Journal: Nature Communications

Article Title: Pancreatic tumor eradication via selective Pin1 inhibition in cancer-associated fibroblasts and T lymphocytes engagement

doi: 10.1038/s41467-022-31928-7

Figure Lengend Snippet: a Establishment and validation of CAFs via high expressions of both FAP-α and α-SMA. b Cellular uptake of Cy5-labeled formulations on Pan02 cells, NIH-3T3 cells, and CAFs, measured by flow cytometry ( n = 3 biologically independent experiments; error bars, mean ± SD). c Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, imaged by fluorescent microscopy. Scale bars are 20 μm. Three independent repetitions got similar results. d Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, quantified by flow cytometry ( n = 6 biologically independent samples; error bars, mean ± SD). Cytotoxicity on Pan02 cells ( e ) and CAFs ( f ) ( n = 3 biologically independent experiments; error bars, mean ± SD). g Western blotting detection of Pin1 and relative proteins from Pan02 cells after treating with PBS, AG17724 (0.5 μM), or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. h Quantitative analysis of the western blotting results from Pan02 cells (Three independent repetitions got similar results.). i Western blotting detection of Pin1 and relative proteins from CAFs treated with PBS, AG17724, or antiCAFs-DMS after treating with PBS, AG17724 (0.5 μM) or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. j Quantitative analysis of the western blotting results from CAFs (Three independent repetitions got similar results.). One-way analysis of variance followed by Turkey posttests. Source data are provided as a Source Data file.

Article Snippet: Anti-FAP-α antibody (MAB9727-100, 0.25 μg/mL as working concentration.) and anti-phospho-CDC2/CDK1 (Y15) antibody (AF888-SP, 0.2 μg/mL as working concentration.) were purchased from R&D SYSTEMS.

Techniques: Biomarker Discovery, Labeling, Flow Cytometry, Microscopy, Western Blot

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 methods

Article Title: Pooled CRISPR screens with imaging on microRaft arrays reveals stress granule-regulatory factors

doi: 10.1038/s41592-020-0826-8

Figure Lengend Snippet: a, Diagram of microRaft isolation and sgRNA sequencing design. MicroRafts are removed from the array using a motorized microneedle fitted over a microscope objective. Dislodged rafts collected with a magnetized wand are placed into individual tubes for DNA extraction and a barcoded targeted-PCR of the sgRNA insert. b, Top, images of individual, stress induced microRafts with 5 different sgRNAs targeting EIF2AK1. Scale bar = 20 μm. Bottom left, representative image of G3BP1 antibody staining in sodium arsenite stressed HEK293T after EIF2AK1 depletion by siRNA or non-targeting siRNA control (NTC). Data are mean ± s.d. across n = 3 wells / condition (4 images / well). Scale bar = 20 μm. Bottom right, Western blot validation of siRNA-mediated depletion of EIF2AK1 in HEK293T. c, siRNA depletion of target RBPs in bulk cells. Stress granule / nuclei area was normalized to the non-targeting control (NTC) for each experiment. RBPs are ordered on the x-axis by the lowest normalized stress granule area. RBPs shown here had significant reduction (P < 0.05, unpaired two-tailed t test, d.f. = 4, 95% confidence interval) of stress granule area relative to NTC in at least 2 of the 3 biological replicates. *Genes previously reported to modify stress granule abundance. Data are mean ± s.d. across n = 3 wells / condition (4 images / well). d, Validation of SNRNP200, ZCCHC14, and PUF60 (top to bottom). Left, image of identified microRaft colony. Middle, representative image of stress induced G3BP1-positive (red) granule and DAPI (blue) after protein depletion by siRNA in HEK293T or HEK293T-G3BP1-GFP cells (false colored red for consistency). Scale bar = 20 μm. Quantification below image is mean stress granule area ± s.d. across n = 3 wells / condition (4 images / well) normalized to NTC. Right, Western blot validation of target protein depletion by siRNA. e , Protein-protein interaction network of the 23 RBP targets identified to modulate stress granule abundance. Network visualized in Cytoscape . Protein-protein interactions curated from BioPlex data and recent publications .

Article Snippet: EIF2AK1 (Bethyl #A302–685A, RRID:AB_10754970), PUF60 (Bethyl #A302–817A, RRID:AB_10631036), ZCCHC14 (Bethyl #A303–096A, RRID:AB_10895018), SNRNP200 (Bethyl #A303453A, RRID:AB_10949362), GAPDH (Abcam #ab8245, RRID:AB_2107448).

Techniques: Isolation, Sequencing, Microscopy, DNA Extraction, Staining, Control, Western Blot, Biomarker Discovery, Two Tailed Test, Protein-Protein interactions

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

Article Title: An antibody targeting an immune checkpoint molecule BTN2A2 enhances anti-tumor immunity

doi: 10.1016/j.neo.2025.101161

Figure Lengend Snippet: hBTN2A2-Ig preparation and functional validation. (A) The extracellular segment of hBTN2A2 nucleotide sequence was identified by ordinary PCR and agarose gel electrophoresis. The purified hBTN2A2-Ig protein was verified by Coomassie blue-stained SDS- PAGE, Western blot with anti-IgG2a antibody and anti-hBTN2A2 antibody. (B–G) The effects of hBTN2A2-Ig protein on murine T cell activation and proliferation in vitro. Splenocytes from C57BL/6 mice were incubated on 96-well plate precoated with 0.5 μg/ml of anti-CD3 antibody and the indicated dose of hBTN2A2-Ig or control Ig. The splenocytes were stained with anti-CD4, CD8, CD44, CD62L, Ki67 antibodies, followed by analyzed for the expression of CD69 18 h later (B–C) and the percentages of CD44 lo CD62L hi naive and CD44 hi CD62L lo effective memory CD4+ and CD8+ T cells 72 h later (D–E) , and T cell proliferation by CFSE dilution assay and Ki67 staining 5 days later (F–G) . The data are representative of three independent experiments with similar results. ( n = 3 each experiment) * P < 0.05, ** P < 0.01 and *** P < 0.001, compared with control Ig.

Article Snippet: The recombinant mouse IgG2a Fc protein (control Ig) was purchased from BioXCell (West Lebanon, NH) [ , ].

Techniques: Functional Assay, Biomarker Discovery, Sequencing, Agarose Gel Electrophoresis, Purification, Staining, SDS Page, Western Blot, Activation Assay, In Vitro, Incubation, Control, Expressing, Dilution Assay

Journal: Methods and Protocols

Article Title: Novel In Vivo Mouse Cryoablation Model to Explore Unique Therapeutic Approaches for Premalignant Columnar Lesions

doi: 10.3390/mps4010006

Figure Lengend Snippet: Preliminary results using the cryoablation procedure. ( A ) The experimental set-up of the validation of the cryoablation procedure is according to with minor adjustments. ( B ) Fluorescent image of untreated K5-GFP mice stomach. ( C ) Fluorescent image of vehicle-treated K5-GFP mice, 14 days post-cryoablation. ( D ) Fluorescent image of K5-GFP mice, 14 days post-cryoablation, treated with mNoggin (orally, 1× day). The delimitation of the original SCJ that separated the squamous and the columnar epithelium before the ablation is indicated by the dotted white line. i.p.: intraperitoneal injection; PO: Per os. , oral administration.

Article Snippet: Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA; cat. no.: 10010023); Formalin solution, neutral buffered, 10% (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: HT501128-4L); Ethanol (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: 51976-500ML-F); Xylene (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: 108298); Hematoxylin solution according to Mayer (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: 51275); Eosin Y solution 1% (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: 1170811000); DPX Mountant (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: 06522-100ML); Tissue-Plus TM O.C.T. compound (Fisher Scientific, Waltham, MA, USA; cat. no.: 23-730-571); DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, Waltham, MA, USA; cat. no.: 62247); Recombinant mouse noggin protein (R&D Systems, McKinley, MN, USA; cat. no.: 1967-NG); Tamoxifen (Sigma-Aldrich, St. Louis, MO, USA; cat. no.: T5648).

Techniques: Biomarker Discovery, Injection

Journal: Nucleic Acids Research

Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

doi: 10.1093/nar/gkx1073

Figure Lengend Snippet: Overview and validation of the H-SPA method. ( A ) Sequences of the cleavable randomized gaps used in this study. ( B ) Schematic representation of the experimental strategy. ( C ) Denaturing gel electrophoresis showing the limited digestion of double stranded substrates. The uncleaved duplexes are indicated with the asterisk and the cleaved fragment with the tilde. The reaction conditions are indicated: w: 37°C; c: 16°C and EDTA: 0.1 mM. ( D ) Correlation of heptamer counts for the construct R4b (positions 9–15) between two untreated controls (top left), two human RNase H1 treated samples (top right) and a control and a treated sample (bottom).

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Biomarker Discovery, Nucleic Acid Electrophoresis, Construct, Control

Journal: Nucleic Acids Research

Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

doi: 10.1093/nar/gkx1073

Figure Lengend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Sequencing, Construct

Journal: Nucleic Acids Research

Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

doi: 10.1093/nar/gkx1073

Figure Lengend Snippet: RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 .

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Sequencing, Construct, Binding Assay

Journal: Nucleic Acids Research

Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

doi: 10.1093/nar/gkx1073

Figure Lengend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Refining, Sequencing

Journal: Nucleic Acids Research

Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

doi: 10.1093/nar/gkx1073

Figure Lengend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Functional Assay, Reverse Transcription, Comparison, Preserving, Sequencing, Binding Assay

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) QMS workflow to identify the PAK4 interactome. MS analysis was performed using MCF7 cells stably expressing FLAG-PAK4 or FLAG-BAP (control). WC lysate and Cyt and Nuc subcellular fractions were analyzed in four independent biological replicates. After IP with anti-FLAG antibody and elution from FLAG beads with FLAG peptides, all samples were digested with trypsin and each labeled with a different iTRAQ 8-plex reagent. The eight iTRAQ labeled samples (four replicates of each FLAG-PAK4 and FLAG-BAP) were pooled and subjected to nano-LC-MS/MS analysis, followed by statistical and bioinformatic analysis. (B) Verification of subcellular fractionation. Lysates from WC, Cyt and Nuc fractions of FLAG-PAK4 and FLAG-BAP stably transfected MCF7 cells were analyzed by immunoblotting. Vinculin was used as a cytoplasmic marker; pRb as nuclear marker. (C) Schematic of the number of proteins identified by QMS in the different fractions before and after cut-off. Top: Total number of proteins recognized by QMS in each cellular fraction; Middle: Number of proteins in each fraction after cut-off; Bottom: Total number of unique proteins in all the fractions after cut-off. The cut-off criteria for specific FLAG-PAK4 associated hits was a combination of 5% FDR and above the 99.9% confidence interval of FLAG-BAP. (D) Venn diagram showing the number of specific PAK4 interacting proteins in WC and subcellular fractions.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Stable Transfection, Expressing, Control, Labeling, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Fractionation, Transfection, Western Blot, Marker

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Whole cell lysates derived from MCF7 cells stably expressing FLAG-PAK4 or FLAG-BAP were used for validation of QMS hits. After anti-FLAG IP and elution with FLAG peptides, samples were subjected to immunoblot analysis for the indicated proteins. Anti-FLAG (4 th row) and anti-PAK4 (5 th row) antibodies were used as controls. The left input panel shows immunoblotting of the two lysates. (B) PAK4 interactome networks obtained from the STRING database with the clusters identified by AutoAnnotate and visualized by Cytoscape. Diamond nodes: PAK4 interactors identified in the whole cell, or in both cytoplasmic and nuclear fractions or in all three fractions; Circle nodes: interactors identified in the cytoplasmic fraction or in both whole cell and cytoplasmic fraction; Squared nodes: interactors identified in the nuclear fraction or in both whole cell and nuclear fraction; Gray nodes: previously described PAK4 interactors.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Derivative Assay, Stable Transfection, Expressing, Biomarker Discovery, Western Blot

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Several subunits of the Arp2/3 and CCT complexes were identified in the PAK4 interactome. White nodes: proteins passed QMS cut-off; Grey nodes: proteins appeared in MS but did not pass the QMS cut-off; Darker grey node: not in the MS list. (B) After GFP-Trap IP of H1299 cell lysates transiently expressing EGFP (control) or EGFP-PAK4, samples were subjected to immunoblot analysis for the indicated proteins. The upper panel is blotted with anti-CCTε, the middle panel with anti-ARPC2, while anti-GFP was used to control the IP efficiency in the lower panel. Input lanes are direct immunoblot of the used cell lysates. (C) After anti-CCTε IP of H1299 cell lysates transiently expressing EGFP-PAK4, blots were probed with an anti-GFP antibody in the upper panel. Anti-CCTε was used to control the IP efficiency in the lower panel. Input lane shows direct immunoblotting of the used lysate.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Expressing, Control, Western Blot

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) PAK4 mediated phosphorylation was analyzed by an in vitro kinase assay using recombinant HIS-PAK4 together with the Arp2/3 complex (left panel) or GST-VCA (right panel) as substrates, with GST as a negative control and GST-RAF1 (332–344) as a positive control (upper panels). The lower panels display the protein loading in the assays by Coomassie Brilliant Blue staining. (B) HIS-PAK4 phosphorylation of the WASP VCA domain was analyzed using an anti-N-WASP pSer484/Ser485 antibody after a kinase assay using recombinant HIS-PAK4 with GST-VCA as a substrate. GST serves as a negative control, while the anti-RAF1 pSer338 antibody was used as a positive control to detect GST-RAF1 phosphorylated by PAK4 (upper panel). The lower panel shows the loading of HIS-PAK4 protein and GST-fusion proteins used in the assay by silver staining. (C) HIS-PAK4 was pulled-down in the presence of GST-VCA or the Arp2/3 complex with Ni-NTA agarose and input (I), supernatant (S) and pellet (P) analyzed by silver staining. (D) IP of EGFP control or EGFP-PAK4 transiently expressed in H1299 cells analyzed by immunoblotting using an anti-N-WASP antibody (upper panel right two lanes). The left two lanes show immunoblotting of the input lysates. Anti-GFP was used to control the expression and IP efficiency in the lower panel. (E) N-WASP was immunoprecipitated with an anti-N-WASP antibody from lysates of H1299 cells transiently expressing EGFP-PAK4 and samples were analyzed by immunoblot using an anti-GFP antibody with the lysate input to the left (upper panel). Anti-N-WASP was used to control the expression and IP efficiency in the lower panel. (F) PAK4 was immunoprecipitated with an anti-PAK4 antibody from lysates of MCF7 cells, with rabbit IgG as a control, samples were analyzed by immunoblot using an anti-N-WASP antibody with the lysate input to the left (upper panel). Anti-PAK4 blotting was used to control IP efficiency in the lower panel. (G) N-WASP was immunoprecipitated with an anti-N-WASP antibody from lysates of H1299 cells, with rabbit IgG as a control, samples were analyzed by immunoblot using an anti-PAK4 antibody with the lysate input to the left (upper panel). Anti-N-WASP blotting was used to control IP efficiency in the lower panel. (H) PAK4, N-WASP and F-actin co-localized in the cell periphery after re-plating. FLAG-PAK4 was labeled with an anti-FLAG mab (Green), N-WASP with an anti-N-WASP antibody (Red), F-actin with SiR-actin (Purple) and Nuclei with Hoechst (Blue), Scale bar: 10 μm.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Negative Control, Positive Control, Staining, Silver Staining, Control, Western Blot, Expressing, Immunoprecipitation, Labeling

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Immunoblotting of lysates from control siRNA transfected cells and PAK4 knockdown (siPAK4) cells with anti-N-WASP and anti-N-WASP pSer484/Ser485 antibodies. PAK4 knockdown efficiency was detected with an anti-PAK4 antibody and vinculin was used as a loading control. (B) Actin polymerization reactions were performed with actin, Arp2/3 complex, GST–VCA and GST-PAK4 KD (kinase domain) in different combinations as indicated. The amount of polymerized actin over time is indicated by the increase in fluorescence intensity. (C) G-actin and F-actin were separated by centrifugation in H1299 cell lysates with or without PAK4 siRNA-mediated knockdown. Left panel: Cytochalasin D (Cyto D) treatment was used as a control to block actin polymerization. Immunoblot analysis with an anti-actin antibody shows the amount of G-actin (G) and F-actin (F) for each condition. Right panel: PAK4 knockdown efficiency was assessed by immunoblotting using vinculin as a loading control (top). In addition, the total amounts of actin were analyzed (bottom). (D) siRNA knockdown of PAK4 alters the cellular morphology and F-actin distribution in H1299 cells. Nuclei were stained with Hoechst (Blue) and F-actin with Phalloidin (Red). Scale bar: 10 μm.

Article Snippet: For endogenous protein-protein interactions, 1000 μg protein lysate from MCF7 or H1299 cells were immunoprecipated by a rabbit anti-PAK4 antibody (6508) or by a rabbit anti-N-WASP antibody (HPA005750, Atlas Antibodies) using rabbit IgG as control.

Techniques: Western Blot, Control, Transfection, Knockdown, Fluorescence, Centrifugation, Blocking Assay, Staining

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. ( B ) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose . ( C ) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. ( D and E ) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD + and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. ( F ) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, untreated; IR, 7-gray ionizing irradiation; H 2 O 2 , oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress conditions. Further details are shown in table in fig. S3B and in table S1. ( G ) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. ( H ) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. Inputs (I) and elutions (E) are shown.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Affinity Purification, Binding Assay, Variant Assay, Immunoprecipitation, CRISPR, Expressing, Mutagenesis, Irradiation, Western Blot, Purification, Filtration, Chromatography

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Levels of three mH2A isoforms in WCE of NIH3T3 cells transfected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. ( B ) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7 −/− MEFs cultured under normal or GS conditions during the indicated times. ( C ) Venn diagrams showing the intersection of SIRT7-associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). ( D ) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7 −/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log 2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. ( E ) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). ( F ) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. ( G ) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (* P < 0.05 and *** P < 0.005).

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Transfection, shRNA, Cell Culture, Purification, Derivative Assay, Protein-Protein interactions, ChIP-qPCR, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Two Tailed Test

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Heat map showing RNA expression changes relative to NT (as log 2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. ( B ) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7 −/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log 2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a difference between WT and KO log 2 FC (GS versus NT) of >0.3 (table S3). ( C ) mH2A1 ChIP-seq signals across ctgf , a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. ( D ) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7 −/− MEFs was rescued by retroviral-mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (* P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [ gbp6 , −6 kb; necab1 , −3 kb; lair1 , −27.5 kb; ctgf , +600 base pairs (bp); adra2a , −5 kb; and nrip3 , +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (* P <0.05, ** P < 0.01, and *** P < 0.005). a.u., arbitrary units. ( E ) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: RNA Expression, RNA Sequencing, Expressing, ChIP-sequencing, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Two Tailed Test, ChIP-qPCR

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Model validation studies in WT and Sirt7 −/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. ( B ) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7 −/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generated from three replicates. Probabilities are those associated with one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.005). ( C ) Levels of SirT7 in liver samples from Wt and Sirt7 −/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. ( D ) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. ( E ) Autophagy activity in the Wt and Sirt7 −/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed with a two-tailed t test (* P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). ( F ) Similar analysis ( n = 5) of Beclin-1 as in (E). ( G ) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Generated, Fractionation, Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Control, In Vivo, Protein-Protein interactions

Journal: Journal of Cancer

Article Title: Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

doi:

Figure Lengend Snippet: Sample mixture loaded on each gel : Each sample was composed of equal amounts of RARRES1 knock-down PWR-1E and control lysates. Cy3 and Cy5 dyes were equally swapped among RARES1 knock-down and control samples. The internal standard was labeled with Cy2 and consisted of 12.5 µg of each knock-down and control. The sample loaded on the pick-gel was composed of 300 µg of each sample and control.

Article Snippet: Western Blotting was performed as described previously using the following antibodies: Rabbit monoclonal to PP2A (cat. ab32141, Abcam, Cambridge, MA); Rabbit polyclonal to PSD93 (cat. ab2930, Abcam, Cambridge, MA); Goat polyclonal to RARRES1 (R&D systems, Mineapolis, MN); Mouse monoclonal to VCP (cat. ab11433, Abcam, Cambridge, MA).

Techniques: Knockdown, Control, Labeling

Journal: Journal of Cancer

Article Title: Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

doi:

Figure Lengend Snippet: DIGE data : Representation set of one of the 4 biological replicates. 25 µg of control PWR-1E cells lysates labeled with 200 pmoles of Cy3 (green); 25 µg of RARRES1 knock-down PWR-1E cells labeled with 200 pmoles of Cy5 (red) and a normalization pool composed of a 50:50 mixture of sample and control labeled with Cy2 (blue). The first dimension separation was performed on a 24 cm non-linear IPG strips and the second dimension separation was accomplished on a 5-20% gradient polyacrylamide gel with 2 built-in spot picking references. Gels were scanned at a 100 µm resolution using excitation and emission wavelength that corresponds to each dye. Combined Cy2, Cy3, and Cy5 signals are represented in the top gel.

Article Snippet: Western Blotting was performed as described previously using the following antibodies: Rabbit monoclonal to PP2A (cat. ab32141, Abcam, Cambridge, MA); Rabbit polyclonal to PSD93 (cat. ab2930, Abcam, Cambridge, MA); Goat polyclonal to RARRES1 (R&D systems, Mineapolis, MN); Mouse monoclonal to VCP (cat. ab11433, Abcam, Cambridge, MA).

Techniques: Control, Labeling, Knockdown

Journal: Journal of Cancer

Article Title: Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

doi:

Figure Lengend Snippet: Sypro Ruby Pick gel : loaded with 300µg of PWR-1E RARRES1 knock-down protein extracts and 300µg of control sample. The green dots represent the proteins that were detected by the Decyder software. Annotated proteins are those that were picked for mass spectrometry analysis. These spots were identified as differentially-expressed across the 4 biological replicates in the analytical gels. The coordinates of these spots were recorded, matched, and excised from the Pick gel for identification.

Article Snippet: Western Blotting was performed as described previously using the following antibodies: Rabbit monoclonal to PP2A (cat. ab32141, Abcam, Cambridge, MA); Rabbit polyclonal to PSD93 (cat. ab2930, Abcam, Cambridge, MA); Goat polyclonal to RARRES1 (R&D systems, Mineapolis, MN); Mouse monoclonal to VCP (cat. ab11433, Abcam, Cambridge, MA).

Techniques: Knockdown, Control, Software, Mass Spectrometry

Journal: Journal of Cancer

Article Title: Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

doi:

Figure Lengend Snippet: Differentially expressed proteins : a) 2- Cropped DIGE; b) three-dimensional view, and c) logarithmic representation of the 4 four biological replicates of Dlg-2 upregulation, PP2A and VCP down-regulation as a result of RARRES1 knock-down in PWR-1E cells.

Article Snippet: Western Blotting was performed as described previously using the following antibodies: Rabbit monoclonal to PP2A (cat. ab32141, Abcam, Cambridge, MA); Rabbit polyclonal to PSD93 (cat. ab2930, Abcam, Cambridge, MA); Goat polyclonal to RARRES1 (R&D systems, Mineapolis, MN); Mouse monoclonal to VCP (cat. ab11433, Abcam, Cambridge, MA).

Techniques: Knockdown

Journal: Journal of Cancer

Article Title: Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

doi:

Figure Lengend Snippet: Western Blot Validation : of a) RARRES1 knock-down, b) Dlg2 homologue, c) PP2A, d) VCP and e) GAPDH loading control. Lane 1 scrambled siRNA nucleofected PWR-1E lysates; lane 2 RARRES1 siRNA nucleofected PWR-1E lysates.

Article Snippet: Western Blotting was performed as described previously using the following antibodies: Rabbit monoclonal to PP2A (cat. ab32141, Abcam, Cambridge, MA); Rabbit polyclonal to PSD93 (cat. ab2930, Abcam, Cambridge, MA); Goat polyclonal to RARRES1 (R&D systems, Mineapolis, MN); Mouse monoclonal to VCP (cat. ab11433, Abcam, Cambridge, MA).

Techniques: Western Blot, Biomarker Discovery, Knockdown, Control