Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Expressing, Construct

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: RNA Sequencing, Comparison

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Expressing

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Isolation, Marker, Injection, Dissection

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Functional Assay, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a – d ) Sensitivity of WT and Rnf168 −/− MEFs to ICRF-193 ( a , b ) or etoposide ( c , d ) was determined using clonogenic assays. ( e , f ) Sensitivity of the human breast cancer cell lines T47D and MDA-MB-231 to etoposide was determined using clonogenic assays. ( a , c , e ) Data are presented as the mean±s.e.m. ( a , c , n >4; e , n =4). * P <0.05 for Rnf168 −/− MEFs compared with WT MEFs and T47D and MDA-MB-231 cells knocked down for RNF168 (sh.RNF168) compared with their respective controls (Sh.Ctr: ShRNA control). ( b , d ) Representative pictures of dishes showing surviving WT and Rnf168 −/− colonies post ICRF-193 ( b ) or etoposide ( d ) treatment. ( f ) Representative pictures of dishes showing surviving colonies of etoposide treated T47D and MDA-MB-231 cells.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: shRNA, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 1 PARL over-expression leads to increased processing of Pink1-66. (a) Schematic representation of human Pink1. The predicted matrix targeting sequence (MTS), the transmembrane domain (TMD), the kinase domain and the putative PARL processing site are indicated. Comparison of the TMDs of human (Hs) and D. melanog- aster (Dm) Pink1 using the EMBOSS pair- wise alignment algorithm reveals significant sequence conservation. The hydrophobicity plot of the relevant region is shown [using the scale of Kyte and Doolittle (1982), with a window size of 7] indicating the potential TMD boundaries. (b) Co-expression of hu- man Pink1 with PARL leads to an increased processing of the full-length 66 kDa form (open triangle). A catalytically inactive PARL mutant (SA) shows no activity. Subcellular fractionation reveals that Pink1-66 and the processed Pink-55 (filled triangle) are found in mitochondria (m) and also the non-mito- chondrial soluble fraction (c). IRES GFP and the cellular markers VDAC, AIF and actin were used as transfection and loading con- trol. t, total cell extract (10% loaded). (c) Quantification of Pink1 66/55 kDa ratio in the mitochondrial fraction upon PARL wt and SA over-expression (n = 3, means ± SEM). Significant changes versus mock are indi- cated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). (d) Titration of PARL cDNA significantly increases processing of Pink1-66 (n = 3, means ± SEM). Significant changes versus mock are indicated (*p < 0.05; One-way ANOVA with Bonferroni’s post-test).

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Over Expression, Sequencing, Comparison, Expressing, Mutagenesis, Activity Assay, Fractionation, Transfection, Titration

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 2 PARL is required for Pink1 processing. (a) Left panel: PARL knockdown by two non-overlapping siRNAs (PARL-1 and -2) blocks Pink1-66 processing and reduces the level of Pink1-55, when com- pared to a non-targeting siRNA (nt). Other proteins like VDAC, AIF, actin and IRES GFP are not affected. t, total cell extracts; m, mito- chondrial pellet and c, non-mitochondrial fraction. Right panel: Quan- tification of the Pink1 66/55 kDa ratio in the mitochondrial fractions upon knockdown of endogenous PARL by siRNAs (means ± SEM, n = 3). Significant changes versus mock are indicated (**p < 0.01; One-way ANOVA with Bonferroni’s post-test). Right bottom panel: PARL knockdown was monitored in the mitochondrial fraction. (b) Left panel: Doxycyclin-induced expression of PARL-specific shRNA in Hek293- shRNA cells for 6 days diminishes Pink1-66 processing. For PARL

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Knockdown, Expressing, shRNA

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 4 Both uncoupling of the mitochondrial membrane potential and PARL ablation lead to the accumulation of Pink1-66 at the outer mitochondrial membrane. (a) Upper panel: Uncoupling of the mitochondrial membrane potential in Hek293-shRNA cells (by CCCP) leads to a Pink1 66/55 kDa ratio as observed upon PARL knockdown (shRNA). Total cell extracts (t), mitochondrial fractions (m) and non-mitochondrial soluble fractions (c) were analyzed. Lower panel: Quantification of Pink1 66/55 kDa ratio of the indicated fractions is shown (means ± SEM, n = 3). Mitochondrial Pink1 66/ 55 kDa ratio is significantly lower after PARL shRNA and CCCP treatment than after treatment either with PARL shRNA or CCCP alone (*p < 0.05; **p < 0.01; paired t-test). (b) Upper panel: Outline of the Pink1 construct with a juxtamembrane factor Xa protease cleavage site (Pink1Xa). Lower panel: Mitochondria isolated from cells expressing Pink1Xa were subjected to digestion by factor Xa protease. Factor Xa cleaves Pink1-66 (open triangle) but not Pink1-55 (filled triangle) generating a 52 kDa form (grey triangle). In mitochondria isolated from cells expressing the PARL-specific shRNA or CCCP-treated cells, the factor Xa-sensitive outer membrane form of Pink1-66 increased. Note that upon PARL knockdown Pink1Xa was cleaved by an alternative protease within the N-terminal MTS leading to a 64 kDa form (labeled with an asterisk), which is not observed upon expression of Pink1 wild type.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Membrane, shRNA, Knockdown, Construct, Isolation, Expressing, Labeling

Journal: Journal of neurochemistry

Article Title: The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking.

doi: 10.1111/j.1471-4159.2011.07253.x

Figure Lengend Snippet: Fig. 5 Pink1 TMD mutations block PARL-catalyzed processing. (a) TMD sequences of Pink1 wild type (wt), Pink1G107L, Pink1G109L, Pink1R98W and Pink1I111S are shown. Mutated residues are highlighted in bold. (b) and (c) Western analysis of Pink1 mutants expressed in Hek293-shRNA cells after doxycyclin-induced knockdown of PARL (shRNA) and CCCP treatment (total cell extracts are shown). Notably, for Pink1G107L, Pink1G109L and Pink1R98W, the Pink1-66 precursor was modified by an unknown post-translational modification leading to a 68 kDa species (labeled by an asterisk). Quantification of the Pink1 66/55 kDa ratio in the indicated fractions is shown (means ± SEM, n = 3). Pink1-55 levels are lower for all probed mutants tested. Pink1G107L and Pink1R98W processing is insensitive to PARL ablation showing highly reduced processing of these mutants by PARL.

Article Snippet: The following antibodies were used at dilutions recommended by the manufacturer: polyclonal rabbit Pink1 (BC100-494) (Novus Biologicals, Littelton, CO, USA), polyclonal rabbit voltage-dependent anion-selective channel protein (VDAC) (Pierce, Rockford, IL, USA), monoclonal mouse apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse FLAG (M2) (Sigma, St Louis, MO, USA), polyclonal rabbit PARL and a-actin (Abcam, Cambridge, MA, USA).

Techniques: Blocking Assay, Western Blot, shRNA, Knockdown, Labeling

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) In uninfected skin, rare and faint nuclear HIF-1α staining occurred in the epidermis whereas the dermis was negative for HIF-1α (A1). (B to L) In all cutaneous infections, a strong nuclear HIF-1α signal was detected in keratinocytes, predominantly of the spinal cell layer (examplified in L). Moreover, endothelial cells of dermal capillaries frequently stained positive (arrows in B1, C1, D1, E1, G, I1). Arrowheads: (B2) border zone of the positive abscess; (C2): positive neutrophils; (F): positive dermal lympho-histiocytic infiltrates; (F1): large positive nuclei of a multi-nuclear giant cell; (F2): skin lesion surrounded by a HIF-1 positive inflammatory infiltrate; (H): positive sub-corneal neutrophils (higher power in H1); (I): positive intra-epidermal infiltrate; (K): intradermal nodules containing leishmaniae. (A) Control: uninfected sample; (B–E): bioptic samples of patients suffering from infections with humanpathogenic bacteria [(B) S. aureus , (C) S. agalactiae and S. aureus , (D) A. baumanii and E. coli and (E) B. burgdorferi ], (F, G) with humanpathogenic Herpes-viruses [(F) Varicella zoster virus (VZV) and (G) Human Herpes Virus-8 (HHV 8)], (H–J) with humanpathogenic fungi [(H+I) T. rubrum and (J) C. albicans ] or (K, L) with the protozoic pathogen L. donovani . Magnifications: all 250×, except for A1, B1, B2, C1, D1, E1, F2, I1 (all 400×), and C2, F1, H1, L (all 1,000×).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Staining, Control, Bacteria, Virus

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (A: S. aureus ATCC 33592, MOI 20, infection time: six hours; B: S. epidermidis ATCC 12228, MOI 20, infection time: six hours; C: E. coli ATCC 25992, MOI 10, infection time: four hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (D: P. aeruginosa ATCC 27853, MOI 10, infection time: four hours; E: S. agalactiae SK 43, MOI 200, infection time: five hours; F: C. albicans ATCC 90028, MOI 20, infection time: six hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Six hours upon infection, HIF-1 activation was analyzed in (A) Western blots (loading control: actin) and by determining (B) the induction of the HIF-1-dependent luciferase activity. HeLa-229 cells were transfected with a 4xHRE-luc reporter construct 24 hours before infection (triplicate means given; for details see ). Induction was determined by chemiluminescence. Bacterial induced (C) VEGF and (D) HIF-1α transcription. Total mRNA was prepared six hours upon infection, transcribed into cDNA and quantified by real-time Light-Cycler-PCR (ratio: VEGF/actin or HIF-1α/actin transcripts; triplicate means given). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant) ; positive control: DFO (200 µmol/L). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Luciferase, Activity Assay, Transfection, Construct, Negative Control, Mutagenesis, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin) of HeLa 229 cells with the following bacterial strains: (A) S. aureus 8325-4 (wt), S. aureus ermB Ω hemB 8325-4 (SCV), S. aureus SA113 (wt), S. aureus SA113 Δ ypf::ermB (reduced in lipoteichoic acid synthesis), S. aureus SA113 Δ tagO (defective in producing wall teichoic acid); (B) S. aureus ATCC 25923 (wt), S. epidermidis ATCC 12228 (wt), S. aureus Newman (wt), S. aureus Newman mAH12 (defective in producing the extracellular adherence protein EAP). Negative control: uninfected cells, positive control: DFO (200 µM). Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin). The following patient isolates were used: S. aureus patient isolate 1 (wt; S. aureus A22223 I), S. aureus patient isolate 2 (clinical SCV; S. aureus A22223 II), S. aureus patient isolate 3 (wt; S. aureus OM 1a), S. aureus patient isolate 4 (clinical SCV; S. aureus OM 1b). Negative control: uninfected cells, positive control: hypoxia. Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Control and infected HeLa-229 cells were incubated with the hypoxic marker pimonidazole hydrochloride which was visualized by using specific Cy3 labelled antibodies (for details see ). Scale bare: 40 µm. (B) pO 2 levels were quantified in the medium of control and infected cells (triplicate means given). (C) Hela-229 cells were incubated in gas permeable (gas-perm) and conventional (conv) culture dishes and HIF 1α protein was analyzed in Western blots (loading control: actin). (D) pO 2 levels in the medium of control or S. aureus infected HeLa-229 cells (means of doubles). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Control, Infection, Incubation, Marker, Western Blot, Negative Control, Mutagenesis

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) HIF 1α immunohistochemistry and (B) hematoxilin-eosin staining of renal mouse abscesses induced by intravenous infection with S. aureus seven days upon infection (following sections). HIF 1α positive nuclei are most abundant within the abscesses borders, some interstitial staining also occurs within the renal parenchyma. In addition, renal tubules in the vicinity of the abscess stain also positively (halfed arrow in B). A: arcuate artery, G: glomerulus, V: arcuate vein; arrowheads: margin of abscesses. Magnifications: (A) 250×, (B) 100×.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Immunohistochemistry, Staining, Infection

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Survival of NMRI mice after intraperitoneal infection with S. aureus (wt; S. aureus 8325-4; n = 8) or S. aureus Δ hemB (SCV, S. aureus erm Ω hemB 8325-4; n = 8). Note the higher susceptibility of mice infected with S. aureus 8325-4 compared to the mice infected with S. aureus Δ hemB . *significant difference: P<0.001. (B) Inhibition of DFO induced HIF-1 activation in HeLa-229 cells by 17-DMAG. Cells were incubated with 17 DMAG (10 µmol/L) for 16 hours following induction of HIF 1 activation by the iron chelator DFO (200 µmol/L) for six hours. HIF 1α protein was analyzed in Western blots (loading control: actin). Negative control: uninfected cells. (C) Survival rate of NMRI mice after intraperitoneal infection with S. aureus ( S. aureus 8325-4). One group of mice was treated 24 h and 16 h before infection with the HIF-1 inhibiting compound 17-DMAG (25 mg/g body weight). Note the higher survival rate of 17-DMAG-treated mice (n = 45) compared with control mice (n = 45). *significant difference: P = 0.005 (Kaplan-Meier analysis).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Inhibition, Activation Assay, Incubation, Western Blot, Control, Negative Control

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 maintains BTIC self-renewal. ( a ) Morphology of self-renewing spheres and immunofluorescence of neuronal and glial lineage markers in differentiated cells. Scale bar=200 μm. ( b ) Immunofluorescence of ING5 in undifferentiated (upper panels) and cells differentiated for 5 days (lower panels). Scale bar=20 μm. ( c , d ) The mRNA levels ( c ) and protein levels ( d ) of ING5 decrease during differentiation in BT 189 cells. n =4. ( e ) Immunoblotting of ING5 protein in the BT 12, and BT 134 lines during differentiation. ( f ) ING5 overexpression increases sphere formation rates and average volumes in BTIC sphere formation assays ( n =3, ** P <0.01, * P <0.05). Scale bar=400 μm. ( g ) ING5 knockdown by siRNA decreases sphere formation rates and sphere sizes of BTICs ( n =4, ** P <0.01, * P <0.05). Scale bars=400 μm. ( h ) (Left) Sphere formation rates for cell lines stably expressing shRNAs against ING5 (shR1 and shR2-ING5) or control non-targeting shRNA (shR-ctr). ( n =4, ** P <0.01, * P <0.05). (Right) Fluorescence of the RFP reporter in stable cell lines superimposed with differential interference contrast (DIC) images. Scale bar=100 μm. ( i ) RT-qPCR analysis of stem cell core transcription factors and stem cell markers after ING5 overexpression. ( j , k ) Western blot analysis of the neural stem cell marker Nestin and neuronal lineage marker Tubb3 in response to ING5 overexpression ( j ) and knockdown ( k ).

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Western Blot, Over Expression, Knockdown, Stable Transfection, Expressing, Control, shRNA, Fluorescence, Quantitative RT-PCR, Marker

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 increases the stem cell pool and inhibits differentiation. ( a ) Flow cytometry analysis of CD133/CD44 positive cells in iPB cell lines (top panels) and CD44 positive cells in shRNA cell lines (bottom panels), gated by isotype control. ( b ) (Left) Mitotic pair analysis of the three division modes: symmetric proliferating (sym-pro), symmetric differentiating (sym-diff) and asymmetric (asym) cell division, in iPB cell lines. Over 150 pairs were counted for each group in one experiment. n =3, * P <0.05. (Right) An example of cell division symmetry based on the distribution of stem cell factor Nestin with the Red arrow indicating symmetric differentiating division and the white arrow asymmetric division. ( c ) Morphological changes of iPB cells before (Day 0) and after (Days 1–3) differentiation induced by 1% FBS. Scale bar=100 μm. ( d ) After differentiation for 5 days, immunofluorescence of Nestin and Tubb3 in shRNA cell lines with an RFP reporter. Scale bar=200 μm. ( e ) Western blot of Nestin and Tubb3 in differentiated shRNA cell lines corresponding to cells shown in d .

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Flow Cytometry, shRNA, Control, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 activates mitogenic pathways to promote self-renewal. ( a ) The sphere formation rates of iPB-ctr and iPB-ING5 overexpressing cells at three successive passages in the absence of EGF and FGF treatment. ( n =3, ** P <0.01, *** P <0.001). ( b ) DIC images of spheres from the tertiary sphere passage in iPB cells. Scale bar=400 μm. ( c ) Sphere formation rate under treatment with protein kinase inhibitors ( n =3, * P <0.05). ( d ) Protein and phosphorylated protein levels of effectors in the PI3K and MEK pathways. Cells were treated with PX-866 at 1 μ M and PD184352 at 2 μ M for 48 h. ( e ) Immunofluorescence of Nestin and Tubb3 in differentiated iPB control (Left panels) or iPB-ING5 cells (Right panels) treated with 1 μ M PX-866 or 2 μ M PD184352. Scale bar=200 μm. ( f ) Flow cytometry analysis of the CD133 positive population in PX-866 (1 μ M ) and PD184352 (2 μ M ) treated BTIC 189 cells, gated by isotype control.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Control, Flow Cytometry

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: The FSH pathway transduces effects of ING5 on stem cell properties. ( a ) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations ( n =3, * P <0.05 and ** P <0.01 compared to iPB-ctr/DMSO; # P <0.05 and ## P <0.01 compared to iPB-ING5/DMSO). ( b ) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. ( c ) RT-qPCR of genes related to hormone and steroidogenesis functions. ( n =3, * P <0.05, ** P <0.01) ( d ) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. ( e ) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. ( f ) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. ( g ) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. ( h ) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines ( n =3, * P <0.05 and ** P <0.01 compared to untreated shR-ctr; # P <0.05 compared to untreated shR-ING cells). ( i ) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells ( n =3, ** P <0.01). ( j ) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both ( n =3, * P <0.05). ( k ) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Tube Formation Assay, Blocking Assay, Quantitative RT-PCR, Expressing, Immunostaining, Flow Cytometry, Control, Immunofluorescence, Inhibition, Recombinant, shRNA

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. ( a ) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) ( n =3, ** P <0.01). ( b ) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. ( c ) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. ( d ) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n =114). ( e , f ) ING5 expression levels negatively correlate with survival of the Proneural subtype ( n =24) and the Classical subtype ( n =30) of GBM patients. ( g ) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n =61). ( h ) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Western Blot, FLAG-tag, Binding Assay, Immunoprecipitation, Expressing, Clinical Proteomics, Membrane, Protein-Protein interactions, Activation Assay

Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 1 mTORC2 affects rearrangement of the F-actin cytoskeleton in mouse fertilized eggs. (a) The development model of mouse fertilized eggs. G1 phase: 12–21 hours after injecting hCG; S phase: 21–27 hours after injecting hCG; G2 phase: 27–30 hours after injecting hCG; M phase: 30–33 hours after injecting hCG. (b) Staining for F-actin (red) and DNA (blue) revealed the organization of the F-actin cytoskeleton in mouse fertilized eggs transfected with RICTOR shRNA (F-actin: yellow arrow). Scale bar: 20 μm. (c) Western immunoblotting detection of mTORC2 in 200 mouse fertilized eggs treated with shRNA targeted against mTORC2. WB: Western blotting

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Staining, Transfection, shRNA, Western Blot

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 2 Akt1 regulates cell division and F-actin rearrangement in mouse fertilized eggs. (a) Western immunoblotting detection of Akt1 in 200 mouse fertilized eggs treated with siRNA targeted against Akt1. WB: Western blotting. (b) The division rate in cultured mouse embryos after 0.03 ng of mRNA-encoding Akt1-WT, myr-Akt1, Akt1-KD or Akt1 siRNA injection. The percentage of cells undergoing cell division and cell survival was calculated after manual counting under a dissecting microscope 35 hours after injection of human chorionic gonadotropin in female mice. The numbers of eggs undergoing cell division (hatched bars) or survival are given above each bar graph. (c) Cortical remodelling of F-actin is induced by Akt1 activation. Mouse fertilized eggs were stained with rhodamine-phalloidin (red fluorescence) to visualize F-actin cytoskeleton. Various scenarios were studied, which included cells injected with mRNA-encoding Akt1-WT, myr-Akt1, Akt1-KD or siRNA targeted against Akt1. Fertilized eggs were fixed and labelled with rhodamine-phalloidin (10 or 20 μmol/L; F-actin labelling, indicated by white arrows) and with Hoechst 33258 (1 mg/ml; DNA labelling) and imaged by laser-scanning confocal microscope. Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Western Blot, Cell Culture, Injection, Microscopy, Activation Assay, Staining, Fluorescence

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 4 Girdin is essential for rearrangement of the F-actin cytoskeleton and the development of mouse fertilized eggs. (a) Depletion of Girdin in mouse one-cell staged fertilized eggs by siRNA. Total cell extracts from control siRNA- and Girdin siRNA- injected one-cell staged fertilized eggs were subjected to Western blot analyses and immunodetection with anti-Girdin, anti-p-Girdin, anti-Akt1 and anti-actin antibodies. (b) The cleavage rate in cultured mouse embryos after Girdin siRNA injections shows that the total number of eggs undergoing cell division is given above each bar graph from three independent experiments. (c) F-actin cytoskeleton of embryos derived from fertilized eggs treated with Girdin siRNA. Mouse fertilized eggs were treated with control or Girdin siRNAs and fixed 48 hours later, followed by staining with rhodamine-phalloidin and anti-Girdin antibody. In situ validation of the interaction between Girdin and polymerized F-actin by confocal microscopy is shown. One-cell stage mouse fertilized eggs were treated with 10 or 20 μmol/L Girdin siRNA. Immunolocalization of Girdin is revealed by green staining (antibody), and immunolocalization of actin is revealed by red staining. Control fertilized eggs (21 hours after hCG): one-cell arrested embryos derived from fertilized eggs that were injected with Girdin siRNA 21 hours after hCG and cultured for 24 hours; control two-cell embryo: in control fertilized eggs, F-actin forms a regular ring in the cell cortex. In embryos derived from fertilized eggs treated with girdin siRNA, polymerized F-actin was observed in the cytoplasm and formed irregular patches that were scattered randomly in the cortex. Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Control, Injection, Western Blot, Immunodetection, Cell Culture, Derivative Assay, Staining, In Situ, Biomarker Discovery, Confocal Microscopy

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 3 The mTORC2/Akt1 pathway rearranges the F-actin cytoskeleton of one-cell stage fertilized eggs. Microinjection of Rictor shRNA then with myr-Akt1 mRNA into mouse one-cell stage embryos. Staining for F-actin (red) revealed the organization of the F-actin cytoskeleton in mouse fertilized eggs (F-actin is shown by the yellow arrow). Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Microinjection, shRNA, Staining

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 5 The Akt1/ Girdin pathway regulates rearrangement of the F-actin cytoskeleton of one-cell stage fertilized eggs. (a) One-cell stage mouse embryos were first microinjected with Akt1-WT mRNA, myr-Akt1 mRNA or siRNA, then stained with rhodamine-phalloidin (20 μmol/L; actin labelling as shown in red) and anti-P-Girdin (1:50; Girdin labelling; as shown in green). Merged colour detection between P-Girdin and polymerize F-actin appears as yellow stained images. The areas of co-localization are highlighted with yellow arrowheads. Scale bar: 20 μm. (b) 200 fertilized eggs were treated with mRNA coding for Akt1- WT, myr-Akt1, Akt1-KD or siRNA against Akt1. Western immunoblot analyses assessed following immunoreaction with anti-P-Girdin (upper panel) and anti-Girdin (lower panel) antibodies are shown. (c) One-cell stage mouse embryos were first microinjected with 0.03 ng of myr-Akt1 mRNA as described in the Materials and Methods, and then 1–2 hours later with Girdin siRNA, following which specimens were stained with rhodamine-phalloidin (20 μmol/L; actin labelling shown in red). Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Staining, Western Blot

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 6 The mTORC2/Akt1/Girdin pathway rearranges the F-actin cytoskeleton in one-cell stage fertilized eggs. (a) The effect of gene knockdown on the expression of Rictor and the phosphorylation of Girdin-1417. Extracts of mouse fertilized eggs were resolved by 6% SDS-PAGE, transferred to nitrocellulose and probed with phosphor- Girdin-Ser1417 antibody and Girdin antibody. (b) We examined Girdin phosphorylation in the fertilized eggs treated with Rictor shRNA, WT-Akt1 mRNA, Rictor shRNA and WT-Akt1 coinjection. In WT-Akt1 mRNA treated cells, we noted that Girdin was highly phosphorylated at 1417. But cells treated with Rictor siRNA and WT-Akt1 exhibited decreased phosphorylation of Girdin 1417

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Knockdown, Expressing, Phospho-proteomics, SDS Page, shRNA

Journal: Journal of Bone and Mineral Research

Article Title: Nfat1 Regulates Adult Articular Chondrocyte Function through Its Age-Dependent Expression Mediated by Epigenetic Histone Methylation

doi: 10.1002/jbmr.397

Figure Lengend Snippet: Specific primers used for quantitative real-time RT-PCR

Article Snippet: Mouse lysine-specific histone demethylase-1 (Lsd1) shRNA lentiviral particles (Santa Cruz, sc-60971-V) containing a pool of three Lsd1-specific constructs were used to knock down Lsd1 expression.

Techniques:

Journal: Journal of Bone and Mineral Research

Article Title: Nfat1 Regulates Adult Articular Chondrocyte Function through Its Age-Dependent Expression Mediated by Epigenetic Histone Methylation

doi: 10.1002/jbmr.397

Figure Lengend Snippet: Knockdown of Lsd1 using Lsd1 shRNA lentiviral particles (Santa Cruz) in E16.5 articular chondrocytes results in an upregulation of Nfat1 expression through increased H3K4me2 at the Nfat1 promoter. (A) qPCR analyses (left panel) and Western blots (right panel) confirm the efficiency of Lsd1 knockdown in Lsd1 RNAi-treated E16.5 articular chondrocytes. “Neg ctl” represents a negative control blot using a protein sample from the scrambled control group but without using primary antibody against Lsd1. (B) ChIP assays using an anti-H3K4me2 antibody with qPCR analyses of co-immunoprecipitated Nfat1 DNA using three different primers (P1–3) designed from the sequences around the TSS of the Nfat1 gene. Lsd1 RNAi up-regulates H3K4me2 level in the Nfat1 promoter region, which is detected by P1–3 primers, but not by Untr17 negative control primers (data not shown). (C) qPCR shows that RNAi-mediated knockdown of Lsd1 up-regulates Nfat1 expression in E16.5 articular chondrocytes. The qPCR level of each control group has been normalized to “1.0” in A–C. n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001. (D) A schema for Lsd1-mediated epigenetic regulation of Nfat1 transcription.

Article Snippet: Mouse lysine-specific histone demethylase-1 (Lsd1) shRNA lentiviral particles (Santa Cruz, sc-60971-V) containing a pool of three Lsd1-specific constructs were used to knock down Lsd1 expression.

Techniques: Knockdown, shRNA, Expressing, Western Blot, Negative Control, Control, Immunoprecipitation

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress–Induced IRE1α Activation Mediates Cross-Talk of GSK-3β and XBP-1 To Regulate Inflammatory Cytokine Production

doi: 10.4049/jimmunol.1401399

Figure Lengend Snippet: ER stress–induced IRE1α activation regulates differential proinflammatory cytokine gene expression via activation of GSK-3β and XBP-1. (A) RAW 264.7 cells were transfected with specific siRNA against IRE1α, XBP-1, or control siRNA (scramble). Under these conditions, the activation of GSK-3β in TM-treated cells was determined by Western immunoblot analysis. (B) Wild-type (WT) or IRE1α-, (C) XBP-1–, and (D) GSK-3β–deficient MEFs were treated with TM for 9 h or indicated times and then activation of GSK-3β was determined by Western immunoblot analysis. α-Tubulin served as the standard. (E) GSK-3β–deficient MEFs were treated with TM for 3 h and then the level of XBP-1(s) mRNA was determined by quantitative real-time PCR. (F–I) RAW 264.7 cells were transfected with specific siRNA against IRE1α, XBP-1, or control siRNA (scramble), and the level of TNF-α and IL-1β was determined by quantitative real-time PCR (F and H) and ELISA (G and I) from RAW 264.7 cells treated with (F and G) TM (1 μg/ml) or (H and I) LPS (1 μg/ml). GAPDH served as the standard. Data represent means ± SD of three independent determinations. Blots shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Transfection Small interfering RNA (siRNA) against mouse IRE1α, XBP-1, and GSK-3β were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Gene Expression, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress–Induced IRE1α Activation Mediates Cross-Talk of GSK-3β and XBP-1 To Regulate Inflammatory Cytokine Production

doi: 10.4049/jimmunol.1401399

Figure Lengend Snippet: The expression of TNF-α, but not of IL-1β, is dependent on IRE1α endonuclease activity. RAW 264.7 cells were treated with TM (10 μg/ml) for 1 or 3 h in the absence or presence of STF083010. (A) Activation of XBP-1 and CHOP mRNA expression were determined by RT-PCR. GAPDH served as the standard. The expression of (B) IL-1β and TNF-α, (C) XBP-1 splicing, and (D) CHOP mRNA was determined by quantitative real-time PCR. GAPDH served as the standard. Data represent means ± SD of three independent determinations. **p < 0.01, ***p < 0.001.

Article Snippet: Transfection Small interfering RNA (siRNA) against mouse IRE1α, XBP-1, and GSK-3β were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activity Assay, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress–Induced IRE1α Activation Mediates Cross-Talk of GSK-3β and XBP-1 To Regulate Inflammatory Cytokine Production

doi: 10.4049/jimmunol.1401399

Figure Lengend Snippet: Schematic representation of the proposed pathways for the transcriptional regulation of IL-1β and TNF-α during IRE1α activation in response to ER stress. IRE1α-mediated GSK-3β activation results in the selective transcription of IL-1β and inhibition of XBP-1 splicing. In contrast, IRE1α-mediated XBP-1 activation results in the transcription of TNF-α.

Article Snippet: Transfection Small interfering RNA (siRNA) against mouse IRE1α, XBP-1, and GSK-3β were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Inhibition