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: STAR Protocols

Article Title: Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

doi: 10.1016/j.xpro.2022.101321

Figure Lengend Snippet:

Article Snippet: DNA Clean & Concentrator-25 (Capped) , Zymo Research , Cat# D4033.

Techniques: Recombinant, Electroporation, Modification, SYBR Green Assay, Transfection, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, DNA Extraction, Software, Real-time Polymerase Chain Reaction, Protein Purification, Plasmid Preparation, Synthesized, Flow Cytometry, Spectrophotometry

Journal: Molecular cell

Article Title: Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF

doi: 10.1016/j.molcel.2019.07.039

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA Polymerase I, Large (Klenow) Fragment , New England Biolabs , Cat. # M0210.

Techniques: In Vitro, RNA Binding Assay, Recombinant, Staining, Flow Cytometry, Sample Prep, Expressing, Imaging, Knock-In, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

doi: 10.1038/ncomms11235

Figure Lengend Snippet: ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.

Article Snippet: Accuprime DNA Polymerase was obtained from Invitrogen (Grand Island, NY).

Techniques: Activity Assay, Fluorescence, Negative Control, Positive Control, Emulsion, Functional Assay, Flow Cytometry

Journal: Nature Communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

doi: 10.1038/ncomms11235

Figure Lengend Snippet: ( a ) Overview of the microfluidic polymerase enrichment strategy. A pool of polymerase genes containing functional (green) and non-functional (blue) members are expressed in E. coli and encapsulated in w/o droplets generated in a microfluidics device. Polymerases are liberated from their bacteria by heat lysis and incubated at 55 °C to allow for primer extension. Using a second microfluidics device, droplets are emulsified into a bulk aqueous phase to generate water-in-oil-in-water compartments (w/o/w). Fluorescent w/o/ws are FACS sorted and the vectors encoding functional polymerases are recovered. ( b ) Vector design. The 9n-GLK vector was engineered to contain a unique NotI restriction site. Control digestion showing that NotI only cuts PCR-amplified DNA from the 9n-GLK vector. ( c ) Following a complete cycle of selection and amplification (see ) PCR-amplified DNA was digested with NotI to measure the enrichment of 9n-GLK from libraries that were doped at levels of 1:100, 1:1,000 and 1:10,000 (9n-GLK to 9n). NotI digestion of the PCR-amplified DNA reveals an enrichment of ∼1,200-fold per round of microfluidics selection.

Article Snippet: Accuprime DNA Polymerase was obtained from Invitrogen (Grand Island, NY).

Techniques: Functional Assay, Generated, Bacteria, Lysis, Incubation, Plasmid Preparation, Control, Amplification, Selection

Journal: Nature Communications

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

doi: 10.1038/ncomms11235

Figure Lengend Snippet: ( a ) Constitutional structure for the linearized backbone of threose nucleic acid (TNA). ( b ) Positions 409, 485 and 664 mapped onto the structure of 9n DNA polymerase (PDB: 4K8X). Polymerases isolated after one round of selection were analysed for TNA synthesis activity in the absence of Mn 2+ . Activity is defined as the amount of full-length product generated in 18 h. Basal activity of wild-type 9n polymerase (dashed grey line). ( c ) Time course of TNA synthesis for 9n-YRI and 9n-NVA polymerases compared with wild-type 9n. ( d ) Fidelity analysis of 9n-YRI polymerase in the presence and absence of manganese ions yields a mutational profile of 8 errors per 100 bases and 2 errors per 1,000 bases, respectively.

Article Snippet: Accuprime DNA Polymerase was obtained from Invitrogen (Grand Island, NY).

Techniques: Isolation, Selection, Activity Assay, Generated

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: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 1. RECQ1 and RECQ4 helicases are recruited to human origins of DNA replication. (A and B) Genomic regions containing the lamin B2 origin (A) and two GM-CSF replication origins (B) are shown together with the locations of sets of primers (converging arrow pairs) used for quantitative real-time PCR analysis. (C) Quantification of cross-linked lamin B2 origin DNA immunoprecipitated by ChIP from T98G and IMR-90 cells, using the antibodies indicated at the bottom. The inset shows the results of Western analysis after immunoprecipitation of the cross-linked material with specific antibodies against the five human RecQ proteins. ORC2-specific immunoprecipitation served as a positive control, and rabbit IgG immunoprecipitation served as a negative control. Whole T98G cell lysates (WCL) were used to confirm the endogenous expression of the five RecQ helicases. (D) Quantification of cross-linked GM-CSF1 and GM-CSF2 origin DNAs immunoprecipitated by ChIP from T98G cells, using the antibodies indicated at the bottom. Fold enrichments of origin sequences were determined versus nonorigin control sequences, and the dashed line indicates the threshold enrichment level obtained by using a negative-control antibody (normal rabbit IgG). Results are reported as means standard errors of the means (SEM) (indicated by error bars) for at least three independent experiments.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Positive Control, Negative Control, Expressing, Control

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 2. Comparison of association of RECQ1 and RECQ4 heli- cases with the lamin B2 origin in untreated versus HU-treated cells. T98G cells were treated with 2 mM HU for 24 h. The bar graph shows the quantification of cross-linked lamin B2 origin DNA immunopre- cipitated from T98G glioblastoma cells, using antibodies specific to the proteins shown across the bottom. Fold enrichments of origin se- quences were determined versus nonorigin control sequences, and the dashed line indicates the threshold enrichment level obtained by using a negative-control antibody (normal rabbit IgG). Results are reported as means SEM for at least three independent experiments.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Comparison, Control, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 3. Cell cycle-dependent association of RECQ1 and RECQ4 with the human lamin B2 replication origin. (A) Western blot analysis of whole-cell extracts of asynchronous T98G cells (AS) or cells from different times after synchronization and release. (B) Cell cycle phase timing was determined by flow cytometry profiling of synchronized T98G cells that had been cultured without serum for 72 h and then sampled over a 28-h time course after the addition of serum. PI, propidium iodide. (C) Quantification of cross-linked lamin B2 origin DNA immunoprecipitated by ChIP from synchronized cells. The key on top indicates the antibodies used for ChIP analyses. Fold enrichments of lamin B2 origin region (B48) DNA over control B13 region DNA are reported for each antibody, where the dashed line indicates the threshold enrichment obtained using a negative-control normal rabbit IgG antibody. Histogram bars report the mean SEM for at least three independent experiments for each antibody and cell cycle fraction. (D) Cell cycle-dependent subcellular distribution of RECQ1 and RECQ4. Whole-cell lysates were fractionated to generate cytosolic (S2), soluble nuclear (S3), and chromatin-enriched (P3) fractions, in which protein levels were assessed by Western blotting. Antibodies against p84 and -tubulin were used as controls for nuclear and cytoplasmic localization, respectively.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Western Blot, Cytometry, Cell Culture, Immunoprecipitation, Control, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 4. G1- and S-phase-specific loading of RECQ4 and RECQ1 on the lamin B2 origin. Paired columns show flow cytometry profiles of synchronized T98G cells at different times after release from serum starvation, together with bar graphs of cross-linked lamin B2 origin DNA immunoprecipitated by ChIP with antibodies to each of the proteins shown across the bottom of the bar graph at that time point. Histograms report the mean SEM for at least three independent experiments at each time point, where fold enrichment of lamin B2 origin region (B48) DNA was determined as described in the legend to Fig. 3.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Cytometry, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 5. Association of RECQ1 and RECQ4 with beta-globin replica- tion origin as a function of origin timing. (A) Genomic region containing the beta-globin replication origin, together with the locations of sets of primers (converging arrow pairs) used for quantitative real-time PCR analysis. (B) Flow cytometry profiling of synchronized K562 cells that had been cultured with mimosine for 24 h and then sampled 3 (early S) and 9 (late S) h after the removal of the drug is shown at the top. The histograms below quantify cross-linked lamin B2 and beta-globin origin DNAs im- munoprecipitated by ChIP from early- and late-S-phase-synchronized K562 cells. (C) Flow cytometry profiling of synchronized HeLa cells that had been cultured with mimosine for 24 h and then sampled 3 (early S) and 9 (late S) h after the removal of the drug is shown at the top. The histograms below quantify cross-linked lamin B2 and beta-globin origin DNAs immunoprecipitated by ChIP from early- and late-S-phase-syn- chronized HeLa cells. Histograms report the means SEM for at least three independent experiments, where fold enrichment of lamin B2 and beta-globin origin region DNAs was determined as described in the leg- ends to Fig. 1 and 2.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Real-time Polymerase Chain Reaction, Flow Cytometry, Cell Culture, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 6. Downregulation of RECQ1 and RECQ4 inhibits cellular proliferation and DNA synthesis. (A) Colony-forming efficiency of T98G cells after siRNA-mediated transfection. Plate photos show representative colony formation after plating of cells transfected with siRNA pools directed against the gene named across the top. The cell number plated is indicated by the code along the left, as follows: I, 200 cells/well; II, 400 cells/well; and III, 800 cells/well. Colonies were stained and counted after 7 days of growth to determine colony-forming efficiencies (means SEM) for three independent experiments. (B) Western blot analysis of RNAi-mediated depletion of RECQ1 or RECQ4 from T98G cells transfected with an siRNA pool against RECQ1, RECQ4, or luciferase (Luc) at 72 h posttransfection. -Tubulin was used as a blot control. (C) Flow cytometry profiles of DNA content (x axis; propidium iodide [PI] staining) versus BrdU incorporation (y axis; anti-BrdU immunostaining) 72 h after siRNA transfection. Boxes are labeled to indicate cell cycle phases, where the mean percent S-phase cells is shown in parentheses across the top of each histogram. The bar graph at bottom reports the percentages of G0/G1, S-phase/BrdU-positive, and G2/M cells in cultures that had been transfected with a RECQ1, RECQ4, or luciferase (control) siRNA pool (indicated in the key). Results shown are the means SEM for three independent experiments.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: DNA Synthesis, Transfection, Staining, Western Blot, Luciferase, Control, Flow Cytometry, BrdU Incorporation Assay, Immunostaining, Labeling

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 7. RECQ1 and RECQ4 depletion reduces newly synthesized nascent DNA from early-firing human replication origins. Fold enrichment of nascent DNA from lamin B2 (A) and two GM-CSF (B) origin regions versus that from adjacent nonorigin regions in neutral sucrose gradient fractions was determined for T98G cells depleted of RECQ1 (middle row) or RECQ4 (bottom row) by RNAi transfection and compared to that for controls (luciferase RNAi-transfected cells). Graphs report the means SEM for at least three independent experiments.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Synthesized, Transfection, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 8. RECQ1 and RECQ4 depletion impairs replication factor loading onto chromatin at the start of S phase. (A) Experimental outline for synchronization and release of human T98G cells to generate highly enriched S-phase cell populations. Asynchronous cells were grown for 72 h without serum and then refed, RNAi transfected, and harvested after 24 h for blot analysis. (B) Western blot analysis of whole and fractionated cell extracts from synchronized T98G cells. Left and right panels show the subcellular distribution of RECQ1 (left column, top), RECQ4 (right column, top), and four additional replication proteins, together with an -tubulin control (both columns), in whole-cell lysates (WCL) and in cytosolic (S2), soluble nuclear (S3), and chromatin-enriched (P3) fractions prepared from RECQ1-, RECQ4-, or control (luciferase)-depleted cells.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Transfection, Western Blot, Control, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 9. Depletion of human RECQ1 or RECQ4 affects DNA replication dynamics. (A) An outline of the experimental protocol is shown at the top, in which asynchronous cells were labeled consecutively with IdU (green) and then with CldU (red) for 40 min each prior to isolating and stretching DNA for immunostaining as described in Materials and Methods. Shown below this are representative images of replication tracks in control (luciferase)-, RECQ1-, and RECQ4-depleted cells. All track photos are shown at identical magnifications, where the white scale bar (lower right) is 10 m long, representing approximately 39 kb of DNA. (B) RECQ1 or RECQ4 depletion reduces the probability of origin firing in stretched DNA samples. Origin firing events among all tracks labeled during the protocol for panel A were identified as CldU-only (red only) or CldU-IdU-CldU (red-green-red) triple-segment tracks (see diagram at top). The mean percentages of new origin firing events defined by these two track types among all labeled tracks are shown for three independent experiments in which 200 to 450 tracks/experiment were typed for control-, RECQ1-, or RECQ4-depleted cells. Error bars show standard deviations. The P values, calculated using Student’s t test, are 0.074 between luciferase and RECQ1 and 0.021 between luciferase and RECQ4. (C) RECQ1 depletion, but not RECQ4 depletion, slows ongoing replication forks. The bar graph summarizes mean lengths of first-label IdU (green) segments labeled for 40 min in two-segment (green-red) tracks to ensure that fork rate measurements were made from active replication forks (see the diagram in panel A). Track lengths were measured in m, using AxioVision software (Carl Zeiss), for three independent experiments in which 150 to 370 first-segment track lengths were measured for each sample. Error bars show 95% confidence intervals for sample means. The statistical significance of differences in mean track lengths was determined by a two-sample Kolmogorov-Smirnov test. A representative P value is shown for a control (luciferase)- versus RECQ1-depleted sample pair.

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: Labeling, Immunostaining, Control, Luciferase, Software

Journal: Molecular and Cellular Biology

Article Title: Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

doi: 10.1128/mcb.01290-09

Figure Lengend Snippet: FIG. 10. Model of cell cycle-dependent loading of RECQ1 and RECQ4 proteins onto DNA replication origins. RECQ4 is recruited to origins in late G1 as part of pre-RC assembly. At the G1/S transition, CDC6 release signals preinitiation complex (pre-IC) formation. RECQ1, as well as additional RECQ4, is recruited in early S phase, after the release of ORC1. Both RECQ1 and RECQ4 are no longer detected on the lamin B2 origin by mid-S phase, when either or both may be associated with active replisomes. This cell cycle phase-dependent loading and the subsequent loss of RECQ1 and RECQ4 for origins of replication suggest specific roles for each protein in replication initiation and, potentially, other specific aspects of DNA replication, such as fork progression (see text for additional discussion).

Article Snippet: Polyclonal antibodies against RECQ1 (BL2074) and WRN (NB 100-471) were purchased from Bethyl Laboratories and Novus Biologicals, respectively.

Techniques: