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: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: rs3212247 is responsible for haplotype‐specific difference in transcriptional activity. (A), Map of single‐nucleotide polymorphisms (SNPs) in the promoter and 5'UTR of Receptor‐interacting protein kinase 3 genotyped in 200 healthy individuals. The eight SNPs (indicated with boxes), which were predicted to be possible functional at RegulomeDB, comprise a single haplotype and define the 1.8 and 0.2 kb in promoter and 5'UTR, respectively. (B), Firefly luciferase expression from constructs transfected into AC16. Both the major (Hap1) and minor (Hap2) haplotypes of the 1.8 kb in promoter region were subcloned into PGL3‐basic vector. (C‐I), Luciferase assays for rs3212247, rs3834521, rs3759625, rs3212249, rs3759630, rs3212246 rs3212250‐51 (rs3212250 and rs3212251 were combined as a haplotype as they are close enough) were conducted using AC16. Approximately 250‐bp regions encompassing corresponding variants were cloned into PGL3‐basic vector. Luciferase activity was measured 48 h after transfection and was normalized against Renilla luciferase activity. Values are mean ± SE of three independent experiments each corresponding to at least six replicates. ** P < 0.01, **** P < 0.0001, NS, not significant

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Activity Assay, Functional Assay, Luciferase, Expressing, Construct, Transfection, Plasmid Preparation, Clone Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Characteristics of RIP3 variants identified by Sanger sequencing

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: SOX17 binds to the rs3212247‐T allele and mediates allelic expression. (A), Firefly luciferase assays were performed with 250‐bp rs3212247‐T or ‐C allele constructs cotransfected into AC16 cells with SOX17 vector encoding sox17. Luciferase activity was measured 48 h after transfection and was normalized against Renilla luciferase activity. (B), AC16 cells were transfected with pcDNA3.1 and SOX17 respectively. Protein was harvested 48 h after transfection and Receptor‐interacting protein kinase 3 (RIP3) expression was normalized against GAPDH. * P < 0.05

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Expressing, Luciferase, Construct, Plasmid Preparation, Activity Assay, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: In vivo plasma concentrations of Receptor‐interacting protein kinase 3 (RIP3) in control group (A) HF NYHA class II group (B) HF NYHA class III group (C) HF NYHA class IV group (D) and among these four groups (E). The data are presented as box (25th percentile, median and 75th percentile)

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: In Vivo, Clinical Proteomics, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Association between RIP3 variants and heart failure

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Association of the RIP3 polymorphisms with cardiac mortality or transplantation in chronic heart failure

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Transplantation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Effect of SOX17 on Regulation of Receptor‐interacting protein kinase 3 (RIP3) Transcription. The variant rs3212247 in RIP3 promoter region is a T‐ to‐C change and is predicted to locate in the binding site of SOX17. Rs3212247‐C allele destroys the SOX17 binding site in the promoter region of RIP3 and subsequently results in increased transcription and translation of RIP3, followed by increased necroptosis of cardiomyocyte under various external stimuli, which leads to poor prognosis of heart failure (HF)

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Variant Assay, Binding Assay

Journal: Nature Communications

Article Title: Targeting the disrupted Hippo signaling to prevent neoplastic renal epithelial cell immune evasion

doi: 10.1038/s41467-025-57697-7

Figure Lengend Snippet: A Western blots showing successful overexpression of the wild-type YAP1 (YAP) and constitutively active YAP1 (YAP S127A ) in HK2 cells. Each experiment was repeated at least three time and a representative image was presented to show the relative level of YAP1 protein. B Fluorescent i mmunocytochemistry showing the expression and subcellular location of YAP (red) in HK2-MX, HK2-YAP, and HK2-YAP S127A cells; Each experiment was repeated independently at least three time and a representative image was presented to show the location and relative level of YAP1 protein. Nuclei were stained with DAPI (blue); scale bar: 10 μm. C Cell growth curves showing that YAP activity promotes the proliferation of HK2 cells. Each time point represents the mean ± SEM ( n = 4~6 biological replicates). The cell numbers on day 12 were analyzed with One-Way ANOVA followed by the Tukey multiple comparisons test. MX v.s YAP: p = 0.2869 (ns); MX vs. YAP S127A : P = 0.0002 (***); YAP vs. YAP S127A : P = 0.0039. D Overexpression of YAP & YAP S127A stimulates multilayer growth of HK2 cells. Scale Bar: 100 μm. E The anchorage-free growth ability of modified HK2 cells. Left panel: Representative images showing colony formation in HK2 control cells (MX) and HK2 cells expressing YAP S127A , scale bar: 500 μm. Right panel: A violin plot (with minima, maxima, median, and percentile etc.) showing colony numbers formed by HK2-MX, HK2-YAP, and HK2-YAP S127A cells ( n = 5 biological replicates). Data were analyzed with One-Way ANOVA followed by the Tukey multiple comparisons test. MX vs YAP: P = 0.0085 (**); MX vs. YAP S127A : P < 0.0001 (****); YAP vs. YAP S127A : P < 0.0001. F A representative image showing the orthotopic inoculation of modified HK2 cells under mouse kidney capsules. G A representative image showing the orthotopic tumor formed by HK2-YAP S127A cells implanted under mouse kidney capsules. H Representative images of H-E staining and immunohistochemistry (IHC) from six biological repeats showing the histological morphology of orthotopic tumors and expression of Ki67, Vimentin, and YAP proteins in orthotopic tumors. IPC: implanted cells, R: renal tissue, scale bar: 50 μm. I Survival rate of mice injected with HK2-MX cells ( n = 5, no tumor) and HK2-YAP S127A cells ( n = 10, all with metastatic tumor). J Representative H-E and IHC images showing the diverse histology of tumor (two H – E staining images) and expression of Ki67, Vimentin, and YAP1 proteins in tumors (three IHC staining images) derived from mice injected with HK2-YAP S127A cells. Scale bar: 50 μm. Source data for panels ( A , C , E , and I ) are provided as a Source Data file.

Article Snippet: MDSC were isolated with a mouse tumor cell isolation kit (Miltenyi Biotec, Auburn, CA) and purified using an EasySepTM Mouse MDSC (CD11b + Gr1 + ) Isolation Kit (STEMCELL Technologies Inc, Cambridge, MA).

Techniques: Western Blot, Over Expression, Expressing, Staining, Activity Assay, Modification, Control, Capsules, Immunohistochemistry, Injection, Derivative Assay

Journal: Nature Communications

Article Title: Targeting the disrupted Hippo signaling to prevent neoplastic renal epithelial cell immune evasion

doi: 10.1038/s41467-025-57697-7

Figure Lengend Snippet: A UMAP plot showing the overview of 18 major cell types identified in renal tissues in control (CTL, non-induced mice) and Pax8-rtTA;Tet-on-YAP S127A mice induced with doxycycline for seven days (D7) and four months (M4). B Violin plots of representative marker genes across the identified cell clusters. C UMAP plots showing the cellular components of renal tissues in Pax8 -rtTA;Tet-on-YAP S127A mice induced with doxycycline for seven days (D7) and four months (M4). D Dynamic alteration of major cell components in renal tissues in control and doxycycline-induced Pax8 -rtTA;tet-on-YAP S127A mice during hyperactivated YAP1-induced tumorigenesis. *: significantly different when compared to CTR. Statistical difference were performed with scCODA with FDR = 0.05. Please note that mice in the M4 group were generated using a low dose/chronic induction protocol in order to avoid acute neoplasia-associated kidney failure.

Article Snippet: MDSC were isolated with a mouse tumor cell isolation kit (Miltenyi Biotec, Auburn, CA) and purified using an EasySepTM Mouse MDSC (CD11b + Gr1 + ) Isolation Kit (STEMCELL Technologies Inc, Cambridge, MA).

Techniques: Control, Marker, Generated

Journal: Nature Communications

Article Title: Targeting the disrupted Hippo signaling to prevent neoplastic renal epithelial cell immune evasion

doi: 10.1038/s41467-025-57697-7

Figure Lengend Snippet: A UMAP plots showing the expression of YAP1 in different types of cells in the renal tissues. Please note that YAP1 is primarily expressed in epithelial cells. B GSEA analyzes showing the enrichment of genes associated with YAP1 conserved signature in renal epithelial cells induced with Dox for seven days (D7) or four months (M4). C UMAP plots showing the changes of epithelial cell subpopulations in control (CTR) and YAP S127A -induced (for 7 days and 4 months) renal tissues. Please note that the numbers of cells in cluster 5 (C5) and cluster 11 (C11) are significantly increased after doxycycline induction of transgene expression. D GSEA analyzes showing the enrichment of genes associated with YAP1 conserved signature in C5 and C11 subpopulations after inducing transgene expression with doxycycline for 7 days (D7) or 4 months (M4). E Heatmap demonstrates significantly altered genes in the C5 cluster of proximal tubal epithelial cells after inducing transgene with doxycycline for 7 days (D7) or 4 months (M4). Renal epithelial cells from the non-induced mice were used as control (CTR). The significantly altered genes involved in extracellular matrix reprogramming are listed on the left side of the heatmap. F Gene ontology enrichment analysis showing alteration of signaling pathways involved in the transformation of cluster 5 (C5) cells in D7 early lesions and M4 pRCC tumor tissues. P .adjust is Benjamini-Hochberg adjusted p values. And P values were derived from GSEA permutation based on an adaptive multi-level split Monte-Carlo scheme implemented in fgsea R package. G Heatmap demonstrates significantly altered genes in the C11 cluster of proximal tubal epithelial cells after inducing transgene with doxycycline for 7 days (D7) or 4 months (M4). Renal epithelial cells from the non-induced mice were used as control (CTR). The significantly altered genes involved in extracellular matrix reprogramming are listed on the left side of the heatmap. H Gene ontology enrichment analysis showing alteration of signaling pathways involved in the transformation of cluster 11 (C11) cells in D7 early lesions and M4 pRCC tumor tissues. P.adjust is Benjamini-Hochberg adjusted p values. And P values were derived from GSEA permutation based on an adaptive multi-level split Monte-Carlo scheme implemented in fgsea R package. I Heatmap showing expression of genes encoding for chemokines, cytokines, and growth factors that are associated with MDSC accumulation and immune suppression in C5 and C11 epithelial cells. Please note that mice in the M4 group were generated using a low dose/chronic induction protocol in order to avoid acute neoplasia-associated kidney failure.

Article Snippet: MDSC were isolated with a mouse tumor cell isolation kit (Miltenyi Biotec, Auburn, CA) and purified using an EasySepTM Mouse MDSC (CD11b + Gr1 + ) Isolation Kit (STEMCELL Technologies Inc, Cambridge, MA).

Techniques: Expressing, Control, Protein-Protein interactions, Transformation Assay, Derivative Assay, Generated

Journal: Nature Communications

Article Title: Targeting the disrupted Hippo signaling to prevent neoplastic renal epithelial cell immune evasion

doi: 10.1038/s41467-025-57697-7

Figure Lengend Snippet: A , B Heatmap demonstrates significantly altered genes in myeloid cells in renal tissues of Pax8 -rtTA;Tet-on-YAP S127A mice induced with doxycycline for seven days (D7, A ) or four months (M4, B ). Myeloid cells in renal tissues of non-induced mice were used as control (CTR). Significant genes involved in renal tissue microenvironment reprogramming are listed on the left side of the heatmap. C , D Gene set ontology enrichment analysis showing significantly altered signaling pathways in myeloid cells in renal tissues of Pax8 -rtTA;Tet-on-YAP S127A mice induced with doxycycline for seven days (D7, C ) or four months (M4, D ). Myeloid cells in renal tissues of non-induced mice were used as control (CTR). Representative pathways involved in renal tissue microenvironment reprogramming are listed in the plots. P.adjust is Benjamini-Hochberg adjusted p values. And P values were derived from GSEA permutation based on an adaptive multi-level split Monte-Carlo scheme implemented in fgsea R package. E UMAP projection of eight clusters identified in the myeloid cells (clusters are labeled in UMAP). F Violin plots of representative marker genes across identified myeloid cell clusters. G UMAP plots showing changes in myeloid cell subpopulations in renal tissues of Pax8-rtTA ;Tet-on-YAP S127A mice induced with doxycycline for seven days (D7) and four months (M4). Renal tissues from non-induced mice were used as a control (CTR). Please note the drastic accumulation of myeloid-derived suppressor cells (MDSCs) in the renal tissue of Pax8- YAP S127A mice. H Dynamic change of myeloid cell subpopulations in renal tissue of PAX8-rtTA;Tet-on-YAP S127A mice induced with doxycycline for seven days (D7) and four months (M4). Renal tissues from non-induced mice were used as a control (CTR). *: significantly different when compared to CTR. Statistical difference were performed with scCODA with FDR = 0.05. Please note that the vast majority of increased myeloid cells are MDSCs. I ) UMAP plots showing high expression of key genes associated with MDSC functionality in renal tissues of Pax8-YAP S127A mice induced with doxycycline for seven days (D7) and four months (M4). Renal tissues from non-induced mice were used as a control (CTR). Please note that mice in the M4 group were generated using a low dose/chronic induction protocol in order to avoid acute neoplasia-associated kidney failure.

Article Snippet: MDSC were isolated with a mouse tumor cell isolation kit (Miltenyi Biotec, Auburn, CA) and purified using an EasySepTM Mouse MDSC (CD11b + Gr1 + ) Isolation Kit (STEMCELL Technologies Inc, Cambridge, MA).

Techniques: Control, Protein-Protein interactions, Derivative Assay, Labeling, Marker, Expressing, Generated

Journal: Nature Communications

Article Title: Targeting the disrupted Hippo signaling to prevent neoplastic renal epithelial cell immune evasion

doi: 10.1038/s41467-025-57697-7

Figure Lengend Snippet: A Radar plots showing the functional clusters of T cells in renal tissue with or without induction with doxycycline. The molecular features of reference cells are labeled in black. Molecular features of T cell clusters in our control and transgenic animal models are labeled in red. CD8_Teff/mem: CD8+ effector/memory T cells; Tex: CD8+ exhaust T cells; Tpex: CD8+ pro-exhaust T cells; CD8_Early Activ: CD8+ Early active T cells; CD8_Naivelike: CD8+ Naïve like T cells; CD4_Naivelike: CD4+ Naïve-like T cells; Tfh: follicular Helper T cells; Th1: type 1 helper T cells; Treg: Regulatory T cells. B Dynamic alternation of T cell subpopulations in control (CTR), early renal epithelial neoplastic lesions (D7), and pRCC (M4). *: significantly different when compared to CTR. Statistical difference were performed with scCODA with FDR = 0.05. a: p = 0.064818. C – G Notched Box plots (with minima, maxima, center, and percentile, etc.) were generated at single-cell level to demonstrate the upregulation of cell surface immune suppressive molecules in Tex cells ( C ), apoptosis-associated molecules in tumor-infiltrating T lymphocytes ( D ), alterations of the intrinsic transcription factors that drive the expression of cell surface immune suppressive molecules ( E ), upregulation of cell surface immune suppressive molecules in Tfh cells ( F ), and upregulation of molecules associated with Treg suppression of T effectors ( G ). CTR: n = 95; D7: n = 199; M4: n = 260. Please note that mice in the M4 group were generated using a low dose/chronic induction protocol in order to avoid acute neoplasia-associated kidney failure. H FACS analyzes showing the impact of MDSC on T cell expansion. T cells were activated with a combination of IL2 (10 ng/ml), anti-CD3 (α-CD3, 1 ug/ml), and anti-CD28 (α-CD28, 1 ug/ml). T Cell expansion in the co-culture was determined by a carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay. Representative FACS graphs showing the expansion of Cd4 + T cells (left panel) and Cd8 + T cells (right panel) (both isolated from mouse spleen) co-cultured with or without 1:1 MDSCs (isolated/purified from Pax8 -YAP S127A mice) for 72 h. The same amount of bone marrow cells (BMC) was used as negative control. “Non-stimulated”: T cells without stimulation/activation (negative control). “MDSC (or BMC): T = 1: 1”: MDSCs (or BMCs) and activated T cells were cocultured with a ratio of 1:1. Please note that addition of MDSCs, not BMCs, blocked the expansion of activated Cd4+ and Cd8+ T cells in the co-culture system.

Article Snippet: MDSC were isolated with a mouse tumor cell isolation kit (Miltenyi Biotec, Auburn, CA) and purified using an EasySepTM Mouse MDSC (CD11b + Gr1 + ) Isolation Kit (STEMCELL Technologies Inc, Cambridge, MA).

Techniques: Functional Assay, Labeling, Control, Transgenic Assay, Generated, Expressing, Co-Culture Assay, Dilution Assay, Isolation, Cell Culture, Purification, Negative Control, Activation Assay

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: FGF19 signaling, along with Wnt activation and TGF β inhibition, promotes proliferation of hPSC-derived hepatoblasts. a) Flow cytometry analysis showing the proportion of Ki67+, and ALB+ cells at day 15, day23, and day29 of differentiation. b) Quantification of Ki67 positive cells in day 15, day 21or 23, and day 29 H9 hepatoblasts by flow cytometry. One-way ANOVA. DATA are represented as mean ± SEM ( n = 11 for day 15, n = 25 for day 21 or 23, n = 19 for day 29). c) RT-qPCR analysis of the expression of the indicated gene in each population at different stages (ES cells, endoderm, day 15, day 23, day 29) of H9-derived hepatoblasts. AL adult liver, FL fetal liver. One-way ANOVA. DATA are represented as mean ± SEM ( n = 5). d) Quantification of cells in day 29+6 H9 hepatoblasts after treatment with different indicated cytokine/ small molecule combinations. SB/CHIR/FGF19 group showed significantly higher values compared to all other groups (p< 0.001). One-way ANOVA. DATA are represented as mean ± SEM ( n = 6). e) Flow cytometry analysis showing the proportion of Ki67+ and ALB+ cells at day 29+6 of differentiation in SB/CHIR or SB/CHIR/FGF19. f) Quantification of Ki67 positive cells in day 29+6 H9-derived hepatoblasts after treatment with SB/CHIR or SB/CHIR/FGF19. Two-tailed Student’s test ( n = 4 and 9). g) Microscopic analysis showing the co-expression of ALB (pink) and Ki67 (green) in H9-derived hepatoblasts. Scale bar: 200µm. h) Flow cytometry analysis showing the proportion of ALB+ and AFP+ cells at day 29+6 of differentiation under SB/CHIR/FGF19 treatment.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Activation Assay, Inhibition, Derivative Assay, Flow Cytometry, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Thyroid hormone and Wnt signaling promote hepatic maturation and zonation of hPSC-derived hepatocytes. a) Flow cytometry analysis showing the population of ALB+ and AFP+ cells in cAMP or T3-treated hepatic aggregates. b) Quantification of the cell populations indicated in . Two-tailed Student’s test ( n = 5). c) RT-qPCR analysis of AFP expression in each population at day 6 and day 18 of H9-derived hepatic aggregates. AL adult liver, FL fetal liver. One-way ANOVA. DATA are represented as mean ± SEM ( n = 3-5). d) Schematic differentiation of 3D hepatic aggregates. e) Flow cytometry analysis showing the population of ALB+ and AFP+ cells in XAV939 or CHIR99021-treated hepatic aggregates (left). Quantification of positive cells in each quadrant, indicated by colours (right) ( n = 5). f) RT-qPCR analysis of the expression of the indicated genes in each population at day 56 of H9-derived hepatic aggregates under different culture conditions. AL adult liver, FL fetal liver. One-way ANOVA. DATA are represented as mean ± SEM ( n = 3-8). g) Immunostaining analysis showing the proportion of ALB and CYP3A4 in H9-derived hepatic aggregates at day 56 treated with XAV939 or CHIR99021. Scale bar: 50µm.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Derivative Assay, Flow Cytometry, Two Tailed Test, Quantitative RT-PCR, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Global gene expression analysis reveals zonation features in hPSC-derived hepatocytes. a) tSNE plot of 3D hepatic aggregates treated with XAV939 or CHIR99021. b) tSNE plots showing cells in various cell cycles. c) tSNE plots displaying the expressions of selected hepatic markers. d) UMAP projection of integrated single-cell transcriptomic profiles from human adult liver cells and 3D hepatic aggregates derived from H9 hPSCs. Clusters corresponding to hPSC-derived hepatocytes are demarcated by dotted circles (Clusters 10, 15, and 19), whereas clusters representing primary human hepatocytes from adult liver tissue are enclosed with solid circles (Clusters 1, 2, 4, 7, 17, and 18). e) Pathway enrichment analysis comparing hPSC-derived hepatocyte clusters (Clusters 10, 15, and 19) to adult primary hepatocyte clusters (Clusters 1, 2, 4, 7, 17, and 18), highlighting signaling pathways significantly downregulated in hPSC-derived populations.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Gene Expression, Derivative Assay, Protein-Protein interactions

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Comparative transcriptomic analysis of H9-derived hepatic aggregates and adult human liver. a. tSNE projection of integrated single-cell transcriptomic data from five adult human liver tissue samples (total liver homogenates) and H9-derived hepatocytes treated with either XAV or CHIR. A total of 9,115 cells were analyzed, encompassing 7,562 genes, and computationally resolved into 23 transcriptionally distinct clusters. Top left: Visualization of clusters enriched in human liver-derived cells (pink dots). Top right: Overlay of hPSC-derived hepatocyte populations: XAV-treated (pink dots) and CHIR-treated (yellow dots) to assess integration and cellular identity. Bottom left: Cluster assignment map displaying numerical labels for all 23 clusters. Bottom right: Cell type annotation based on canonical marker gene expression, allowing inference of hepatocyte, cholangiocyte, endothelial, and non-parenchymal cell lineages across clusters. b. UMAP projection of human adult liver cells and 3D H9-derived hepatic aggregates labeled by cell cluster. c. Pathway enrichment analysis comparing H9-derived hepatocyte clusters (Clusters 10 and 19) to adult primary hepatocyte clusters (Clusters 1, 2, 4, 7, 17, and 18), highlighting signaling pathways significantly upregulated in hPSC-derived populations.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Derivative Assay, Marker, Gene Expression, Labeling, Protein-Protein interactions

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Generation and functional characterization of mature hepatocytes from cryopreserved HBs a) Schematic differentiation protocol of hPSC-derived hepatocytes from cryopreserved hepatoblasts. b) Flow cytometry analysis showing the population of ALB+ and AFP+ cells with XAV939 or CHIR99021-treated hepatic cells over day-24 period. c) Microscopic (left) and Immunostaining (right) analysis, showing the proportion of ALB (green), and AFP (red) with XAV939 or CHIR99021-treated hepatic cells. Scale bar: 200µm (left), 50µm (right). d) Quantification of fold change in cell numbers with XAV939 or CHIR99021-treated hepatic cells treated in the presence or absence of FGF19. Student’s t-test. DATA are represented as mean ± SEM ( n = 7). e) Microscopic analysis showing hepatic aggregates treated with XAV939 or CHIR99021. Scale bar: 200µm. f) Immunostaining analysis showing the proportion of ALB/AFP (top) and ALB/CYP3A4 (bottom) in H9-derived hepatic aggregates treated with XAV939 or CHIR99021. Scale bar: 50µm. g) CYP3A4 activity in H9- and iPSC-derived hepatic aggregates at day12, 18 and 24 of maturation treated with XAV939 or CHIR99021. CHIR-treated groups exhibited higher CYP3A4 compared to both HB and XAV-treated groups. DATA are represented as mean ± SEM. One-way ANOVA. Statistical significance compared to HB and XAV groups is indicated in Figure (H9: n = 6, iPS n = 5). h) Representative kinetics of the oxygen consumption rate (OCR) in H9-derived day 24 mature hepatic aggregates cultured in indicated conditions (left). Comparison of each parameter in the day 24 mature hepatic aggregates (right). One-way ANOVA. DATA are represented as mean ± SEM from 4 technical replicates of 5 biologically independent experiments. i) Representative transmission electron microscope images of mitochondria in different treated hepatic aggregates under the indicated conditions. Scale bar: 5µm (top), 1µm (bottom). j) Quantification of cristae found in mitochondria under the indicated conditions. Data are from 3 biologically independent experiments, Student’s t-test. DATA are represented as mean ± SEM.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Functional Assay, Derivative Assay, Flow Cytometry, Immunostaining, Activity Assay, Cell Culture, Comparison, Transmission Assay, Microscopy

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Functional differentiation of cryopreserved H9-derived hepatic progenitors into mature hepatocytes. a. Schematic differentiation protocol of hPSC-derived hepatocytes from cryopreserved hepatoblasts. b. RT-qPCR analysis of gene expression in hepatic cells treated with XAV939 or CHIR99021 at different time point of differentiation. AL adult liver. One-way ANOVA. DATA are represented as mean ± SEM ( n = 4-6). c. Immunostaining analysis in hepatic aggerates treated with XAV939 or CHIR99021 showing the indicated proteins. Scale bar: 50µm.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Functional Assay, Derivative Assay, Quantitative RT-PCR, Gene Expression, Immunostaining

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Functional characterization of primary human hepatocytes (HepaSH) via metabolic profiling a. CYP3A4 activity in different treated PH-derived hepatic aggregates at day6, 12, 18 and 24. Day6 CHIR showed significantly higher values compared to all other groups One-way ANOVA. DATA are represented as mean ± SEM (n = 5). Significant differences are indicated in the figure. b. Representative kinetics of the oxygen consumption rate (OCR) measured in primary hepatocyte aggregates cultured in indicated conditions. c. Comparison of each parameter in primary hepatocyte aggregates. One-way ANOVA. DATA are represented as mean ± SEM from 4 technical replicates of 5 biologically independent experiments.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Functional Assay, Activity Assay, Derivative Assay, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: Wnt signaling modulation differentially regulates functional maturation and mitochondrial gene expression in hepatic aggregates derived from cryopreserved H9-derived hepatoblasts. a. Schematic differentiation protocol of H9-derived hepatocytes from cryopreserved hepatoblasts. b. Microscopic analysis showing hepatic aggregates in aggrewells (left), embedded in collagen (middle), and single cells in collagen (right) scale bars: 200µm. c. Flow cytometry analysis showing the population of ALB + and AFP + cells with XAV939 or CHIR99021-treated hepatic cells. d. RT-qPCR analysis of gene expression in hepatic cells treated with XAV939 or CHIR99021 across various culture formats. AL adult liver. One-way ANOVA. DATA are represented as mean ± SEM ( n = 3-5).

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: Functional Assay, Gene Expression, Derivative Assay, Flow Cytometry, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

doi: 10.1101/2025.06.13.659636

Figure Lengend Snippet: In Vivo functional assessment of H9-derived hepatocytes following transplantation. a. Human ALB secretion in the kidneys of NSG mice 4 weeks after transplantation with hepatic cells treated with XAV939 or CHIR99021. ALB secretion were compared among groups at week 4 using One-way ANOVA. Statistical significance is indicated in the figure. DATA are represented as mean ± SEM (n = 5). b. Confocal image of histological section in the kidney of NSG mouse after 4weeks of transplantation expressing human ALB and Ku80. Scale bar: 50 µm (top and middle), 400 µm (bottom). c. Human ALB secretion in TK-NOG mice post-transplantation of hepatic cells treated with XAV939 or CHIR99021. One-way ANOVA. DATA are represented as mean ± SEM (n = 15 in XAV and CHIR, n = 10 in HB). d. Confocal image of histological section in the liver of TK-NOG mouse after 16 weeks of transplantation of human primary hepatocytes (top row) and H9-derived XAV-treated hepatic cells (bottom row), expressing human ALB (green) and Ku80 (white) Scale bar: 100 µm (top), 50µm (bottom). ALB (green) and CYP3A4 (magenta). Scale bar: 200µm.

Article Snippet: To initiate definitive endoderm differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher: 11875093) supplemented with glutamine (2 mM, Thermo Fisher: 25030081), 4.5X10 -4 M monothioglycerol (MTG; Sigma: M6145), activin A (100ng/ml, R&D: 388-AC/CF), and CHIR99021 (2µM, TOCRIS: 4423).

Techniques: In Vivo, Functional Assay, Derivative Assay, Transplantation Assay, Expressing

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 1A. To determine which cell type binds mCRP in the brain after the intraperitoneal (i.p.). injection, double immunostaining of mCRP and different types of cell markers was conducted in the cortex and hippocampal DG region. Cell markers, including the neuronal marker NeuN, the microglial marker Iba1, the astrocyte marker GFAP, vascular marker Lectin and the endothelial cell marker CD31 and CD144, were applied individually. Correlation coefficients and R values were calculated for each protein pair detected and are shown. 1B Immunofluorescence analysis of isolated brain microvessels from mice treated with mCRP (purple) were stained with antibodies for endothelial cell components CD144 (red), vWF (green), CD31 (red) and its phosphorylation pCD31 (green). Nuclear stained with DAPI. The bar is 20 µm. 1C. Representative images of cortex stained with CD31 (red) and mCRP (green) and merged (yellow) are shown to observe mCRP deposits in CD31 positive regions. The yellow fluorescence intensities were quantified and compared among different genotype mice with i.p. mCRP treatment. p = 0.0016, n = 11-14 mice in each group. 1D. Representative images of double immunostaining of phos-CD31 (pCD31, green) and CD31 (red) and the merged images (yellow), nuclei stained with DAPI, on the cortex of WT mice, ApoE -/- mice and mice expressing different ApoE genotypes after i.p. treatment with PBS vs. mCRP are shown. Total CD31 and pCD31 were quantified by fluorescence intensity and the pCD31/CD31 ratio (ApoE4 p < 0.001, ApoE -/- p < 0.001) total CD31 (WT p = 0.03, ApoE3 p = 0.03, ApoE4 p = 0.001), n = 7-19 in each group. 1E. Western blots showed that mCRP increased the level of pCD31 and decreased the CD31 expression levels in the hippocampal region in ApoE4 mice but not in ApoE3 or ApoE2 mice. 1F. Proximity ligation assay (PLA) was performed on the cortex (upper panel) and hippocampal CA3 region (lower panel) to examine the binding of mCRP and CD31 in WT mice and mice expressing different ApoE genotypes after i.p. injection of PBS vs. mCRP. Positive PLA fluorescence signals are shown in orange; nuclei were stained with DAPI (blue). Quantifications of orange fluorescence were conducted and are shown: WT vs. ApoE4 p < 0.0001, ApoE2 vs. ApoE4 p < 0.0001, and ApoE3 vs. ApoE4 p = 0.0002 for cortex; ApoE2 vs. ApoE4 p = 0.0004 and ApoE3 vs. ApoE4 p = 0.0018 for CA3. n = 8 in each group. 1G. Representative images of primary CD31 + BECs from WT mice treated with mCRP in vitro at day 5 are shown. PLA (orange) was performed to detect colocalization/binding between mCRP (purple) and CD31 (green). pCD31 (red) was detected by a specific antibody, and the nuclei were stained with DAPI. The scale bar is 10 µm. 1H. To determine whether the direct effects of mCRP on endothelia are dose-dependent and/or time-dependent, primary CD31 + BECs were treated with various concentrations of mCRP for 1, 4, 8 and 24 h. Quantification is shown of the mCRP deposits on the surface of cells (upper left panel), the binding affinity of mCRP with CD31 (lower left panel), the CD31 expression level (upper right panel) and the ratio of pCD31 normalized against total CD31 (lower right panel) under different concentrations of mCRP and time course. mCRP (10 µg/ml) showed maximum binding to CD31 after 8 hours of incubation ( p = 0.0002). mCRP decreased the expression of CD31 within the first hour of incubation ( p = 0.04) and increased the levels of pCD31 after incubation for up to 24 hours ( p < 0.001) in a dose-dependent manner. At least three independent experiments were conducted for each condition. 1I. Primary BECs were transfected with CD31-targeting siRNA to effectively knockdown CD31 expression ( p < 0.001) before adding mCRP (10 µg/mL) stimulation. Fluorescence immunostainings with antibodies, mCRP (purple), CD31 (green) and pCD31 (red) were shown. Nuclear was stained with DAPI. The quantifications were shown that compared to control cells, BECs after silencing CD31 with siRNA had lower mCRP binding ( p = 0.04). 1J. Primary BECs were incubated with CD31-specific antibody followed by adding mCRP (10 µg/mL) stimulation. A significant decreased pCD31 levels were observed in BECs after blocking CD31 by the antibodies ( p < 0.001). Data are shown as the mean ± SEM. One or two -way ANOVA with Tukey’s post hoc test and Pearson or Spearman correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The scale bar is 50 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Injection, Double Immunostaining, Marker, Immunofluorescence, Isolation, Staining, Phospho-proteomics, Fluorescence, Expressing, Western Blot, Proximity Ligation Assay, Binding Assay, In Vitro, Incubation, Transfection, Knockdown, Control, Blocking Assay

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 2A. Representative images of double immunostaining of ApoE (green) and CD31 (red) and the merged images (yellow) of the cortex sections of WT vs. different ApoE knock-in mice are shown. The ApoE + fluorescence intensity was compared between PBS and mCRP-treated mice and reached a significant difference only in the ApoE2 mice ( p = 0.02). n = 11-14 mice in each group. 2B. PLA was applied to detect the binding of ApoE and CD31 on cortex sections from different ApoE genotype mice treated with PBS vs. mCRP. Positive PLA fluorescence signals are shown in orange, and nuclei are stained with DAPI (blue). Quantifications of orange fluorescence intensity of ApoE-CD31 binding in the cortex are shown and reached a significant difference only in ApoE2 mice ( p = 0.04). PLA (mCRP-CD31) levels were negatively correlated with PLA (ApoE-CD31) in the brain (r = -0.51, p = 0.005). n = 8 in each group. 2C. Representative images of primary CD31 + BECs treated with 0.3 µM ApoE2, ApoE3 or ApoE4 protein are shown. The interaction/binding between ApoE (purple) and CD31 (green) was detected by PLA (orange). The positive PLA signal and CD31 protein level were quantified and compared. ApoE4 protein had the lowest binding with CD31 compared to ApoE2 ( p = 0.0002) and ApoE3 ( p = 0.0013) proteins. In addition, ApoE4 exhibited the most significant decrease in CD31 expression in CD31 + BECs compared to ApoE2 ( p < 0.001) and ApoE3 ( p = 0.04). The scale bar is 50 µm. 2D. To examine how different ApoE isoform proteins influence the effect of mCRP on endothelia, primary CD31 + BECs were preincubated with different concentrations of ApoE2, ApoE3 or ApoE4 protein for one hour followed by the addition of 10 µg/mL mCRP for 6 hours. Quantification of the CD31 expression level (upper left panel), the binding of ApoE-CD31 (upper right panel) and mCRP-CD31 (lower left panel) and the pCD31/CD31 ratio (lower right panel) were conducted. *Three ApoE isoform proteins were compared for each concentration, and the statistical significance is shown. #The comparison among different doses of protein for one isoform of ApoE; at least three independent experiments were conducted. Data are shown as the mean ± SEM. One or two -way ANOVA with Tukey’s post hoc test and Pearson correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Double Immunostaining, Knock-In, Fluorescence, Binding Assay, Staining, Expressing, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 3A. After the i.p. treatment of PBS (top column) vs. mCRP (bottom columns), 3D images of cortex sections stained for CD31 (red) in WT, different ApoE-expressing and ApoE -/- mice were used to examine cerebrovasculature. The length of CD31-positive microvasculature in the cortex was quantified and compared between the PBS and mCRP groups in WT ( p = 0.01), ApoE3 ( p < 0.0001), ApoE4 ( p < 0.0001) and ApoE -/- ( p = 0.04) mice. # Comparison of ApoE genotypes in the PBS group, ApoE4 vs WT p = 0.04, ApoE2 p < 0.0001, ApoE3 p < 0.0001. n = 7-19 mice in each group. 3B. To examine the impact of elevated peripheral mCRP on cerebrovascular damage, we conducted immunostaining of a vascular damage biomarker, von Willebrand factor (vWF, green), and CD31 (red), in the cortex of WT vs. different ApoE knock-in mice. Representative images of each genotype treated with PBS vehicle (left columns) and mCRP (right columns) are shown. The numbers of vWF-positive endothelial cells in the cortex were compared among different genotype mice in the absence and presence of i.p. mCRP and showed statistical significance only in the ApoE4 group ( p = 0.04). A negative correlation between the length of CD31 + microvessels and the expression of vWF in the cortex was shown (r = -0.38, p = 0.0006). n = 5-6 in each condition. 3C. Western blots of different inflammatory and vascular-related proteins, including phosphorylated eNOS (p-eNOS), eNOS, NF-κB and CD144, in the cortex were conducted to examine the effects of mCRP on these proteins in each ApoE genotype. Peripheral mCRP significantly increased the expression of p-eNOS ( p = 0.04) and NF-κB ( p = 0.006) only in ApoE4 mice. 3D. Primary BECs were transfected with CD31-targeting siRNA to effectively knockdown CD31 expression ( p < 0.001) before adding mCRP (10 µg/mL) stimulation. Representative images of primary BECs silencing of CD31 with mCRP treatment were stained with antibodies vWF(green), NF-κB (red), p-eNOS (green). The nuclei was stained with DAPI. The quantifications were shown that lower levels of vWF ( p < 0.001), NF-κB ( p = 0.003) but higher level of p-eNOS ( p = 0.005) were found in siRNA group, compared with control group after mCRP treatment. At least three-time experiments were conducted. 3E. Different correlation analyses were conducted to examine the relationship between elevated peripheral mCRP and CD31 + microvessels in the brain. The graphs show that the length of CD31 + microvessels (y-axis) was positively associated with the CD31 level (r = 0.54, p < 0.0001) and the levels of PLA (ApoE-CD31) (r = 0.27, p = 0.03) but negatively associated with the levels of PLA (mCRP-CD31) (r = -0.65, p < 0.0001) and pCD31 (r = - 0.27, p = 0.04). Data are expressed as the mean ± SEM. Two-way ANOVA with Tukey’s post hoc test and Pearson or Spearman correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001. The scale bar is 50 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Staining, Expressing, Comparison, Immunostaining, Biomarker Discovery, Knock-In, Western Blot, Transfection, Knockdown, Control

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 4A and 4B. Representative images of i.p. mCRP-induced neuronal tau phosphorylation (pTau) in the cortex (A) and the CA3 region of the hippocampus (B) are shown. Double immunostaining of pTau (stained with PHF1, green) and neuronal marker (stained with NeuN, red); nuclei were stained with DAPI. Quantification of PHF1 levels in NeuN-positive cells showed statistical significance after i.p. mCRP in the cortex ( p = 0.006) and in the CA3 region ( p = 0.02) only in ApoE4, but not ApoE2 and ApoE3, mice. n =11-14 in each group. 4C. Representative images of CD68 + active microglia (red), Iba1 + microglia (green) and GFAP + astrocytes (purple) in the cortex of WT and ApoE knock-in mice after the i.p. treatment of PBS vs. mCRP are shown. Quantification of the microglial biomarkers CD68 ( p = 0.02) and Iba1 ( p = 0.0005) in the cortex showed significant differences between the PBS and mCRP groups only in ApoE4 mice, but the astrocyte biomarker GFAP ( p = 0.12) only showed tendency in the ApoE4 mice. n =11-14 in each group. 4D. Double immunostaining of CD8 (red), CD3 (green), and double-positive cells (yellow) was performed to study the transcytosis of T lymphocytes in the cortex after i.p. treatment with PBS (left columns) vs. mCRP (right columns) in WT and different ApoE knock-in mice. Quantification of CD8 + T lymphocytes and CD8 + /CD3 + T lymphocytes in the cortex and the comparisons between PBS vs. mCRP treatment in each genotype was conducted. mCRP significantly increased the number of T lymphocytes only in the WT ( p = 0.05) and ApoE4 ( p = 0.001) mice. n = 7-8 in each condition. 4E. Different correlation analyses using the data from all the mice used in the experiments were conducted to examine the relevance of different factors of the mCRP vs. ApoE to CD31 binding and the development of AD related pathology in the brain. The first row graphs show that the CD31 level (r = -0.48, p < 0.0001) was inversely related to PHF1 level in the brain, but PLA (ApoE-CD31) was not; the levels of PLA (mCRP-CD31) (r = 0.45, p = 0.02) and pCD31 (r = 0.40, p = 0.002) were positively correlated with PHF1 levels. The second row graphs show that the CD31 level (r = -0.35, p = 0.007) was inversely correlated with CD68/Iba1-positive cells in the brain, but PLA (ApoE-CD31) was not; the levels of PLA (mCRP-CD31) (r = 0.52, p = 0.02) and pCD31 (r = 0.45, p = 0.003) were positively correlated with double staining of CD68/Iba1 + cells. The third row graphs show that the number of CD8 + /CD3 + T lymphocytes (y-axis) was negatively associated with CD31 level (r = -0.28, p = 0.03) and positively associated with the levels of pCD31 (r = 0.54, p < 0.0001); however, there was no association with PLA (ApoE-CD31) or PLA (mCRP-CD31). The forth row graphs show that the length of CD31 + microvessels (x-axis) was negatively correlated with the levels of pTau (r = -0.56, p < 0.0001), the numbers of CD68 + /Iba1 + cells (r = -0.47, p = 0.0001), the numbers of CD8 + /CD3 + T lymphocytes (r = -0.48, p < 0.0001) and tended to be correlated with the levels of Aβ1-42 (r = -0.21, p = 0.06) in the brain. Data are expressed as the mean ± SEM. One or two-way ANOVA with Tukey’s post hoc test and Pearson or Spearman correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001. The scale bar: 50 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Phospho-proteomics, Double Immunostaining, Staining, Marker, Knock-In, Biomarker Discovery, Binding Assay, Double Staining

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: The human temporal cortex of healthy controls (n = 8) and AD patients (n = 10) was used for immunostaining. ApoE4 carriers (red dots) and ApoE2 carriers (green dots) are illustrated. 5A. Representative images of CD31 + microvessels (red), mCRP (green) and the merged images (yellow) in the temporal cortex are shown. The levels of microvessel mCRP and mCRP-CD31 binding were quantified and compared. AD brains had significantly higher mCRP immunostaining in microvessels (yellow) than control brains ( p =0.01). AD brains tended to have lower levels of microvessel CD31 expression ( p = 0.09) and higher levels of mCRP-CD31 binding detected by using PLA ( p = 0.09) than control brains. 5B. PLA was also performed to detect interaction/binding between ApoE and CD31 in the temporal cortex of healthy controls and AD patients. Representative images of positive PLA (orange), ApoE (green) and CD31 (purple) staining are shown. The levels of microvessel ApoE and ApoE-CD31 were quantified and compared. AD brains had significantly lower levels of microvessel ApoE expression ( p = 0.02) and ApoE-CD31 binding ( p = 0.03) than control brains. 5C. Western blots for pCD31 and CD31 expression in the temporal cortex were performed and quantified after normalization against β-actin and compared between AD and controls. AD brains had higher levels of pCD31 ( p = 0.02), tended to have lower levels of total CD31 ( p = 0.07), and had a higher pCD31/CD31 ratio ( p = 0.03) than control brains. 5D. Double immunostaining of temporal cortex sections with CD31 (red), pCD31 (green) and the merged images (yellow) were conducted to examine CD31 phosphorylation and microvessel integrity. AD brains had higher brain pCD31 levels ( p = 0.05), pCD31/CD31 ratio ( p = 0.03), and microvessel pCD31 ( p = 0.01). Measurement of the lengths of CD31 + microvessels from 3D images revealed shorter lengths in AD brains than in controls ( p < 0.001). 5E. Correlation analyses were conducted to investigate the mCRP-ApoE-CD31 pathway and brain AD pathology in humans. In the first row, the microvessel pCD31 level (y-axis) was positively associated with mCRP (r = 0.67, p = 0.003) and mCRP-CD31 binding (PLA) (r = 0.53, p = 0.01) and negatively associated with ApoE-CD31 binding (PLA) (r = -0.49, p = 0.04). Microvessel length (y-axis) was negatively associated with pCD31 (r = - 0.55, p = 0.02) but positively associated with ApoE-CD31 (r = 0.69, p = 0.002). In the second row, the Braak stage (y-axis) was negatively associated with CD31 expression (r = -0.49, p = 0.04), ApoE-CD31 binding (r = -0.61, p = 0.007) and microvessl length (r = - 0.70, p = 0.001); in contrast, the Braak stage was positively associated with mCRP-CD31 binding (r = 0.51, p = 0.03) and microvessel pCD31 expression (r = 0.76, p = 0.0003). In the third row, the CAA (y-axis) was negatively associated with CD31 expression (r = - 0.50, p = 0.03), ApoE-CD31 binding (r = -0.66, p = 0.003) and microvessl length (r = - 0.67, p = 0.003); in contrast, CAA was not associated with the level of mCRP-CD31 binding but was positively associated with microvessel pCD31 expression (r = 0.54, p = 0.02). In the fourth row, the Mini-Mental State Exam (MMSE) score (y-axis) was positively associated with CD31 expression (r = 0.54, p = 0.03), the level of ApoE-CD31 binding (r = 0.52, p = 0.04) and microvessl length (r = 0.83, p < 0.0001); in contrast, MMSE score tended to be negatively associated with mCRP-CD31 binding (r = -0.47, p = 0.07) and significantly associated with the level of microvessel pCD31 + cells (r = -0.69, p = 0.003). Data are expressed as the mean ± SEM. Student’s t-test and Pearson or Spearman correlation tests were used. * p < 0.05, ** p < 0.01, *** p < 0.001. The scale bars are 100 µm, 20 µm, and 10 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Immunostaining, Binding Assay, Control, Expressing, Staining, Western Blot, Double Immunostaining, Phospho-proteomics

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 6A. The schematic showed CD31 + brain endothelial cells (BECs) were isolated by flow sorting and subjected to RNA sequencing from the mouse brain treated with i.p. PBS vs. mCRP. 6B-D. GSEA based on their differential gene expression was conducted to study the prominent pathways enriched in gene lists from different ApoE genotypes for AD pathogenesis. For the comparison between mCRP and PBS in each genotype, genes were ranked by GSEA based on their differential expression level. Four pathways, including 1) oxidative phosphorylation, 2) mitochondrial metabolism and respiration, 3) Alzheimer’s disease and 4) mTORC1 signaling, were upregulated by mCRP in ApoE4 mice but were downregulated in ApoE2 mice, while ApoE3 mice were in the middle (6B). In contrast, another four pathways, including 1) the histone lysine methyltransferase pathway, 2) synapse formation, 3) Notch signaling and 4) vasculogenesis, were upregulated by mCRP in ApoE2 mice but downregulated in ApoE4 mice, while ApoE3 mice were in the middle (6C.) The top or bottom of the ranked gene list in each ApoE comparison was evaluated using the enrichment score (green line) to determine whether a candidate pathway was significant. Black vertical lines mark positions where members of a particular pathway appear in the ranked list of genes. NES, normalized enrichment score; FDR, false discovery rate and q values. 6D shows the heatmaps of z-scored gene intensities of the differentially expressed genes enriched in the pathways/modules of oxidative phosphorylation and Alzheimer’s disease for ApoE2 and ApoE4 CD31 + BECs after the i.p. treatment. The key factors involved in the Alzheimer’s disease pathway that overlapped with the oxidative phosphorylation pathway were ranked and labeled by different intensities of yellow color. 6E. The RNAseq data were used for the functional enrichment analysis of the different signatures between ApoE4 and ApoE2 after the i.p. treatment of PBS vs. mCRP by using the ToppCluster tool (FDR correction, p < 0.05). The red/green box represents the top genes with up/downregulated expression in the ApoE4 mCRP vs PBS comparisons and down/upregulated expression in the ApoE2 mCRP vs PBS comparisons. The genes were connected to form a hub biological process and pathway (blue box). 6F. The CD31-related pathways are listed and shown according to NES comparison between mCRP vs PBS in each ApoE genotype (left panel). The network of CD31 and ApoE together with other components involved in biological processes, including vasculogenesis, leukocyte migration and Ras signaling pathways, is illustrated (right panel). Data are expressed as the mean ± SEM. One or two-way ANOVA with Tukey’s post hoc test was used. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Isolation, RNA Sequencing, Gene Expression, Comparison, Quantitative Proteomics, Phospho-proteomics, Labeling, Functional Assay, Expressing, Migration, Protein-Protein interactions

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: This study demonstrated a novel pathological mechanism, the competition of ApoE and mCRP to CD31binding, for cerebrovascular neuroinflammation resulting in an early stage of AD pathogenesis in the brain. During the chronic stage of peripheral inflammation, pCRP proteins disassociate into mCRP. mCRP binds to CD31 on microvessels to increase CD31 phosphorylation (pCD31), cause damage to the cerebrovasculature and induce extravasation of T lymphocytes into the brain, leading to AD pathogenesis (ApoE4>ApoE3>ApoE2). This process is antagonized by ApoE-CD31 binding (ApoE2>ApoE3>ApoE4) to block mCRP-CD31 binding and differentially regulate pathways (mitochondrial function, epigenetics and vasculogenesis) to intervene in the neurodegenerative process of AD.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Phospho-proteomics, Binding Assay, Blocking Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Personalized cardiovascular risk assessment in Rheumatoid Arthritis patients using circulating molecular profiles and their modulation by TNFi, IL6Ri, and JAKinibs.

doi: 10.1016/j.biopha.2024.116357

Figure Lengend Snippet: Fig. 5. Serum from RA patients with high CVD risk induced the upregulation of CVD-related markers in endothelial cells (ECs) and monocytes, which was prevented by biological and targeted synthetic DMARDs.- (A and C) Heat map showing the changes promoted in CV- related proteins on endothelial cells (A) and monocytes (C) treated in vitro with serum from RA patients belonging to cluster 3 (showing the highest inflammatory profile) in comparison with serum from HD. Levels of inflammatory proteins are expressed as NPX (normalized protein expression, arbitrary units on a Log 2 scale), and have undergone a clustering analysis to aid interpretation. Proteins showing significant alterations (FDR-adjusted p value <0.05) are detached in bold. (B and D) Predicted and validated protein-protein interactions among proteins potentially modulated by RA-C3 serum, using the STRING platform. Protein networks showing the relationship between differen tially expressed proteins are displayed. Below are displayed tables containing functional enrichment analysis of biological processes by using the Gene ontology platform. (E and G) Changes in cardiovascular mediators’ levels in cell lysates of endothelial cells and monocytes cultured with HD or RA-C3 serum, either, in the presence or in the absence of TNFi, IL6Ri or JAKinibs. *p<0.05, **p<0.01, ***p<0.001. (F and H) Venn diagram showing the common and differential proteins modulated in endothelial cells and monocytes by TNFi, IL6Ri or JAKinibs.

Article Snippet: Neutrophils and monocytes purified from HD and primary human umbilical vein endothelial cells (HUVECs) were subjected to a 12-h treatment (neutrophils) or 24-h treatment (monocytes and HUVECs)at 37 ◦C. with the serum from HD, the serum of RA patients that had suffered previous CV events (RA-CVD), or the serum from patients belonging to the cluster 3 (RA-C3), either in the presence or absence of TNFi (Etanercept, - European Pharmacopoeia reference standard, Strasbourg, France- 10 ug/mL), IL6Ri (Sarilumab, -Selleckchem, Planegg, Germany- 10 ug/mL,) or JAKinibs (Baricitinib, -Tocris Bioscience, Bristol, UK- 10 ug/mL).

Techniques: In Vitro, Comparison, Expressing, Protein-Protein interactions, Functional Assay, Cell Culture

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Personalized cardiovascular risk assessment in Rheumatoid Arthritis patients using circulating molecular profiles and their modulation by TNFi, IL6Ri, and JAKinibs.

doi: 10.1016/j.biopha.2024.116357

Figure Lengend Snippet: Fig. 4. Changes in clinical and molecular profiles of rheumatoid arthritis patients treated with b-DMARDs or ts-DMARDs. (A) RA patients’ (n = 83) changes in clinical features at 6 months of TNFi, IL6Ri and JAKinibs therapies, including Disease Activity Score (DAS28), Clinical Disease Activity Index (CDAI), and Simple Disease Activity index (SDAI). At the initial timepoint (t0), each data point corresponds to an individual patient, with a subsequent connection established to the respective measurement obtained at the 6-month interval. (B) Heat map showing differential levels of circulating inflammatory proteins in plasma of patients with RA after 6 months of TNFi, IL6Ri and JAKinibs therapies (Δ T6-T0). Levels of inflammatory proteins are expressed as log 2,and have undergone a clustering analysis to aid interpretation. (C) RA patients’ changes in NETosis bioproducts (elastase and nucleosomes) and lipoperoxides after 6 months of TNFi, IL6Ri and JAKinibs therapies. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001.

Article Snippet: Neutrophils and monocytes purified from HD and primary human umbilical vein endothelial cells (HUVECs) were subjected to a 12-h treatment (neutrophils) or 24-h treatment (monocytes and HUVECs)at 37 ◦C. with the serum from HD, the serum of RA patients that had suffered previous CV events (RA-CVD), or the serum from patients belonging to the cluster 3 (RA-C3), either in the presence or absence of TNFi (Etanercept, - European Pharmacopoeia reference standard, Strasbourg, France- 10 ug/mL), IL6Ri (Sarilumab, -Selleckchem, Planegg, Germany- 10 ug/mL,) or JAKinibs (Baricitinib, -Tocris Bioscience, Bristol, UK- 10 ug/mL).

Techniques: Activity Assay, Clinical Proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Personalized cardiovascular risk assessment in Rheumatoid Arthritis patients using circulating molecular profiles and their modulation by TNFi, IL6Ri, and JAKinibs.

doi: 10.1016/j.biopha.2024.116357

Figure Lengend Snippet: Fig. 6. Serum from RA patients with high CVD risk induced NETosis and neutrophils activation, which was prevented by b-DMARDs and ts-DMARDs.- (A- B) Bar graphs showing the changes promoted in gene expression levels of several activation markers on neutrophils treated in vitro with serum from RA patients belonging to cluster 3 (showing the highest inflammatory profile) either in the presence or in the absence of TNFi, IL6Ri or JAKinibs inhibitors. All experiments were compared with neutrophils treated with HD serum, set at 100% in each panel. (Significance at p<0.05, a, vs neutrophils treated with HD serum; b, vs neutrophils treated with RA-C3 serum). (C) Representative micrographs of neutrophil extracellular traps (NETs) from HD neutrophils treated in vitro with serum from RA patients belonging to cluster 3 (showing the highest inflammatory profile) either in the presence or in the absence of TNFi, IL6Ri or JAKinibs inhibitors. NETs were visualized by using a Nikon Eclipse-Ti-S fluorescence microscope 20x objective. Scale bar 10 micrometers. (D) Concentration of cell-free nucleosomes and cell-free elastase in supernatants from cell cultures of HD neutrophils performed as above described (Significance at p<0.05, a, vs neutrophils treated with HD serum; b, vs neutrophils treated with RA-C3 serum).

Article Snippet: Neutrophils and monocytes purified from HD and primary human umbilical vein endothelial cells (HUVECs) were subjected to a 12-h treatment (neutrophils) or 24-h treatment (monocytes and HUVECs)at 37 ◦C. with the serum from HD, the serum of RA patients that had suffered previous CV events (RA-CVD), or the serum from patients belonging to the cluster 3 (RA-C3), either in the presence or absence of TNFi (Etanercept, - European Pharmacopoeia reference standard, Strasbourg, France- 10 ug/mL), IL6Ri (Sarilumab, -Selleckchem, Planegg, Germany- 10 ug/mL,) or JAKinibs (Baricitinib, -Tocris Bioscience, Bristol, UK- 10 ug/mL).

Techniques: Activation Assay, Gene Expression, In Vitro, Fluorescence, Microscopy, Concentration Assay