaplan-Meier survival curve of the subset of the whole cohort of patients who did not undergo adjuvant chemotherapy after surgical resection (N = 36). SMPD3 expression was not associated with survival (P = 0.8001). F-G) Kaplan-Meier curves depict patient survival based on SMPD3 expression in locally advanced primaries or metastatic PDA from the COMPASS trial for patients who received chemotherapy with modified Folfirinox or Gemcitabine plus Abraxane. SMPD3 expression was stratified into high and low with low expression defined as the maximal chi-squared statistic. H) Log2 of SMPD3 expression by hypoxia status defined by . I) Log2 of SMPD3 expression in defined Waddell structural variation subtypes in COMPASS cases is depicted. J) Kaplan-Meier curves depict patient survival based on cytoplasmic and membranous nSMase2 expression in treatment naïve primary resected PDA. Tumor, and not stromal, cell nSMase2 expression was scored in 143 patients. When comparing survival of PDA patients with low cytoplasmic nSMase2 expression (n=89, mean survival=30.1 months) and high cytoplasmic nSMase2 expression (n=54, mean survival=29.1 months), p=0.627 using the log-rank test. When comparing survival of PDA patients with low membrane nSMase2 expression (n=65, mean survival=25.6 months) and high membrane nSMase2 expression (n=78, mean survival=32.9 months), p=0.052 using the log-rank test.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Biomarker Discovery, Modification, Membrane

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Pathways analysis of pancreatic nSMase2 expression in PDA patients based on treatment with chemotherapy in addition to the effect of nSMase2 expression on chemosensitivity and vasculature integrity. A) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs receiving adjuvant chemotherapy. The threshold line represents statistical significance. B) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs not receiving adjuvant chemotherapy. C) Representative images used to score the TMA for nSMase2 low and nSMase2 high membrane and cytoplasmic labeling. D) Images depict dextran and tomato lectin, a marker of blood vessels in mice, immunofluorescence at 10-11 weeks of age in KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreases. E) Percent pancreatic neoplastic area per field occupied by dextran is graphed for KPC; Smpd3 wt/wt (n=8) and KPC; Smpd3 f/f (n=6) mice. F) Negative log of gemcitabine IC50 for KPC; Smpd3 wt/wt , KPC; Smpd3 f/f , KPC Mock OE, and KPC Smpd3 OE cell lines is graphed. G) Quantification of total HIF1α in nSMase2 high and nSMase2 low primary pancreatic tumors of PDA patients is depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Membrane, Labeling, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 regulates PDA vasculature development. A-B) Heat maps showing VEGF Signaling and HIF1α Signaling pathways in all three PPT cell lines. C) Log of transcript per million (TPM) values for Vegfa gene are graphed for sequenced PPT cell lines. Wilcoxon test was performed. D) Pancreatic CD31 and CK19 immunolabeling at 19-21 weeks of age in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is depicted. Scale bar is 20uM. E) Quantification of pancreatic CD31 immunofluorescence in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is shown. F) Pancreatic CD31 and CK19 immunolabeling at 21 days post orthotopic injection of KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines into Rag1 KO mice 21 days post injection is depicted. Scale bar is 20uM. G) Quantification of pancreatic CD31 immunofluorescence in Rag1 KO mice injected with KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines 21 days post injection is depicted. H) Immunohistochemistry of CD31 in primary tumors from PDA patients is depicted with low and high nSMase2 expression. I) Percent CD31 positive area in primary nSMase2 low (n=19) and nSMase2 high (n=34) pancreatic tumors from PDA patients is shown. J) Immunohistochemistry of HIF1α is depicted in primary tumors from PDA patients with low and high nSMase2 expression. K) Percent nuclear HIF1α epithelial tumor cells in primary nSMase2 low (n=5) and nSMase2 high (n=6) PDA patient pancreatic tumors is shown. L) Ratio of pancreas weight to body weight for gemcitabine and vehicle treated Rag1 KO mice 21 days after pancreatic orthotopic injection of the depicted cell lines is shown. For KPC scrambled shRNA vehicle treated mice, n=4, for KPC scrambled shRNA gemcitabine treated mice, n=9, for KPC Smpd3 shRNA 1 vehicle treated mice, n=10, for KPC Smpd3 shRNA 1 gemcitabine treated mice, n=10, for KPC Smpd3 shRNA 2 vehicle treated mice, n=10, and for KPC Smpd3 shRNA 2 gemcitabine treated mice, n=10. M) Kaplan-Meier survival curves show probability of survival for vehicle-injected KPC; Smpd3 wt/wt mice (n=12, median survival=20.29 weeks), gemcitabine-injected KPC; Smpd3 wt/wt mice (n=19, median survival=23.43 weeks), vehicle-injected KPC; Smpd3 f/f mice (n=15, median survival=23.29 weeks), gemcitabine-injected KPC; Smpd3 f/f mice (n=16, median survival=19.86 weeks). A black line indicates a censored animal.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Protein-Protein interactions, Immunolabeling, Immunofluorescence, Injection, shRNA, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) X-gal staining to detect Rcor2 expression patterns. Whole-embryo staining at E11.5 stage shows Rcor2 is mainly expressed in the CNS. Scale bar, 1 mm. ( b ) Western blot analysis of Rcor2 expression levels during brain development. The decreased expression of Rcor2 with embryonic development is noteworthy. β-Actin is used as an endogenous control. ( c ) In situ hybridization to detect endogenous Rcor2 mRNA expression patterns in cortical development at E11.5, E13.5, E15.5 and E17.5. Insets show high-magnification image of Rcor2 expression in the neocortex at E13.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 100 μm. ( d ) Confocal images of immunofluorescence to detect cellular localization of Rcor2 in the neocortex at E13.5. Rcor2 localized mainly in the nucleus at interphase and metaphase, and localized between separated chromosomes in anaphase of dividing cells in VZ. Dotted lines circle the shape of nuclei. Scale bar, 5 μm. ( e ) Western blot analysis of Rcor2 expression level in Rcor2 fl/fl and Rcor2 cko brains at E13.5 and E15.5, respectively. Rcor2 expression was depleted in Rcor2 cko brains. β-Actin is used as an endogenous control. ( f ) Representative images of Rcor2 fl/fl and Rcor2 cko brain size at different stages of development. Rcor2 cko mice show severe brain growth retardation at E13.5 and E15.5. Scale bar, 1 mm. ( g ) Representative images of Rcor2 fl/fl and Rcor2 cko cortex at E15.5 by Nissl staining. Structural abnormalities of lamination with reduced cortical thickness are observed in Rcor2 cko cortex. Scale bar, 200 μm. ( h ) RT–qPCR analysis of knockdown efficiencies of the two shRNAs targeting Rcor2. Transcripts were normalized to the control group. Data are shown as mean±s.e.m., t -test, **** P <0.0001, n =3. ( i ) Confocal images of E16.5 cortical sections electroporated with shControl (red), shRcor2-a (red) and shRcor2-b (red) plasmids at E13.5. Knockdown of Rcor2 results in impaired cortical development. IZ, intermediate zone. Scale bar, 20 μm. ( j ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after electroporation as shown in i . proportion of RFP + cells in different zones ( y axis). Data are shown as mean±s.e.m., t -test, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, n =3 individual experiments.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Staining, Expressing, Western Blot, Control, In Situ Hybridization, Immunofluorescence, Quantitative RT-PCR, Knockdown, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Immunostaining images of Sox2 at E13.5. Sox2 is dramatically reduced on Rcor2 depletion. VZ, ventricular zone; SVZ, subventricular zone. Scale bar, 20 μm. ( b ) Quantification of Sox2 + cell ratios in VZ/SVZ regions shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Confocal images of Tbr2 expression at E13.5. Tbr2 is dramatically reduced on Rcor2 depletion. Scale bar, 20 μm. ( d ) Quantification of Tbr2 + cell ratios in VZ/SVZ regions shown in c , respectively. Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Immunostaining images of Nestin, Sox2 and Tbr2 in cultured Rcor2 fl/fl and Rcor2 cko NPCs, all of which exhibit significantly reduced expression in the Rcor2 cko NPCs. Scale bar, 20 μm. ( f ) Confocal images of immunofluorescence for Ki67 and PHH3 in Rcor2 fl/fl and Rcor2 cko cortex at E13.5 and E15.5. Ki67 signals (red), but not PHH3 signals (green), are dramatically reduced in Rcor2 cko developing brains. Scale bar, 20 μm. ( g ) Quantification of Ki67 + cells in the VZ/SVZ regions of the developing neocortex as shown in f . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 separate stainings from three independent brains. ( h ) Confocal images of BrdU (green) and Ki67 (red) staining in Rcor2 fl/fl and Rcor2 cko cortex 24 and 48 h after BrdU incorporation. Scale bar, 100 μm. ( i ) Quantification of the cell cycle exit by percentage of BrdU + and Ki67 + NPCs divided by BrdU + cells shown in h . Data are shown as mean±s.e.m., t -test, *** P <0.001 and **** P <0.0001, n =3 individual experiments. ( j , k ) Representative images ( j ) and quantification of ( k ) of Rcor2 fl/fl and Rcor2 cko neurosphere sizes. The neurospheres'radius of Rcor2 cko are much smaller than those of Rcor2 fl/fl , t -test, **** P <0.0001, n =12. Scale bar, 50 μm. ( l ) Representative time-lapse imaging of the RGC dividing process in the sections of the cerebral cortex electroporated with RFP-shControl (upper panels) and RFP-shRcor2 (lower panels). The radial glial dividing pattern is abnormal on Rcor2 knockdown, resulting in cell death. Arrows, mother RGCs. Arrowheads, two daughter cells. Scale bar, 50 μm. ( m ) Representative time-lapse images of Rcor2 fl/fl cortex sections electroporated with EGFP-Control (upper panels) and EGFP-Cre (lower panels). Loss of cells is observed with Rcor2 knockout by Cre recombinase electroporation. Scale bar, 50 μm.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Immunostaining, Expressing, Cell Culture, Immunofluorescence, Staining, BrdU Incorporation Assay, Imaging, Knockdown, Control, Knock-Out, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Confocal images of Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5, which exhibit significant reduction on Rcor2 knockout. Scale bar, 50 μm. ( b ) Quantification of Satb2 + and Tbr1 + cells in Rcor2 fl/fl and Rcor2 cko cortex at E15.5 in a indicates Satb2 and Tbr1 expressions are decreased on Rcor2 depletion during development. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Western blot to analyse Dcx, Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control. ( d ) Representative images of Map2 and Tuj1 immunostaining in cultured neurons directly dissociated from Rcor2 fl/fl and Rcor2 cko brain cortex at E15.5. Decreased expression of both markers and reduced neurofilaments can be observed in Rcor2 cko cultured neurons. Scale bar, 20 μm. ( e ) Confocal images of in-vitro cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation using neuronal marker Map2 and Tuj1 antibodies, both of which are significantly reduced in the differentiated Rcor2 cko NPCs. Scale bar, 20 μm. ( f , g ) RT–qPCR analysis of neuronal markers expression in both Rcor2 fl/fl and Rcor2 cko neocortex at E15.5 stage ( f ) and in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation ( g ). Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-Out, Western Blot, Control, Immunostaining, Cell Culture, Expressing, In Vitro, Marker, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Schematic overview of strategy to generate an Rcor2 Flag knock-in allele by CRISPR/Cas9. The sgRNA sequence site is shown as a green arrowhead. The start codon of Rcor2 is indicated and capitalized. The oligo donor contained 50 bp homologies on both sides flanking the DSB, in which 3 × Flag sequences are labelled as a red box. ( b ) Western blot analysis to validate FLAG, RCOR2 and LSD1 expressions in Rcor2 Flag knock-in neocortex using Flag-M2 antibody. β-Actin was used as an endogenous control. ( c ) Pie chart depicts distribution of Rcor2 occupancies in genome-wide scale in FLAG ChIP-seq results using Rcor2 Flag knock-in neocortex at E13.5. ( d ) WebLogos of consensus binding motifs of Rcor2 generated by Multiple EM for Motif Elicitation (MEME) motif analysis tool. ( e ) GO analysis for Rcor2-binding regions in genome-wide scale revealed by Flag ChIP-seq results using Rcor2 Flag brain. ( f ) GO analysis for LSD1 occupancy in genome-wide scale revealed by LSD1 ChIP-seq results using Rcor2 Flag brain. ( g ) Density plots analysis of H3K4me1 signal change in promoter regions (−2- to ∼0.5 kb from TSS) on Rcor2 depletion. Compared with all genes, the change of H3K4me1 signal is significantly ( P <0.0005, Kolmogorov–Smirnov test) enhanced in the promoter regions of Shh pathway-related genes on Rcor2 depletion. H3K4me1 signal change on Rcor2 depletion ( x axis); H3K4me1 signal density ( y axis). ( h ) Gene tracks of Rcor2, LSD1 and H3K4me1 enrichments by ChIP-seq analysis at core promoter regions of Dlx2 and upstream regulatory regions of Shh, which are closely related to Shh signalling. ( i ) ChIP–qPCR analysis of Rcor2 Flag and Rcor2 cko cortex at E13.5 using specific FLAG-M2 antibody. Significant enrichments of the Rcor2 at the regulatory regions of Dlx2 and Shh gene locus detected in g in the Rcor2 flag samples are worth noting. Fold enrichments of Rcor2 occupancy compared with input ( y axis). Data are shown as mean±s.d., t -test, *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Control, Genome Wide, ChIP-sequencing, Binding Assay, Generated, ChIP-qPCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a , b ) Scatter plot analysis of genome-wide expression profiles of Rcor2 cko versus Rcor2 fl/fl samples at E13.5 ( a ) and E15.5 ( b ). Dots above or below the dash line indicate upregulated or downregulated genes on Rcor2 depletion, respectively. Red dots or green dots highlight the significantly differentially expressed genes on Rcor2 depletion. Raw counts ( x axis); gene expression fold changes on Rcor2 depletion ( y axis). ( c ) Venn diagrams of upregulated genes (left) and downregulated genes (right) in Rcor2 cko samples compared with Rcor2 fl/fl samples. ( d ) The profiles of Rcor2 and H3k4me1 enrichments analysed in ChIP-seq results shown in in regulatory regions of genome-wide scale (red) and of the upregulated genes (purple) according to RNA-seq results. ( e ) Correlation network of overlapped upregulated genes in both E13.5 and E15.5 samples. Lines indicate the correlations between two connected genes with R >0.55. Genes were analysed by GO analysis and divided into different categories.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Genome Wide, Expressing, Gene Expression, ChIP-sequencing, RNA Sequencing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) qPCR analysis of the expression of genes related to the Shh signalling pathway in the cortex of Rcor2 fl/fl and Rcor2 cko brains during development. Significant upregulation of these genes on Rcor2 depletion is noteworthy. Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3. ( b ) Confocal images of Shh and Ptch1 expressions in Rcor2 fl/fl and Rcor2 cko cortex. Enhanced Shh and Ptch1 signals are observed in Rcor2 cko compared with Rcor2 fl/fl neocortex at E13.5 and E15.5. Insets show high-magnification images of the outlined regions. Scale bars, 50 μm. ( c ) Dlx2 expression in Rcor2 fl/fl and Rcor2 cko cortex detected by immunofluorescence analysis at E15.5. Dlx2 + cells were observed in the neocortex on Rcor2 depletion. Scale bars, 50 μm. ( d ) Confocal images of Shh, Ptch1 and Dlx2 expressions in in-vitro -cultured Rcor2 cko NPCs. Scale bar, 20 μm. ( e ) Western blot analysis of expression levels of Dlx2, Shh and Ptch1 in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Expressing, Immunofluorescence, In Vitro, Cell Culture, Western Blot, Control

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Knockdown of Rcor2 impairs cortical neurogenesis, which can be partially rescued by knockdown of Shh during cortical development. In-utero electroporation with RFP-shControl (red)/GFP-shControl (green), RFP-shRcor2 (red)/GFP-shControl (green), RFP-shControl (red)/GFP-shShh (green) or RFP-shRcor2 (red)/GFP-shShh (green) plasmids was performed at E13.5. Cerebral sections were fixed and imaged at E16.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 50 μm. ( b ) Quantification of the percentage of RFP + /GFP + cells in different regions of the developing cortex after electroporation shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01 and *** P <0.001, n =3 individual experiments. ( c ) Inhibition of Shh by Cyclopamine can partially rescue neurogenesis defects caused by Rcor2 downregulation during cortical development. Rcor2 was knocked down at the lateral ventricle in the brain by in-utero electroporation with RFP-shRcor2 plasmids at E13.5. Cerebral sections were collected at E14.5 and then treated with cyclopamine to inhibit Shh activity for 48 h. Scale bar, 50 μm. ( d ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after knockdown of Rcor2 or inhibition of Shh shown in c . Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Representative images depicting neurosphere size is partially rescued in the in-vitro -cultured Rcor2 cko NPCs after treatment with Cyclopamine. Scale bar, 50 μm. ( f ) Histogram depicting cell numbers of in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment for 48 h. Cells (5 × 10 5 ) are seeded initially. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3. ( g ) Confocal images of Tuj1 expression in the differentiated cells from in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment. Tuj1 expressions are partially restored in Cyclopamine-treated Rcor2 cko cells. Scale bar, 20 μm. ( h ) Model of Rcor2 function in neurogenesis in the developing neocortex. Rcor2 safeguards cortical neurogenesis by recruiting LSD1 complex to the regulatory regions of Dlx2 and Shh genes, to inhibit the Shh pathway activation during development. The absence of Rcor2 leads to inhibition release of these genes and thus ectopic activation of Shh signalling in the developing neocortex, resulting in cortical neurogenesis defects.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knockdown, In Utero, Electroporation, Inhibition, Activity Assay, In Vitro, Cell Culture, Expressing, Activation Assay