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: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A ) G-TPP treatment leads to PINK1 stabilization and pS65-Ub induction in HeLa cells. HeLa cells stably expressing untagged Parkin were treated with 10 µM G-TPP for the indicated times. Western blots were prepared with cell lysates and probed with antibodies against PINK1 and pS65-Ub. GAPDH served as a loading control. ( B ) pS65-Ub is induced in G-TPP treated cells and co-localizes with EGFP-Parkin and mitochondria. HeLa cells stably expressing EGFP-Parkin (green) were treated with 10 µM G-TPP for the indicated times and fixed. Cells were stained with antibodies against pS65-Ub (red) and the mitochondrial marker TOM20 (cyan). Scale bars correspond to 10 µM. ( C ) Quantification of Parkin translocation using High Content Imaging. HeLa EGFP-Parkin cells were treated for 4 or 8 h with or without 10 µM G-TPP. CCCP treatment (10 µM for 2 h) was used as a positive control. Cells were fixed, counterstained with Hoechst dye to visualize nuclei, imaged and analyzed using the ratio of cytoplasmic to nuclear EGFP signal . Data was normalized to positive (2 h 10 µM CCCP treatment) and negative (2 h DMSO) controls. G-TPP significantly induced Parkin re-localization to levels similar to or beyond 2 h CCCP treatment. Shown are the mean values of three independent experiments with triplicate wells each ± SEM (one-way ANOVA with Tukey’s posthoc, *** p < 0.0005).

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Stable Transfection, Expressing, Western Blot, Control, Staining, Marker, Translocation Assay, Imaging, Positive Control

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A , B ) HeLa cells stably expressing EGFP-Parkin were treated with 12 different concentrations of G-TPP or 17-AAG in a dose-response format for 2 or 6 h before adding (A) DMSO or (B) CCCP (final assay concentration = 10 µM) for an additional 2 h. Cells were fixed and analyzed for Parkin translocation (black line). Cell number was assessed by counting the number of Hoechst-positive nuclei in each well (gray line). Values were normalized to positive (2 h 10 µM CCCP) and negative (2 h DMSO) controls. In absence of CCCP, only G-TPP but not 17-AAG induced Parkin translocation. In combination with CCCP, increasing doses of 17-AAG led to inhibition of Parkin translocation. Very high concentrations of G-TPP also inhibited Parkin translocation and resulted in cell toxicity. ( C ) HeLa cells expressing untagged Parkin were treated with 1 µM 17-AAG, 10 µM G-TPP or DMSO as a control for 6 h before CCCP (10 µM) or medium containing DMSO was added for 4 h. Cells were harvested and western blots probed with antibodies against PINK1, pS65-Ub, the mitochondrial phosphatase PGAM5. Vinculin was used as a loading control. Compared to controls, 17-AAG pre-treated cells showed lower PINK1 levels that were accompanied by reduced pS65-Ub induction upon CCCP treatment, while G-TPP pre-treatment led to induction of pS65-Ub in the absence of CCCP, as expected.

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Stable Transfection, Expressing, Concentration Assay, Translocation Assay, Inhibition, Control, Western Blot

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A , C ) Fibroblasts were treated with 15 µM G-TPP for the indicated time points. Cells were harvested and western blots were probed with antibodies against (A) PINK1, pS65-Ub and total Ub or (C) autophagy adapter proteins. GAPDH and Vinculin served as loading control. G-TPP treatment led to PINK1 stabilization and pS65-Ub induction in primary skin fibroblasts. p62 levels were induced upon G-TPP treatment, while other adapters seemed decreased. ( B , D ) Human fibroblasts were treated with 15 µM G-TPP for 16 h and fixed and stained with antibodies against (B) pS65-Ub (green) or (D) the autophagy adapters NBR1, NDP52, p62, OPTN and TAX1BP1 (green). Mitochondria were stained with antibodies against TOM20 (red), nuclei were visualized with Hoechst (blue). Scale bars indicate 10 µM. A magnified image of the boxed region, the fluorescence profile along the arrow and the Pearson’s correlation coefficient of adapter protein and mitochondrial stainingare shown to the right. Shown is the mean ± SEM of at least five randomly selected images (unpaired, two-sided t -test, *** p < 0.0005).

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Western Blot, Control, Staining, Fluorescence

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A ) Control cells or PINK1 Q456X fibroblasts were converted to induced neurons (iNeurons). Cells were treated for the indicated times with 10 µM G-TPP and harvested. Western blots were prepared and probed with antibodies against PINK1, pS65-Ub and Parkin. Beta III tubulin served as a control for successful conversion to neuronal cells, GAPDH as a loading control. ( B ) iNeurons were treated with 15 µM G-TPP for 8 h and fixed. Cells were stained with antibodies against pS65-Ub (green) and the mitochondrial marker TOM20 (red) and the neuronal marker Beta III tubulin (cyan). Nuclei were stained with Hoechst 33342 (blue). Scale bars correspond to 10 µM. A magnified image of the boxed region and the fluorescence profile along the arrow are shown to the right.

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Control, Western Blot, Staining, Marker, Fluorescence

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Purified SARS-CoV-2 RdRp proteins expressed in baculovirus-infected insect cells ( Spodoptera frugiperda , Sf) analysed by SDS–PAGE and Coomassie staining. 7H8: nsp7-His 6 -nsp8, nsp12-HF: nsp12-His 6 -3xFlag, nsp12-F/7H8: nsp12-3xFlag/nsp7-His 6 -nsp8, nsp12-HF/7L8: nsp12-His 6 -3xFlag/nsp7-GGSGGS-nsp8, nsp12-HF/7/8: nsp12-His 6 -3xFlag/nsp7/nsp8. ( B ) Bacterially expressed and purified SARS-CoV-2 nsp7, nsp8 and nsp12 proteins analysed by SDS–PAGE and Coomassie staining. The proteins were expressed as 14His-SUMO fusion proteins in E. coli . 14His-SUMO was removed by a SUMO-specific protease during purification generating native N-termini. ( C ) Gel-based primer-extension assay to test RNA-dependent RNA synthesis using the RdRp preparation Sf nsp12-F/7H8. The substrate consists of a 10 nt RNA primer annealed to the 3′ end of a 35 nt RNA template. The 5′ end of the template strand is labelled with a Cy3 fluorophore. Reaction products were analysed by native PAGE and visualisation of Cy3 fluorescence. Formation of duplex RNA by RdRp was observed over time. Controls: a preformed Cy3-labelled dsRNA with the same size as the reaction product (dsRNA), the primed substrate (no protein). ( D ) Schematic diagram illustrating the FRET-based RdRp strand displacement assay. The RNA substrate is composed of a Cy3 fluorophore-containing template strand, an annealed primer and an annealed quencher strand with a 5′ flap. RdRp activity synthesises RNA by extending the primer strand and displaces the quencher strand. The displaced quencher strand can no longer anneal to fully synthesised duplex RNA leading to an increase in Cy3 fluorescent signal. ( E ) FRET-based strand displacement assay using the indicated concentrations of Sf nsp12-F/7H8.

Article Snippet: SARS-CoV-2 RdRp is the target of remdesivir, the only antiviral approved for human use to treat COVID-19 by regulatory agencies [ ].

Techniques: Purification, Infection, SDS Page, Staining, Primer Extension Assay, Clear Native PAGE, Fluorescence, Activity Assay

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Logistics of the screen. A custom chemical library consisting of over 5000 compounds was screened against RdRp activity using the FRET-based strand displacement assay in a 384-well format. RdRp was prepared by preincubation of Sf nsp12-HF/7L8 and Sf 7H8 in a 1 : 3 ratio for 30 min at room temperature. RdRp was dispensed into compound-containing 384-well plates and incubated for 10 min. Reactions were started by the addition of a substrate mix and florescence monitored in 90 s intervals. ( B,C ) Results of the HTS screen performed at 1.25 µM ( B ) and 3.75 µM ( C ) compound concentration. The normalised reaction velocity plotted against the compound number is shown. Validated hits are shown in red. ( D ) Kinetic curves with >15% reduction in reaction velocity or >10% reduction in fluorescent signal at the endpoint were inspected manually. As example, kinetic data for compound GSK-650394 is shown (red curve, data from surrounding wells in black). ( E ) Summary of the HTS hit selection strategy. From over 5000 compounds tested in the screen, 64 were considered primary hits after manual inspection of HTS reactions, which showed a reduction in reaction velocity below 85%, or a reduction in endpoint signal below 90%. Out of these, 46 primary hits were eliminated as they likely represent nonspecific modes of enzymatic inhibition such as colloidal aggregation or interference with the substrate structure. As part of this analysis promiscuous compounds that were identified as hits in other SARS-CoV-2 HTS [ , ] were removed with the exception of five suramin and suramin-like compounds, which were also identified in the SARS-CoV-2 nsp13 helicase HTS (Zeng et al. ). In vitro validation of the effect of suramin and suramin-like compounds on the activity of SARS-CoV-2 helicase and SARS-CoV-2 RdRp can be found in Zeng et al. . A total of 18 compounds (including five suramin and suramin-like compounds) were selected as hits, of which 14 were included in further in vitro validation in this work.

Article Snippet: SARS-CoV-2 RdRp is the target of remdesivir, the only antiviral approved for human use to treat COVID-19 by regulatory agencies [ ].

Techniques: Activity Assay, Incubation, Concentration Assay, Selection, Inhibition, In Vitro

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Viral inhibition experiments workflow graphical representation. In brief, 24 h after Vero E6 cells were seeded in a 96-well format, they were treated with selected drugs at specified concentrations. Then, cells were infected with a SARS-CoV-2 isolate at a MOI of 0.5 PFU per cell. Twenty-two hours later, cells were fixed and analysed by immunofluorescence staining and imaging. ( B ) SARS-CoV-2 antiviral activity of GSK-650394, C646, BH3I-1, MDK-83190 and cefsulodin. Representative overlaid images of viral N protein immunofluorescence (green) and DNA dye DRAQ7 staining (red). ( C ) Dose–response curve analysis. Viral infection values represent the area of viral plaques visualised by viral nucleocapsid protein staining (green curves) and cell viability was measured in the same experiment as the area of cells stained with the DNA dye DRAQ7 (red curves). Data are plotted as percentage relative to DMSO only control wells (100%). Data represent the mean and standard deviation (SD) of 3 replicates. Areas were calculated using FIJI and EC 50 values were calculated using Prism software.

Article Snippet: SARS-CoV-2 RdRp is the target of remdesivir, the only antiviral approved for human use to treat COVID-19 by regulatory agencies [ ].

Techniques: Inhibition, Infection, Immunofluorescence, Staining, Imaging, Activity Assay, Standard Deviation, Software

Journal: Nature cell biology

Article Title: SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

doi: 10.1038/s41556-023-01096-x

Figure Lengend Snippet: Fig. 4 | SARS-CoV-2 ORF6 or NSP13 expression is sufficient to cause CHK1 loss. a, Immunofluorescence (IF) images of Huh7 cells expressing Strep-tagged SARS-CoV-2 ORF6 or NSP13 fixed 48 h post-transfection and stained for DDR markers; GFP was used as control; staining with anti-Strep-tag was used to label transfected cells; nuclei were stained with DAPI. Scale bar, 10 μm. b, The histograms show the percentage of CHK1- or RRM2-expressing cells among the transfected ones (Strep-Tag+) as determined in a; n = 3 independent experiments (n = 4 for GFP-expressing cells in RRM2 analysis). The dot plots show the number

Article Snippet: 3 nature portfolio | reporting sum m ary M arch 2021 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used gH2AX (Ser139) Abcam ab11174 gH2AX (Ser139) Millipore 05-636 53BP1 Bethyl A303-906A 53BP1 Novus NB100-304 ACE2 Abcam ab15348 ATM Abcam ab32420 ATR Santa Cruz sc-1887 Beclin 1 Bethyl A302-566A-T Beta-actin Sigma-Aldrich A2228 BrdU BDbioscience 347580 CD68 Abcam ab125212 CDT1 Cell Signaling #8064 cGAS Cell Signaling #15102 CHK1 Novus NB100-46 CHK1 (2G1D5) Cell Signaling #2360 CHK1 (ST57-09) ThermoFisher MA532180 CHK2 Millipore 05-649 Cleaved Caspase-3 (Asp 175) Cell Signaling 9661 DNA-PK Abcam ab32566 HA-tag Abcam ab236632 Histone H3 Abcam ab10799 Human Cytokeratin 8/18 (EP17/EP30) Dako M3652 ISceI (FL-86) Santa Cruz sc-98269 KAP1 Abcam ab10484 LC3B Sigma-Aldrich L7543 p16 Abcam ab51243 p21 Abcam ab188224 P21 Cell Signaling #2946 P38 MAPK Cell Signaling #9212 P53 Abcam ab1101 p62 Abcam ab240635 pATM (Ser1981) Rockland 200-301-400 pATR (T1989) Abcam ab223258 pCHK1 (S317) Cell Signaling #2344 pCHK2 (Thr68) Cell Signaling #2661 PCNA Bio Rad MCA1558 pDNA-PK (Ser2056) (EPR5670) Abcam ab124918 pKAP1 (S824) Bethyl A300-767A pP38 MAPK (Thr180/Tyr182) Cell Signaling #9211 pP53 (Ser15) Cell Signaling #9284 proSP-C Abcam ab3786 pRPA (S4/S8) Bethyl A300-245A pSTAT1 (Ser 727) Cell Signaling #9177 pSTAT1 (Tyr 701) (58D6) Cell Signaling #9167 RPA Calbiochem NA18-100UG RRM1 Santa Cruz sc-11733 RRM2 Novus NBP1-31661 RRM2 Santa Cruz sc-10844 SARS-CoV2 nucleocapsid Sino Biological 40588-T62 SARS-CoV2 nucleocapsid Sino Biological 40143-R019 SARS-CoV2 nucleocapsid (1A6) ThermoFisher MA5-35941 STAT1 (9H2) Cell Signaling #9176 Strep-tag II epitope Qiagen 34850 Tubulin Sigma-Aldrich T5168 Ubiquitin (P4D1) Santa Cruz sc-8017 Vinculin Sigma-Aldrich V9131 Cy3 D/M Jackson 715-165-150 Cy3 D/R Jackson 711-165-152 Cy3 D/G Jackson 705-165-147 4 nature portfolio | reporting sum m ary M arch 2021 A488 D/M ThermoFisher A21202 A488 D/R ThermoFisher A21206 A488 D/G ThermoFisher A11055 A647 D/M ThermoFisher A31571 A647 D/R ThermoFisher A31573 A647 D/G ThermoFisher A21447 Validation All antibodies were validated by the manufacturer and were previously used in peer reviewed works.

Techniques: Expressing, Immunofluorescence, Transfection, Staining, Control, Strep-tag

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: Biopsy samples were selected by the pathology service at Boston University Henry M. Goldman School of Dental Medicine and tissue sections were prepared and stained. Slides made from one selected subject from 3 to 5 subjects sampled in each category of dysplasia, differentiated oral cancer, and poorly differentiated oral cancer, respectively, are shown. Stained slides were imaged using an automated slide imager, and images were processed using Case Viewer software version 2.2 (Budapest, Hungary). Data indicate that LOXL2 was highly expressed in a variety of cancer cells and associated mesenchymal cells in human oral cancer, while LOX expression was more restricted

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Staining, Software, Expressing

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a PXS-S1C attenuates human tongue tumor growth in mice, and b and c PSX-S1C significantly decreases tumor cell spreading. Tumor volume of mouse tongues were measured every 3 days. Data are means ± SD. ANOVA, p : 0.0001, Tukey’s multiple comparisons test, ** p < 0.001, *** p < 0.0001 indicate difference among the groups ( n = 8 per group). b IVIS imaging for red fluorescent protein-labeled HSC3 cells 21 days after commencing injections of vehicle or PXS-S1C (30 mg/kg). The fluorescence signals were optimized for DsRed protein at excitation 570 nm and emission 620 nm. c Quantification of fluorescence signal area shows a significant difference between PXS-S1C-treated and non-treated groups. Data are mean ± SD. Student’s t -test, * p < 0.05 indicates difference between the groups. d Histology of HSC3 cell orthotopic tongue tumors. Hematoxylin and eosin staining a mouse tongue is shown 18 days after implantation. The photo is representative of histological features of HSC3 orthotopic tumors. The images were taken at ×4 and ×20 magnifications. Scale bar = 100 µm. PXS-S1C attenuates expression of ( e ) Ki67 and ( f ) LOXL2 in LY2 orthotopic tumors in immunodeficient mice. e Immunohistochemistry staining of tongue sections with anti-Ki-67 antibody shows that PXS-S1C reduced Ki-67 staining in orthotopic HSC3 tumors in mice. Scale bar = 100 µm. Data are mean ± SD. ANOVA, p < 0.01, Tukey’s multiple comparisons test, * p < 0.05 indicate difference among the groups ( n = 8 per group). f Staining of tongue tissues with anti-LOXL2 antibody shows that PXS-S1C reduced LOXL2 staining in orthotopic HSC3 tumors in mice. Scale bar = 100 µm. Data are mean ± SD. ANOVA, p < 0.01, Tukey’s multiple comparisons test, * p < 0.05 indicate difference among the groups ( n = 8 per group)

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Imaging, Labeling, Fluorescence, Staining, Expressing, Immunohistochemistry

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a Gross features of tongues and lymph nodes of the mice in all groups. Circles mark grossly oversized lymph nodes. b Number of the mice with abnormal size of lymph nodes (left panel) was reduced by the treatment with PXS-S1C. Number of the mice with normal versus abnormal size of lymph nodes. Normal sized LN in control mice = 0.053 ± 0.01 cm 2 , * p < 0.05. Chi-Square test (4 × 2 analysis) p = 0.008, Chi-Square test, p = 0.008, indicates difference in number of normal and abnormal sized LN among the groups. Average size of the lymph nodes in each group (right panel). ANOVA, p < 0.05, Tukey’s multiple comparison test * p < 0.05 indicates difference between the groups. Fisher’s exact test (2 × 2 analysis). Control vs. LY2, p = 0.001, mice injected with LY2 have larger LNs than controls. Fisher’s exact test (2 × 2 analysis) LY2 vs. both LY2 + PXS-S1C 10 and 30 mg/kg group ogether, p = 0.03, LOX inhibitor reduces the frequency of mice having enlarged LNs. Fisher’s exact test (2 × 2 analysis), LY2 + PXS-S1C 10 mg/kg vs. LOXL2 + 30 mg/kg, p > 0.05 There is no statistical difference between the two different doses of LOX inhibitor. c Histology of LY2 orthotopic tongue tumors. Hematoxylin and eosin staining of LY2 orthotopic tongue tumor. The images are representative of histological features of LY2 tumor. The images were taken at ×4 and ×20 objectives. Scale bar = 100 µm

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Control, Comparison, Injection, Staining

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: PXS-S1C attenuates expression of ( a ) PCNA and ( b ) LOXL2 in LY2 orthotopic tongue tumors in immunocompetent mice after 6 weeks of treatment, scale bar = 100 µm. Data are mean ± SD. ANOVA, p < 0.05, Tukey’s multiple comparisons test, * p < 0.05 indicate difference among the groups ( n = 12 per group). PSX-S1C treatment of LY2 tongue orthotopic tumors in mice alters tongue and cervical lymph node ( c ) tumor cell morphology, ( d ) E-cadherin expression and ( e ) vimentin expression. The images were taken at ×10 and ×20 magnifications. Scale bar = 100 µm. Images are from mice at the 6-week time point

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Expressing

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a Collagen accumulation in orthotopic tongue LY2 tumors by Sirius red staining of LY2 tumors in the tongue. A representative image is shown from one of 12 mice at the 6-week time point. Scale bar = 100 µm. Treatment with PXS-S1C appeared to reduce the amount of collagen, particularly at the apparent interfaces of tumor with surrounding non-tumor tissue. The images were taken at ×10 and ×20 magnifications. b Stimulation of human gingival fibroblast proliferation induced by CM of different oral cancer cell lines was inhibited by PXS-S1C treatment. Gingival fibroblasts were serum depleted for 24 h and then treated with cancer cell CM with and without PXS-S1C (1 µM) in serum-free conditions for 24 h, and DNA accumulation measured by CyQUANT assays: [(HSC3 CM)–(HSC3 + PXS-S1C)]/HSC3 CM × 100]. Data are means ± SD. Experiments were done with six replicate samples for each cell line. ANOVA, p < 0.0001, Tukey’s multiple comparison test. ** p < 0.001 indicate differences among different groups. c LOXL2 is the most abundantly expressed paralogue by HSC3 cells. RNAs isolated from serum-depleted HSC3 cells was subjected to qPCR for all five lysyl oxidase paralogues using Taqman probes. Data are means ± SEM. This experiment was performed three times independently with triplicate samples. The RNA levels were normalized to 18S rRNA. ANOVA One way, P < 0.01 among all LOX family members. d LOXL2 protein is secreted at high levels by HSC3 cells. CM from human HSC3 cells, mouse LY2 cells and normal human gingival fibroblasts were collected under serum-free conditions, concentrated by 25-fold, and subjected to western blotting and visualized with anti-LOXL2 antibody. β-tubulin was used as a loading control. Data are means ± SEM. This experiment was done three times independently. ANOVA, p < 0.002, Tukey’s multiple comparison test * p < 0.05 indicate difference among the groups

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Staining, CyQUANT Assay, Comparison, Isolation, Western Blot, Control

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: LOXL2 inhibitor PXS-S1C attenuates HSC3 CM-stimulated human oral fibroblast ( a ) proliferation, ( b ) phosphorylation of PDGFRβ at the Y771 and Y857 but not Y751 residues, and ( c ) ERK activation, but not AKT. a Human gingival fibroblast proliferation was reduced after 24-h treatment with PXS-S1C (1 µM) or AG 1296 (5 µM) in the HSC3 CM as determined by the CyQUANT assay. Data are means SEM. This experiment was done three times independently with six replicate samples. ANOVA, p < 0.0001, Dunnett’s multiple comparisons test, *** p < 0.0001 indicates significant difference between treated groups; while ## p < 0.001, ### p < 0.0001 indicate significant differences from non-CM group. b and c Gingival fibroblasts were serum depleted and then treated with non-CM, and CM with and without PXS- S1C (1 µM) and cell layer protein samples were subjected to western blot. Data are means ± SEM. The experiment was performed with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05 indicates a significant difference from PXS-S1C treated group. Dunnett’s multiple comparison test, # p < 0.05, ## p < 0.001 indicate significant differences from Non-CM group. d PXS-S1C attenuates PDGF-BB stimulated phosphorylation of all three PDGFRβ phosphorylation sites Y771, Y857 and Y751, and AKT activation in oral fibroblasts. Gingival fibroblasts were serum depleted and then treated with no PDGF-BB, and PDGF-BB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05, ** p < 0.001, and *** p < 0.0001 indicate difference from PXS-S1C-treated group. Dunnett’s multiple comparison test, # p < 0.05, ## p < 0.001, ### p < 0.0001 indicate difference from No PDGF group. e PDGF-AB mimics the effects of HSC3 cell CM on oral fibroblasts in phosphorylation of PDGFRβ. Gingival fibroblasts were serum starved and then treated with no PDGF-AB, and PDGF-AB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05 indicates difference from PXS-S1C-treated group. Dunnett’s multiple comparison test, # p < 0.05 indicates difference from No PDGF group

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Phospho-proteomics, Activation Assay, CyQUANT Assay, Western Blot, Isolation, Comparison

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a PDGF-AB mimics the effects of HSC3 cancer cell CM on oral fibroblasts in phosphorylation of ERK1/2, and not AKT. Oral fibroblasts were serum depleted and then treated with no PDGF-AB, and PDGF-AB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05 indicates difference from PXS-S1C-treated group. Dunnett’s multiple comparison test, # p < 0.05 and ### p < 0.0001 indicate difference from No PDGF group. b PDGF-A and PDGF-B knockdown in HSC3 cells blocks HSC3 CM stimulation of oral fibroblast proliferation. The concentration of PDGF-AB ligand was decreased significantly in CM of knockdown HSC3 cells as compared with non-target control cell CM. PDGF-A and PDGF-B were, respectively, knocked down with independent shRNAs (A1, A2, or A3 for PDGF-A; and B1 and B2 for PDGF’B). The concentration of PDGF-AB ligand in knocked down HSC3 CM was measured using a PDGF-AB-specific ELISA kit. Data are mean ± SD. This experiment was done with triplicate samples. ANOVA, p < 0.0001, Dunnett’s multiple comparisons test # p < 0.05, ## p < 0.001, ### p < 0.0001 indicate difference from Control HSC3 group. c The proliferation of oral fibroblasts treated with knocked down HSC3 medium for 24 h was assessed by CyQUANT assay. Data are mean ± SEM. This experiment was performed three times independently with primary human gingival fibroblasts isolated from three different donors. ANOVA, p : 0.0001, Dunnett’s multiple comparisons test: # p < 0.05, indicate difference from HSC3 Control group. d The degree of PDGF-A or PDGF-B knockdown correlate linearly with decreased proliferative responses to HSC3 CM. The relationship between fibroblast proliferation inhibition and the relative level of PDGF-AB concentration was analyzed using linear regression. Correlation coefficient r : −0.93, R squared: 0.87, p -value: 0.006. Data indicate that PDGF-AB specifically is the ligand in HSC3 CM that stimulates oral fibroblast proliferation. e Carbonyl pull down assay for PDGFRβ in oral fibroblasts treated with HSC3 CM in the absence or presence of PXS-S1C. Human oral fibroblasts were treated with HSC3 CM in the absence or presence of 1 µM PXS-S1C followed by biotin hydrazide derivitization and affinity pulldown with a streptavidin affinity resin (Neutravidin). Input samples and proteins eluted by boiling in SDS–PAGE were subjected to Western blotting for PDGFRβ. Data are representative of two experiments with the same outcome from two different gingival fibroblast donors. f PXS-S1C and BAPN did not inhibit serum-stimulated proliferative response of HSC3 tumor cells. HSC3 cells were serum-depleted overnight and treated with PXS-S1C (1 µM) or BAPN (0.5 mM) in medium containing 2.5% serum for serum stimulation of a proliferative response. Data are means ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparisons * p < 0.05 indicates difference among the groups. g PXS-S1C decreased the expression of LOXL2 in HSC3 cells in vitro. Relative LOXL2 mRNA levels in HSC3 cell line with and without PXS-S1C after 24 h treatment was measured. Data are means ± SEM. This experiment was done three times independently with triplicate samples. ANOVA, p : 0.04, Sidak’s multiple comparisons test * p < 0.05 indicates difference from non-treated HSC3 group. The RNA levels were normalized to 18S rRNA

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Phospho-proteomics, Western Blot, Isolation, Comparison, Knockdown, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, CyQUANT Assay, Inhibition, Pull Down Assay, SDS Page, Expressing, In Vitro

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: LOXL2 secreted by tumor cells ( a ) oxidizes lysine residues on PDGFRβ in proximal fibroblasts ( b ), in addition, to its classical role in collagen maturation (not shown). PDGF-AB secreted by tumor cells is consequently able to more efficiently stimulate PDGF signaling ( c ), resulting in increased ERK1/2 activation and cell proliferation ( d ). LOXL2 production by tumor cells is required for maintaining LOXL2 synthesis is a feed-forward pathway ( e ), whose mechanism remains to be determined

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Activation Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

doi: 10.1007/s00018-017-2681-z

Figure Lengend Snippet: CRT0066101 suppresses proliferation, anchorage-independent growth, and migration of bladder carcinoma cells in vitro. a, b Bladder cancer cells (T24, T24T, TCCSUP, and UMUC1) were treated with CRT0066101 at the indicated concentrations. Cell viability was determined at days 0, 2, and 4 by the MTT assay (a). Dose–response effect of CRT0066101 at 4-day treatment (b). Cell viability values are expressed relative to those for cells with no drug exposure. The IC50 values for CRT0066101 at day 4 in the four cell lines were calculated using GraphPad Prism version 6.01 software. c Differential effect of CRT0066101 on cell survival between human uroepithelial cells and bladder tumor cells as determined by using a Celigo image cytometer. The four bladder cancer cell lines and the human uroepithelial cell line SV-HUC were incubated with CRT0066101 at 5 μM for 6 days. Cell viability was simultaneously stained with a mixture of calcein AM, propidium iodide, and Hoechst 33342 reagents for respective staining of live, dead, and all cells, and the percentage of viable cells was quantified with the Celigo imaging cytometer. *P < 0.01 versus SV-HUC cells. d CRT0066101 abrogates bladder carcinoma cell growth in low attachment (GILA). The four bladder cancer cell lines were distributed in a 96-well flat bottom ultra-low attachment microplate and treated with CRT0066101 at the indicated concentrations. After 5 days of incubation, cell viability was measured as described in “Materials and methods”. NC no cells. *P < 0.01 and **P < 0.05 vs. the untreated control group. e, f CRT0066101 reduces the invasive ability of bladder cancer cells. A two-chamber cell invasion assay was used for assessing the effect of CRT0066101 on the invasion capability of bladder carcinoma cells. TCCSUP cells (e) and UMUC1 cells (f) were treated with CRT0066101 at the indicated concentrations, and cell migration/invasion was then assessed after 2 days of incubation at 37 °C in the chamber, as described in “Materials and methods”. The numeric data for the two cell lines are shown in the two column graphs, respectively. *P < 0.01 and **P < 0.05 vs. the untreated control group

Article Snippet: The human bladder carcinoma cell lines SCaBER, 5637, T24, UMUC3, and TCCSUP, and the SV40-transformed human uroepithelial cell line SV-HUC were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, In Vitro, MTT Assay, Software, Cytometry, Incubation, Staining, Imaging, Control, Invasion Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

doi: 10.1007/s00018-017-2681-z

Figure Lengend Snippet: Immunoblot analysis of PKD1, PKD2, and PKD3 protein levels in human urothelial bladder carcinoma cell lines. Four bladder cancer cell lines (SCaBER, UMUC5, UMUC1, and 5637) were used for analysis of PKD1 protein expression, while 8 bladder tumor cell lines (TCCSUP, T24T, T24, UMUC3, SCaBER, UMUC5, UMUC1, and 5637) were utilized for analysis of PKD2 and PKD3 protein levels. Total cellular protein was extracted from these cell lines, and a total of 80 μg cell extract protein was subjected to SDS-PAGE and immunoblotted with an antibody against PKD1, PKD2, or PKD3, as described in Materials and Methods. β-Actin was used as the control to verify equal protein loading and transfer

Article Snippet: The human bladder carcinoma cell lines SCaBER, 5637, T24, UMUC3, and TCCSUP, and the SV40-transformed human uroepithelial cell line SV-HUC were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Expressing, SDS Page, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

doi: 10.1007/s00018-017-2681-z

Figure Lengend Snippet: Knockdown of PKD2 expression decreases proliferation, anchorage-independent growth, and invasion of bladder carcinoma cells in vitro. a Six cell lines (T24, T24T, TCCSUP, UMUC1, UMUC3, and UMUC5) were transduced with lentivirus coding for human shRNA sequences for PKD2 (shPKD2#1 and shPKD2#2) or for the control (shNTC). Cell growth and proliferation were assessed by MTT assay at days 0, 2, 4, and 6 after cell plating. P < 0.01 for the shPKD2 groups vs. the control shNTC group at day 6 in all the six cell lines tested. b Depletion of PKD2 abrogates bladder cancer cell growth in low attachment. T24T and UMUC1 cell lines were transduced with lentivirus coding for human shRNA sequences for PKD2 (shPKD2#1 and shPKD2#2) or for the control (shNTC). Cell growth in low attachment was assessed by GILA assay at day 5 after cell plating, as described in “Materials and methods”. *P < 0.01 and **P < 0.05 for the shPKD2 groups vs. the control shNTC group. c, d Knockdown of PKD2 expression compromises the invasive ability of bladder carcinoma cells. TCCSUP cells (c) and UMUC1 cells (d) were transduced with lentivirus coding for human shRNA sequences for PKD2 (shPKD2#1 and shPKD2#2) or for the control (shNTC). A two-chamber cell invasion system was set up, as described in “Materials and methods”, and the number of invading cells was counted in ten random microscopic fields (×40). The numeric data for the two cell lines are shown in the two column graphs, respectively. Values are expressed as mean ± SD. **P < 0.05 and *P < 0.01 for the shPKD2 groups vs. the shNTC group in TCCSUP and UMUC1 cells, respectively

Article Snippet: The human bladder carcinoma cell lines SCaBER, 5637, T24, UMUC3, and TCCSUP, and the SV40-transformed human uroepithelial cell line SV-HUC were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Expressing, In Vitro, Transduction, shRNA, Control, MTT Assay

Journal: Molecular cell

Article Title: mRNA location and translation rate determine protein targeting to dual destinations.

doi: 10.1016/j.molcel.2023.06.036

Figure Lengend Snippet: Figure 6. NET1 mRNA location robustly affects NET1 function in cell migration and controls TGF-b-induced NET1 activity (A) FLIM imaging of a RhoA activity biosensor in MDA-MB-231 cells treated with the indicated PMOs or siRNAs. Graph shows FRET efficiency in protrusive regions. n = 32–38 cells from 2 independent experiments. (B) Measurement of focal adhesion size based on paxillin immunofluorescence staining of cells treated with the indicated PMOs or siRNAs. Numbers within each bar indicate the number of focal adhesions measured in 3 independent experiments. (C) Average migration speeds measured by timelapse imaging of mCherry-NLS expressing cells treated with the indicated PMOs or siRNAs. n = 57–78 cells from 3 independent experiments. (D) Phase contrast images of MCF7 cells with or without TGF-b treatment.

Article Snippet: To generate the RhoA FRET biosensor, the RhoA biosensor fragment was PCR amplified from the pLentiRhoA2G plasmid (Addgene #40179) to add BamHI and XhoI sites at the ends.

Techniques: Migration, Activity Assay, Imaging, Staining, Expressing

Journal: Molecular cell

Article Title: mRNA location and translation rate determine protein targeting to dual destinations.

doi: 10.1016/j.molcel.2023.06.036

Figure Lengend Snippet: Figure 7. Proposed model Binding of NET1 to importins or to CASK and NET1 targeting to the nucleus or cytoplasm are determined by the location of the NET1 mRNA and the rate of its translation. Perinuclear mRNA and/or slower translation rate favor interaction of the N-terminal NLSs with import receptors and targeting to the nucleus. Peripheral mRNA and/or a faster translation rate favor CASK binding through the PH domain to competitively retain NET1 in the cytoplasm. Cytoplasmic NET1 can activate RhoA and promote cell adhesion and migration.

Article Snippet: To generate the RhoA FRET biosensor, the RhoA biosensor fragment was PCR amplified from the pLentiRhoA2G plasmid (Addgene #40179) to add BamHI and XhoI sites at the ends.

Techniques: Binding Assay, Migration

Journal: The EMBO Journal

Article Title: Immunity against Moraxella catarrhalis requires guanylate‐binding proteins and caspase‐11‐ NLRP3 inflammasomes

doi: 10.15252/embj.2022112558

Figure Lengend Snippet: Brightfield microscopy analysis of WT and mutant BMDMs left untreated (Med.) or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100). Immunoblot analysis of phospho‐IkB (pIkB), IkB, phospho‐ERK (pERK), ERK, caspase‐11 (Casp‐11) and GAPDH (loading control) of WT and Casp11 −/− BMDMs 0–60 min after infection with M. catarrhalis (Ne11 strain, MOI 50). Immunoblot analysis of caspase‐1 (Casp‐1), Casp‐11 and gasdermin D (GSDMD) in WT BMDMs left untreated (Med.) or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100), 30 min after treatment with LPS + ATP (5 mM) or 5 h after transfection with 5 μg of Escherichia coli LPS in the absence (−) or presence (+) of the NLRP3 inhibitor MCC950 (20 μM). Release of IL‐1β, IL‐18 and LDH from BMDMs after treatment as in (C). Release of KC and IL‐6 in WT and Casp11 −/− BMDMs left untreated (Med.) or infected as in (A). qRT–PCR analysis of the genes encoding Casp‐11, KC ( Cxcl1 ), IL‐1β, IL‐18, IL‐6 and TNF in WT and Casp11 −/− BMDMs left untreated or assessed 4 h after infection with M. catarrhalis (Ne11 strain, MOI 50), relative to Gapdh . Data information: Arrowheads indicate dead cells (A). Each symbol represents an independent biological replicate (D–F). NS, no statistical significance; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (two‐tailed t ‐test (D–F)). Data are from one experiment representative of three independent experiments (A–C) or are pooled from three independent experiments (D–F; mean and s.e.m. in D–F). Scale bar, 20 μm (A).

Article Snippet: The BL21(DE3) E. coli strain (C2527H, NEB) was transformed with pET‐28a(+)‐TEV plasmid containing the sequence for mGBP2 and transformants were selected with 50 μg/ml kanamycin (10106801001, Roche).

Techniques: Microscopy, Mutagenesis, Infection, Western Blot, Control, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: The EMBO Journal

Article Title: Immunity against Moraxella catarrhalis requires guanylate‐binding proteins and caspase‐11‐ NLRP3 inflammasomes

doi: 10.15252/embj.2022112558

Figure Lengend Snippet: Immunoblot analysis of caspase‐1 (Casp‐1), caspase‐11 (Casp‐11) and gasdermin D (GSDMD) in WT and Casp11 −/− BMDMs left untreated (Med.) or assessed 10 h after infection with 12 strains of M. catarrhalis (MOI 50), or 30 min after treatment with LPS + ATP (5 mM). Release of IL‐1β, IL‐18, TNF and LDH from BMDMs after treatment as in (A). Release of IL‐1β, IL‐18 and LDH from WT and Casp4 −/− THP‐1 macrophage‐like cells left untreated (Med.), assessed 6 h after infection with M. catarrhalis (Ne11, MOI 50), following overnight transfection with 5 μg of Escherichia coli LPS or after Pam3CSK4 priming with overnight transfection with 5 μg of poly(dA:dT). IncuCyte live‐imaging analysis of WT and Casp4 −/− THP‐1 macrophage‐like cell viability assessed after treatment as in (C). Data information: Each symbol represents an independent biological replicate (B and C). NS, no statistical significance; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (two‐tailed t ‐test (B–D)). Data are from one experiment representative three independent experiments (A) or are pooled from three independent experiments (B–D; mean and s.e.m. in B–D).

Article Snippet: The BL21(DE3) E. coli strain (C2527H, NEB) was transformed with pET‐28a(+)‐TEV plasmid containing the sequence for mGBP2 and transformants were selected with 50 μg/ml kanamycin (10106801001, Roche).

Techniques: Western Blot, Infection, Transfection, Imaging, Two Tailed Test

Journal: The EMBO Journal

Article Title: Immunity against Moraxella catarrhalis requires guanylate‐binding proteins and caspase‐11‐ NLRP3 inflammasomes

doi: 10.15252/embj.2022112558

Figure Lengend Snippet: A Immunoblot analysis of caspase‐1 (Casp‐1), caspase‐11 (Casp‐11) and gasdermin D (GSDMD) in WT, Nlrp3 −/− , Casp11 −/− and Aim2 −/− BMDMs left untreated (Med.) or 5 h after transfection with 5 μg of LOS from M. catarrhalis (O35E) or 5 μg of LPS from Escherichia coli . B Release of IL‐1β, IL‐18, TNF and LDH from BMDMs after treatment as in (A). C Immunoblot analysis of Casp‐1, Casp‐11 and GSDMD in WT, Nlrp3 −/− and Casp11 −/− BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (O35E and Δ lpxA , MOI 100). D Release of IL‐1β, IL‐18, TNF and LDH from BMDMs after treatment as in (C). E Immunoblot analysis of Casp‐1, Casp‐11 and GSDMD in WT, Nlrp3 −/− and Casp11 −/− BMDMs left untreated (Med.) or assessed 10 h after incubation with 10 μg of OMVs purified from M. catarrhalis (O35E and Δ lpxA ). F Release of IL‐1β, IL‐18, TNF and LDH from BMDMs after treatment as in (E). G Silver‐stained SDS–PAGE of LOS purified from M. catarrhalis (O35E and Δ lpxA ). H, I Transmission electron microscopy of negative stained OMVs purified from M. catarrhalis (O35E and Δ lpxA ). Data information: Each symbol represents an independent biological replicate (B, D, F). NS, no statistical significance; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (one‐way ANOVA with Dunnett's multiple‐comparisons test (B, D, F)). Data are from one experiment representative of two (G–I) or three independent experiments (A, C, E) or are pooled from three independent experiments (B, D, F; mean and s.e.m. in B, D, F). Scale bars, 100 nm (H and I). Source data are available online for this figure.

Article Snippet: The BL21(DE3) E. coli strain (C2527H, NEB) was transformed with pET‐28a(+)‐TEV plasmid containing the sequence for mGBP2 and transformants were selected with 50 μg/ml kanamycin (10106801001, Roche).

Techniques: Western Blot, Transfection, Infection, Incubation, Purification, Staining, SDS Page, Transmission Assay, Electron Microscopy

Journal: The EMBO Journal

Article Title: Immunity against Moraxella catarrhalis requires guanylate‐binding proteins and caspase‐11‐ NLRP3 inflammasomes

doi: 10.15252/embj.2022112558

Figure Lengend Snippet: Confocal microscopy analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20). Quantitation of GBP1‐, GBP2‐, GBP3‐, GBP5‐ and GBP7‐positive M. catarrhalis in WT BMDMs as treated in (A). Confocal microscopy analysis of FLAG‐OVA, FLAG‐GBP1, FLAG‐GBP2, FLAG‐GBP3, FLAG‐GBP5, FLAG‐GBP7 (red) and M. catarrhalis (green) in LA‐4 cells left untreated (Med.), or 16 h after infection with M. catarrhalis (Ne11, MOI 20) primed with IFN‐γ (100 U/ml). Immunoblot analysis of caspase‐1 (Casp‐1), caspase‐11 (Casp‐11) and gasdermin D (GSDMD) in WT, Gbp chr3 ‐KO and Casp11 −/− BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 μg LPS from Escherichia coli . Release of IL‐1β, IL‐18, TNF and LDH from BMDMs after treatment as in (D). Recovery of M. catarrhalis (as colony‐forming units (CFU)) from primary WT, Gbp chr3 ‐KO and Ifnar1 −/− lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100). Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbp chr3 ‐KO and Ifnar1 −/− BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Data information: Arrowheads indicate bacteria colocalised with GBPs (A and C). Each symbol represents an independent biological replicate (E and F). NS, no statistical significance; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (one‐way ANOVA with Dunnett's multiple‐comparisons test (E–G)). To quantify the bacterial number in a field of view, we used the single green channel (anti‐ M. catarrhalis staining), where a single bacterium is defined as an intact circle. To quantify the proportion of GBP‐positive bacteria in a field of view, we used the single red channel (anti‐GBP staining) and compared this to the green channel (anti‐ M. catarrhalis staining). The number of GBP‐positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP‐positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP‐positive bacteria per cell using confocal microscopy (B) or the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) or three independent experiments (A, C, D) or are pooled from two (B) or three independent experiments (E, F; mean and s.e.m. in B, E–G). Scale bars, 5 μm (A, C). Source data are available online for this figure.

Article Snippet: The BL21(DE3) E. coli strain (C2527H, NEB) was transformed with pET‐28a(+)‐TEV plasmid containing the sequence for mGBP2 and transformants were selected with 50 μg/ml kanamycin (10106801001, Roche).

Techniques: Confocal Microscopy, Infection, Quantitation Assay, Western Blot, Transfection, Bacteria, Transmission Assay, Electron Microscopy, Staining

Journal: The EMBO Journal

Article Title: Immunity against Moraxella catarrhalis requires guanylate‐binding proteins and caspase‐11‐ NLRP3 inflammasomes

doi: 10.15252/embj.2022112558

Figure Lengend Snippet: Immunoblot analysis of caspase‐1 (Casp‐1), caspase‐11 (Casp‐11) and gasdermin D (GSDMD) in WT, Gbp Chr3 ‐KO and Casp11 −/− BMDMs left untreated (Med.) or 10 h after transfection with 5 μg of LPS from Escherichia coli or 5 μg of LOS from M. catarrhalis (O35E). Release of IL‐1β, IL‐18, TNF and LDH from BMDMs after treatment as in (A). Confocal microscopy analysis of FLAG‐OVA, FLAG‐GBP1, FLAG‐GBP2, FLAG‐GBP3, FLAG‐GBP5 (red) and M. catarrhalis (green) in LA‐4 cells left untreated (Med.), or 16 h after infection of IFN‐γ‐primed (100 U/ml) LA‐4 cells with M. catarrhalis (O35E or Δ lpxA , MOI 20). Quantitation of FLAG‐OVA, FLAG‐GBP1, FLAG‐GBP2, FLAG‐GBP3 and FLAG‐GBP5 positive M. catarrhalis in LA‐4 cells treated as in (C). Data information: Arrowheads indicate bacteria colocalised with GBPs (C). Each symbol represents an independent biological replicate (B). NS, no statistical significance (one‐way ANOVA with Dunnett's multiple‐comparisons test (B and D)). To quantify the bacterial number in a field of view, we used the single green channel (anti‐ M. catarrhalis staining), where a single bacterium is defined as an intact circle. To quantify the proportion of GBP‐positive bacteria in a field of view, we used the single red channel (anti‐GBP staining) and compared this to the green channel (anti‐ M. catarrhalis staining). The number of GBP‐positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP‐positive bacteria. A total of 100 lung epithelial cells (LA‐4) were analysed to quantify the proportion of GBP‐positive bacteria per cell using confocal microscopy (D). Data are from one experiment representative of two (C) or three independent experiments (A) or are pooled from two (D) or three independent experiments (B; mean and s.e.m. in B and D). Scale bars, 4 μm (C).

Article Snippet: The BL21(DE3) E. coli strain (C2527H, NEB) was transformed with pET‐28a(+)‐TEV plasmid containing the sequence for mGBP2 and transformants were selected with 50 μg/ml kanamycin (10106801001, Roche).

Techniques: Western Blot, Transfection, Confocal Microscopy, Infection, Quantitation Assay, Bacteria, Staining

Journal: The EMBO Journal

Article Title: Immunity against Moraxella catarrhalis requires guanylate‐binding proteins and caspase‐11‐ NLRP3 inflammasomes

doi: 10.15252/embj.2022112558

Figure Lengend Snippet: Immunoblot analysis of caspase‐1 (Casp‐1), caspase‐11 (Casp‐11) and gasdermin D (GSDMD) in WT, Gbp1 −/− , Gbp2 −/− , Gbp3 −/− , Gbp5 −/− , Gbp7 −/− , Gbp4/8/9 −/− , Gbp11 −/− and Casp11 −/− BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 μg LPS from Escherichia coli . Release of IL‐1β, IL‐18, LDH, TNF, IL‐6 and KC from BMDMs after treatment as in (A). Brightfield microscopy analysis of WT and mutant BMDMs after treatment as in (A). Data information: Arrowheads indicate dead cells (C). Each symbol represents an independent biological replicate (B). NS, no statistical significance; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (one‐way ANOVA with Dunnett's multiple‐comparisons test (B)). Data are from one experiment, representative of three independent experiments (A and C) or are pooled from three independent experiments (B; mean and s.e.m. in B). Scale bars, 20 μm (C). Source data are available online for this figure.

Article Snippet: The BL21(DE3) E. coli strain (C2527H, NEB) was transformed with pET‐28a(+)‐TEV plasmid containing the sequence for mGBP2 and transformants were selected with 50 μg/ml kanamycin (10106801001, Roche).

Techniques: Western Blot, Infection, Transfection, Microscopy, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 1. Comparison of protein expression of GDNF and GDNF receptor complex between murine cerebral cortex and cultured cortical neurons and astrocytes. Total RNAs of murine astrocytic (Ast) and neuronal (Neur) cortical cultures and murine cerebral cortex (Cx) were extracted and reverse transcribed using a poly-dT primer. A, D, G, PCR amplification was performed then using a specific set of primers for GDNF (A), GFRa-1 (D), and c-Ret (G). B, E, H, Western blot was performed on a lysate derived from murine cerebral cortex, neurons, or astrocyte cultures showing GDNF (B) and GDNF receptor expression (E, H). C, F, I, Immunocytochemistry analysis of GDNF and GDNF receptor components in cultured astrocytes and neurons. Bright-field photomicrographs of either pure cortical astrocytes or pure cortical neu- rons in culture, after fixation and peroxidase staining for antibodies raised against GDNF (C), GFRa-1 (F), and c-Ret (I).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Comparison, Expressing, Cell Culture, Reverse Transcription, Western Blot, Derivative Assay, Immunocytochemistry, Staining

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 3. rhGDNF does not protect neurons against apoptotic cell death. To induce apoptosis, near pure cortical neuronal cultures (DIV 7) were transferred to a serum-deficient medium (SD). A, B, 24 hr after serum deprivation, the neurons were fixed and stained with DAPI to observe DNA condensation (A) or the DNA of cortical neurons was extracted and loaded onto an agarose gel to evidence DNA fragmentation (B). C, D, Neuronal death percentage was assessed after 24 hr by trypan blue dye staining (mean 6 SEM; n 5 12) after SD (C) or by LDH release (mean 6 SEM; n 5 12) after exposure of mixed cortical cultures (14 DIV) to staurosporine 200 nM (Stp) (D). SD and Stp exposure were performed in the presence ( gray bars) or absence (white bars) of rhGDNF and with or without 1 mg/ml cycloheximide (CHX). In both apoptotic paradigms, MK-801 was systematically added to block the secondary activation of NMDA receptors. * indicates significantly different from SD or STP by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Staining, Agarose Gel Electrophoresis, Blocking Assay, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 2. GDNF induces a phosphorylation of ERKs. Mouse primary cortical neurons (A) or astrocytes (B) were treated with rhGDNF (10 ng/ml) for the indicated times. Aliquots of whole-cell lysates were sepa- rated by SDS-PAGE, transferred to membranes, and incubated with the anti-p-ERKs antibody or anti-total ERK antibody. The immunoblots are representative of three independent experiments.

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Phospho-proteomics, SDS Page, Incubation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 4. rhGDNF is neuroprotective against NMDA-induced neuronal death. A, Cultures were exposed for 24 hr to AMPA (10 mM) or kainate (50 mM) (with 10 mM MK-801 added to block secondary NMDA receptor- mediated toxicity), without (white bars) or with ( gray bars) 10 ng/ml rhGDNF. Medium LDH was assessed at the end of the exposure to excitotoxins (mean 6 SEM; n 5 10 cultures per condition). B, C, Mixed neuron–glia cortical cultures (B) or near pure cortical neuron cultures (C) were exposed for 24 hr to NMDA (12.5 mM) without (white bars) or with ( gray bars) increasing concentration of rhGDNF or MK-801 (10 mM). Medium LDH was assessed at the end of the exposure to excitotoxin (mean 6 SEM; n 5 12 cultures per condition. * indicates significantly different from NMDA; # indicates significantly different from control (sham wash) by ANOVA, followed by Bonferronni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Blocking Assay, Concentration Assay, Control

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 6. GDNF does not protect against neuronal death induced by reactive oxygen species. Mixed neuron–glia cortical cultures were ex- posed for 24 hr to FeCl2 (35 mM) without (white bars) or with ( gray bars) 10 ng/ml rhGDNF and with or without Trolox (100 mM). Medium LDH was assessed at the end of FeCl2 exposure (mean 6 SEM; n 5 12 cultures per condition). * indicates significantly different from FeCl2 by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 5. A GPI-linked protein mediates the neuroprotective activity of rhGDNF. A, Phase-contrast photomicrographs of mixed neuron–glia cortical cultures pretreated or not with PIPLC (0.3 IU/ml for 2 hr at 37°C) after NMDA application (12.5 mM) in the presence or absence of rh- GDNF. Top left, Sham-washed cultures; top right, NMDA-treated cells; bottom left, NMDA-treated cells coincubated with rhGDNF (10 ng/ml); bottom right, cells pretreated with PIPLC and incubated with NMDA and rhGDNF. B, Neuronal death percentage was estimated by LDH release (mean 6 SEM; n 5 12) after a 24 hr exposure to NMDA (12.5 mM) in mixed neuron–glia cortical cultures in the presence ( gray bars) or absence (white bars) of rhGDNF and pretreated or not pretreated with 0.3 IU/ml PIPLC for 2 hr at 37°C. * indicates significantly different from NMDA; # indicates significantly different from NMDA plus GDNF by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Activity Assay, Incubation

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 8. The MEK kinase inhibitor U0126 blocks the rhGDNF- induced reduction of NMDA-evoked calcium influx. A, Mouse primary cortical neurons were treated for 15 min with rhGDNF (10 ng/ml) in the presence of U0126 (1 mM) and with or without NMDA (12.5 mM). Aliquots of whole-cell lysates were separated by SDS-PAGE, transferred to membranes, and incubated with the anti-MAPK antibody ( p-ERKs, ERKs). Representative immunoblots are shown, demonstrating the spe- cific blockade of ERKs phosphorylation by U0126 treatment. B, Neuronal death percentage was estimated by LDH release (mean 6 SEM; n 5 12) after a 24 hr exposure to NMDA (12.5 mM) in mixed neuron–glia cortical cultures in the presence ( gray bars) or absence (white bars) of rhGDNF and treated or not treated with 1 mM U0126. * indicates significantly different from NMDA alone; # indicates significantly different from NMDA plus GDNF by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05). C, Calcium imaging was performed as described in the legend to Figure 7. Neurons (13–14 DIV) were exposed to serum-free medium for 45 min in the absence (control; vehicle treated; white bars) or presence ( gray bars) of rhGDNF (10 ng/ml) and with (hatched bars) or without (unhatched bars) U0126 (1 mM). Time course of the effect of rhGDNF on the NMDA-induced [Ca 21] increase, with or without treatment with 1 mM of U0126. Data are expressed as an NMDA-induced fold [Ca 21]i. Results represent the mean 6 SEM of three separate preparations (3 coverslips; 20 neurons being imaged per coverslip in a single microscopic field). * indicates significantly different from NMDA; # indicates signif- icantly different from NMDA plus GDNF by ANOVA for repeated measures followed by a Bonferroni–Dunn’s test for multiple comparisons ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: SDS Page, Incubation, Western Blot, Phospho-proteomics, Imaging, Control

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 7. rhGDNF pretreatment reduces the NMDA-evoked calcium increase in cortical neurons. A, Intracellular free Ca 21 ([Ca 21]i) was measured using fura-2 fluorescence videomicroscopy. Neurons (13–14 DIV) were exposed to a serum-free medium for 45 min in the absence (control, vehicle treated) or presence of rhGDNF (10 ng/ml). Resting [Ca 21 ]i was recorded, and NMDA (100 mM) was applied. [Ca 21]i was measured and analyzed as described in Materials and Methods. B, Rep- resentative graph of NMDA-induced Ca 21 response in neurons pre- treated with rhGDNF ( gray bars) or vehicle (control; white bars) for 45 min. Time course of the effect of rhGDNF on the NMDA-induced [Ca 21] increase, with (hatched bars) or without (unhatched bars) pretreat- ment with 0.3 IU/ml PIPLC for 2 hr. Data are expressed as an NMDA- induced fold increase in [Ca 21]i. Results represent the mean 6 SEM of three separate preparations (3 coverslips; 20 neurons being imaged per coverslip in a single microscopic field). * indicates significantly different from NMDA; # indicates significantly different from NMDA plus GDNF by ANOVA for repeated measures, followed by a Bonferroni–Dunn’s test for multiple comparisons ( p , 0.05). C, Neurons (13–14 DIV) were exposed to serum-free medium containing MK-801 (10 mM) for 45 min in the absence (control; vehicle treated) or presence of rhGDNF (10 ng/ml). Resting [Ca 21 ]i was recorded, and KCl (50 mM) was applied. Represen- tative graph of KCl-induced Ca 21 response in neurons pretreated with rhGDNF or vehicle (control) for 45 min.

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Control

Journal: eLife

Article Title: Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models

doi: 10.7554/eLife.45457

Figure Lengend Snippet:

Article Snippet: Peptide, Recombinant Protein , Tau-441(A152T) Protein , SignalChem , Cat. T08-56VN Lot. B2157-7 , Human recombinant protein expressed in E. coli cells, tag-free. Accession no. P10636-8..

Techniques: CRISPR, Knock-Out, Western Blot, Staining, Recombinant, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Lysis, Immunoprecipitation, Protease Inhibitor, Software, Imaging, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress

doi: 10.1093/nar/gku505

Figure Lengend Snippet: RPA binding to DNA is directly affected by PAR. ( A ) Anti-PAR antibodies co-immunoprecipitated RPA2 and RPA1 subunits of RPA at higher levels from HU-treated shPARG cells compared to shCTL cells. Cell extracts from shCTL (C) and shPARG (P) cells treated with 2 mM HU for 24 h were immunoprecipitated with anti-PAR antibodies. The presence of PARP-1, RPA1 and RPA2 in the anti-PAR immunoprecipitates (IP PAR, lanes 3 and 4), in the control immunoprecipitates using rabbit anti-mouse antibody (IP IgG, lanes 1 and 2) and in the input (lanes 5 and 6, corresponding to 5% of the lysate) was assessed by western blotting using the indicated antibodies. The asterisk points to a non-specific band. ( B ) PARP-1 PARylated by RPA mostly on RPA1 subunit. The trimeric RPA (2.5 μg, lanes 2, 3, 5 and 6) was incubated with 0 (lanes 2 and 5) or 500 ng of PARP-1 (lanes 3 and 6) for 30 min at 25°C in the presence of 32 P-NAD + and DNAse I-activated calf thymus DNA. As negative control, PARP-1 was incubated with GST (2 μg) (lanes 4 and 7). Reaction products were analysed by 8–20% SDS-PAGE and autoradiography (right panel) of the Coomassie blue-stained and dried gel (left panel). Lane 1: molecular weight marker. ( C ) RPA binds non-covalently to PAR. RPA complex (2.5 μg, lanes 2 and 6), GST (2 μg, lanes 3 and 7), H2A and H3 (1 μg each, lanes 4 and 8) and H2B, H4 (1 μg each, lane 5 and 9) were separated on 8–20% SDS–PAGE and either stained with Coomassie blue (left panel) or blotted onto nitrocellulose membrane, renaturated and incubated with free PAR. Bound PAR was detected by immunodetection using anti-PAR antibody (right panel, PAR-blot). Lane 1: molecular weight marker. ( D ) PARylated proteins are co-immunoprecipitated with RPA2 in HU-treated shPARG cells. Cell extracts from shCTL (C) and shPARG (P) cells treated with 2 mM HU for 24 h were immunoprecipitated with anti-RPA2 antibodies as described in “ Material and Methods ” section. The presence of PARylated proteins, RPA1 and RPA2 in the anti-RPA2 immunoprecipitates (IP RPA2, lanes 3 and 4), the control immunoprecipitates using mouse anti-HA antibody (IP IgG, lanes 1 and 2) and the input (lanes 5 and 6, corresponding to 5% of the lysates) was assessed by western blotting using the indicated antibodies. The asterisk points to non-specific bands. ( E and F ) EMSA analyses showing that RPA binding to ssDNA is favored by low levels but counteracted by high levels of PAR. ( E ) PAR competes with DNA for RPA. RPA (1.7 nM) was incubated with 1 nM of a Cy5.5-oligodT probe (50-mer) and increasing concentrations of PAR (lanes 3–11: from 8 ng in lane 3, doubled each point to 256 ng in lane 12) for 30 min at RT, complexes were separated by 1% native agarose gel electrophoresis and observed at 680 nm on Odyssey Infrared Imaging System. Lane 1: DNA only; lane 2: no PAR. F . High PAR levels dislodge RPA from DNA. RPA (7 nM) was pre-incubated with 2.5 nM of a Cy5.5-oligodT probe for 15 min before the addition of increasing concentrations of PAR (from lane 3 to 11: 4, 8, 16, 32, 48, 64, 96, 128, 192 ng, respectively) and incubation for 30 min at 23°C. Complexes were separated by 1% native agarose gel electrophoresis and observed at 680 nm on Odyssey Infrared Imaging System. Lane 1: DNA only; lane 2: no PAR.

Article Snippet: After washings, membranes were incubated for 1 h with the appropriate secondary antibodies: an Alexa Fluor 680 goat anti-rabbit or goat anti-mouse Alexa Fluor 680 (1:20 000, Invitrogen) or a Donkey anti-mouse IRDye800 (1:10 000, Li-Cor, Bioscience) and revealed on Odyssey Infrared Imaging System (Li-Cor, Bioscience).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Incubation, Negative Control, SDS Page, Autoradiography, Staining, Molecular Weight, Marker, Immunodetection, Agarose Gel Electrophoresis, Imaging