Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Expressing, Construct

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: RNA Sequencing, Comparison

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Expressing

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Isolation, Marker, Injection, Dissection

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

Techniques: Functional Assay, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.23.614610

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

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

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

Journal: Nature Communications

Article Title: Tumor-associated reactive astrocytes aid the evolution of immunosuppressive environment in glioblastoma

doi: 10.1038/s41467-019-10493-6

Figure Lengend Snippet: Microglia loss-of-function model with transcriptional profiling of astrocytes. a Illustration of the workflow to set-up a human slice model combined with microglia depletion and tumor injection. Entry cortex was taken from the operation theatre, sliced within 10 min into 300 µm slices and cultured in serum-free conditions. In a 3 days time-course, slices are incubated with 1 mg/ml of Chlodronate to deplete microglia or control condition. 20,000 primary serum-free cultured ZsGreen tagged tumor cells were injected. After 4 days of tumor growth, astrocytes and microglia were purified for RNA-seq analysis (Detailed workflow is given in the Supplementary Fig. ) b Representative staining of IBA-1 confirmed a robust depletion of microglia in human slices without loss of NeuN expression in neurons. c Representative staining of tumor injection in a time dependent manner. d Analysis of differentially expressed genes of purified astrocytes in Microglia(+) or Microglia(−) condition. Genes are ordered based on fold-change of gene expression between the control (Tumor(−)) and tumor injection (Tumor(+)), with blue indicating an > = 12 fold higher expressed in control samples and red an > = 12 fold higher expression in tumor injected samples. Lines indicate the differences of the fold-change rank (top 30 genes) between Microglia(+) and Microglia(−) condition. The R-code and detailed description is given in the source data. e Gene Set Expression Analysis (GSEA) of ranked gene expression of Microglia(+) or Microglia(−) condition indicate the enrichment differences of the JAK/STAT pathway. f Cytokine protein level in all conditions. Exact values, as well as statistical analysis are given in the source file. g Representative immunostaining of tumor infiltration after 4 days of culture and quantification h , i . Exact values of cell numbers are given in the source file. j Gating strategy to purify astrocytes from FACS data. Detailed plots for gating are given in the Supplementary Fig. . k FACS data analyzed by T-SNE, colors indicate the experimental conditions. I T-SNE map with STAT3-P (left) and Ki-67 (right) intensity, colors indicate the intensity (red: high intensity, blue: low intensity). P -values are determined by one-way ANOVA ( f , h , i ) adjusted by Benjamini–Hochberger ( f , h , i ) or False-Discovery Rate ( e ) for multiple testing. Data is given as mean ± standard deviation

Article Snippet: Anti- Ki67 (Rabbit) , Abcam, Cambridge, UK , Ab15580.

Techniques: Injection, Cell Culture, Incubation, Purification, RNA Sequencing Assay, Staining, Expressing, Immunostaining, Standard Deviation

Journal: Nature Communications

Article Title: Tumor-associated reactive astrocytes aid the evolution of immunosuppressive environment in glioblastoma

doi: 10.1038/s41467-019-10493-6

Figure Lengend Snippet: Reagent sources

Article Snippet: Anti- Ki67 (Rabbit) , Abcam, Cambridge, UK , Ab15580.

Techniques: Conjugation Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK is highly expressed in and required for Th17 cells. (A) Naïve CD4 T cells were cultured under each subset differentiation conditions for 3 days. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were introduced with a control empty vector (control) or a CRE recombinase-expressing vector (RV-Cre) to induce Fak deletion and cultured under Th17-polarizing conditions for 3 days (B) or various subset-polarizing conditions (C) for 3 days. (B) GFP+ cells were sorted. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (C) The expression of GFP, IFN-γ, IL-4, IL-17A, and FOXP3 was analyzed by flow cytometry (top). The statistical analysis was performed on pooled data from five independent experiments (bottom). Error bars represent the standard deviation. The significance of differences between groups was determined by one-way ANOVA (A) and Student t test (B, C) . ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK affects Th17 cell differentiation program. (A, B) Naïve CD4 T cells from Fak fl/fl mice were cultured and sorted as Figure 1B . (A) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (B) Transcript levels of Il17a , Rorc , Il23r , and Foxp3 were measured by RT-qPCR. (C, D) Naïve CD4 T cells were introduced with either the control vector (MSCV-LMP) or Fak shRNA vectors (#1, #2, and #3) and cultured under Th17-polarizing conditions for 3 days. (C) The transcript level of Fak was measured by RT-qPCR (left) and protein level of FAK was measured by immunoblot analysis (right). (D) IL-17A+ cells among the vector-transduced cells (GFP+) were measured by flow cytometry (left). GFP+ cells were sorted and the transcript level of Il17a was measured by RT-qPCR (right). All of RT-qPCR data were normalized to Gapdh . (E–G) Naïve CD4 T cells were transduced with control or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and subjected to RNA-seq analysis. (E) Scatter plot of RNA-seq data. (F) Gene ontology analysis of differentially expressed genes (DEGs) from control and RV-Cre-transduced Th17 cells. (G) Heatmap of immune/inflammatory response-related genes among the DEGs from control and RV-Cre-transduced Th17 cells. All of RT-qPCR data were normalized to Gapdh . Data in (A–D) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Plasmid Preparation, shRNA, Western Blot, Transduction, RNA Sequencing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK deficiency ameliorates the severity of EAE. (A, B) Naïve CD4 T cells from WT and Fak fl/fl Rorc cre mice were cultured under polarizing conditions toward each different subset for 3 days. Transcript levels of Ifng for Th1 cells, Il4 for Th2 cells, Il17a for Th17 cells, and Foxp3 for Treg cells were measured by RT-qPCR (A) and the percentage of IL-17A+ cells under Th17-polarizing conditions was measured by flow cytometry (B) . (C) EAE was induced in control (WT, n = 10) and Fak fl/fl Rorc cre (n = 10) mice as described in the Materials and Methods section. The symptoms of EAE were monitored every day and clinical scores were evaluated after EAE induction. (D, E) Histopathological analysis of lumbar spinal cords from control and Fak fl/fl Rorc cre mice at the peak of the disease. (D) H&E stained sections of spinal cords. Arrows indicate the inflammatory foci. (E) Immunohistochemical staining of myelin basic protein (MBP). Arrows indicate the more preserved myelin in Fak fl/fl Rorc cre mice compared with control mice. (F) IL-17A+ and IFNγ+ cells (left) and FOXP3+ cells (right) among CNS-infiltrating CD4 T cells were measured by flow cytometry. (G–J) The percentage and absolute number of CNS-infiltrating mononuclear cells were measured. (G) CD4+ cells, (H) IL-17A+ cells, (I) IFNγ+ cells, and (J) FOXP3+ cells. (K) Transcript levels of Il17a , Rorc , Il23r , Ifng , and Foxp3 in CNS-infiltrating mononuclear cells were measured by RT-qPCR. Data were normalized to Gapdh . Data in (F–K) are pooled from ten independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Flow Cytometry, Control, Staining, Immunohistochemical staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates the STAT3 signaling pathway in Th17 cells. (A) Naïve CD4 T cells were cultured under various differentiation conditions for the indicated time periods and the transcript levels of Fak , Il17a , Rorc , and Il17f were measured by RT-qPCR. (B) Naïve CD4 T cells were introduced with control vector or Rorc -expressing vector ( Rorc O/X) and cultured in Th17-polarizing conditions for 3 days. Transcript levels of Rorc , Il17a , and Fak were measured by RT-qPCR. (C) Naïve CD4 T cells were cultured under Th17-polarizing conditions with dose-dependent treatment of GSK805 for 3 days. Transcript levels of Il17a and Fak were measured by RT-qPCR. (D, E) Naïve CD4 T cells from Fak fl/fl mice were cultured as described in Figure 1B and GFP+ cells were sorted. (D) Each protein level was measured by immunoblot analysis (left) and the ratios of pSTAT3/STAT3 and pSTAT5/STAT5 were calculated by densitometry (right). (E) Transcript level of Il2 was measured by RT-qPCR (left) and the protein level of IL-2 from the supernatants was measured by ELISA (right). All of RT-qPCR data were normalized to Gapdh . Data in (A, B, D, E) were pooled from three independent experiments, and data in (C) from five. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Control, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates Th17 cell differentiation through the NF-κB pathway. (A) Naïve CD4 T cells from Fak fl/fl mice were transduced with control vector or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and rested in normal media for 2 days and serum-free medium for an additional 8 hours. The cells were restimulated with anti-CD3/CD28 antibodies for the indicated time periods, and nuclear/cytoplasmic extracts were prepared. Each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were transduced with RV-Cre and cultured under Th17-polarizing conditions for 3 days (B) and 16 hours (C) and GFP+ cells were sorted. (B) Transcript level of Socs3 was measured by RT-qPCR. (C) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were reacted with an anti-RelA antibody, and precipitated DNA fragments were measured by qPCR. Isotype-matching IgG was used as a negative control. (D–F) Il17a promoter activity was measured through luciferase assay. (D) EL4 cells were transfected with pGL3- Il17a promoter vector and control or Fak siRNA, and the cells were rested for 20 hours. After transfection, the cells were divided into non-stimulated or stimulated groups, with the stimulated groups received 4 hour stimulation with PMA/ionomycin. (E) EL4 cells were transfected with the pGL3- Il17a promoter vector and rested for 20 hours with or without PDTC treatment (1 μM). (F) EL4 cells were transfected as described in (D) and rested for 20 hours with 1 μM PDTC treatment. (G–I) Naïve CD4 T cells from Rela fl/fl mice were introduced with a control empty vector (WT) or a Cre recombinase expressing vector (p65 KO) to induce Rela deletion and cultured under Th17-polarizing conditions for 3 days. (G) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (H) GFP+ cells were sorted and transcript level of Rela , Il17a , Rorc , and Il23r were measured by RT-qPCR. (I) Naïve CD4 T cells from Rela fl/fl mice were cultured as described in (G) and additionally treated with vehicle (control) or FAK inhibitor (PND1186, 1 μM). IL-17A+ and FOXP3+ cells were measured by flow cytometry. RT-qPCR data in (B, H) were normalized to Gapdh . Data in (A–I) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Transduction, Control, Plasmid Preparation, Cell Culture, Western Blot, Quantitative RT-PCR, Binding Assay, Negative Control, Activity Assay, Luciferase, Transfection, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: A FAK inhibitor blocks differentiation of Th17 cells in vitro. (A–E) Naïve CD4 T cells were cultured under Th17- or Treg-polarizing conditions with dose-dependent treatment of PND1186 for 3 days. IL-17A+ and FOXP3+ cells were measured by flow cytometry (A) and transcript levels of Il17a and Foxp3 were measured by RT-qPCR (C) in Th17 cells. FOXP3+ cells were measured by flow cytometry (B) and transcript level of Foxp3 was measured by RT-qPCR (D) in Treg cells. (E–G) Naïve CD4 T cells were cultured under Th17-polarizing conditions with vehicle (control) or PND1186 (1 μM) treatment for 3 days (E, G) and the indicated time periods (F) . (E) pSTAT3 and pSTAT5 levels were measured by flow cytometry. (F) Nuclear or cytoplasmic extracts were prepared from cultured cells, and each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (G) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were incubated with an anti-RelA antibody and the precipitated DNA fragments were measured by qPCR. An isotype-matching IgG was used as a negative control. RT-qPCR data in (C, D) were normalized to Gapdh . Data in (A–G) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Western Blot, Binding Assay, Incubation, Negative Control, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Airway in adult normal nonsmoker human lung was analyzed for club cell subtypes. Club cells were identified as SCGB1A1 + cells (green) and subsets were identified using various markers in the single cell sequencing. a Secretoglobin family 3A member 1 (SCGB3A1) (red) and secretory leukocyte protease inhibitor (SLPI) (purple). b SCGB1A1 (green), mucin 5B (MUC5B; purple). Club cells that only express SCGB1A1 but no secondary markers are denoted with a white arrow. Club cells expressing multiple markers are denoted with a white arrowhead. Scale bar = 20 μm.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Sequencing, Protease Inhibitor, Expressing

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Differentially expressed genes in progenitor vs. effector club cells a .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Most commonly enriched genes in each club cell subcluster.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Expressing, Binding Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Assessment of epithelial cell differentiation using Slingshot pseudotime value. Shown are epithelial cells differentiating in the secretory pathway including basal cells (BC, pink), intermediate cells (IC, orange), the progenitor club cell cluster (CC1, red), the effector club cell cluster (CC3, yellow), the proliferating club cell cluster (CC2, blue) and mucous cells (MC, green) for nonsmokers (NS) and smokers (S) plotted separately. Superimposed on each tSNE plot, the Slingshot pseudotime analysis shows the path of differentiation assuming basal cells as a starting point. b Pseudotime analysis. The extent of differentiation from the starting point was evaluated using a pseudotime analysis separately for nonsmokers (NS) and smokers (S). The results for each cluster have been plotted to enable pairwise comparisons. Significance of differences in the differentiation of each cluster is indicated by the p value to the right of the graph. p values were determined using a t test applied to the pseudotime values extracted by Slingshot. c Stem-progenitor activity of CC1 correlates with elevated KRT5 expression. Expression levels measured by the number of unique molecular identifiers (UMI) detected per cell during single cell RNA sequencing were plotted for KRT5 and KRT7 as a function of cluster for NS and S. Significant differences between NS and S are indicated by p values. Note that KRT5 levels in CC1 in NS have a mean that is greater than KRT5 expression in IC. KRT5 levels are considered a marker of stem-progenitor cells and are commonly thought to steadily fall as differentiation proceeds (see Supplementary Fig. ). Expression of high levels of KRT5 in CC1 support CC1 as a branch point in the trajectory. At the same time, KRT7, a marker of club cells (see Supplementary Fig. ), is clearly elevated in CC1 confirming the identity of CC1 as club cells rather than basal cells. d Change in branch point in smokers correlates with elevated SPDEF activity. Significant changes in expression levels of genes known to be up- or down-regulated by SPDEF were assessed in CC1 and CC3. Up-regulated genes included CEACAM5 and GCNT3. Down-regulated genes included SCGB1A1, SCGB3A1, LTF, and AQP5. Note that AQP5 is also up-regulated by oxidant stress such as exposure to cigarette smoke, potentially explaining the lack of down-regulation by smoking in CC1 and CC3. Significance of difference for panels c and d was determined based on the combined conditions of an adjusted p value < 0.05 (Bonferroni corrected) and a log fold change > 0.25. Violin plots in panels b – d include box-and-whisker plots showing the mean, 2nd and 3rd quartiles (box), range from minimum to maximum (whiskers), and outliers (dots).

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Differentiation, Activity Assay, Expressing, RNA Sequencing, Marker, Whisker Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Nonsmoker primary small airway epithelium (SAE) basal cells were cultured in air–liquid interface (ALI) culture for 28 days. a Morphology of the cultures at day 7 and 28. Shown are cross sections of the ALI culture, hematoxylin and eosin stain. b , c Multicolor immunofluorescence assessment of club cell subtypes. All club cells were identified using SCGB1A1 marker (green); effector club cells were further identified by the co-expression of MUC5B (panel b red) or SCGB3A1 (panel c red). DAPI identifies the nucleus of all cells. The number of days after establishment of the air liquid interface is noted. Green arrowheads mark cells that are SCGB1A1 + club cells that lack other markers. Orange arrowheads mark cells that are positive for SCGB1A1 and either MUC5B or SCGB3A1. Red arrowheads mark cells that are positive for MUC5B or SCGB3A1, but express little if any SCGB1A1. Scale bars are 50 μm. d Gene expression on ALI over time. TaqMan probes for genes enriched in effector club cells were assessed by qPCR and normalized to an 18S rRNA control. Probes were tested at days 0, 7, 14, and 28 of SAE nonsmoker cells differentiated in ALI. Error bars represent standard deviation among three different ALI cultures. Genes assessed for expression include MUC5B, SCGB1A1, SLPI, PIGR, and LYZ.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Culture, H&E Stain, Immunofluorescence, Marker, Expressing, Gene Expression, Control, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Smoking-related reprogramming of small airway epithelium club cells.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Surface expression of ( A, C ) CCR3 or ( B, D ) CCR10 on murine neutrophils obtained from ( A, B ) the gut, blood, and bone marrow (BM) 3 dpi with STm, or ( C, D ) the bronchoalveolar lavage (BAL), blood, and bone marrow 1 dpi with Ab, analyzed by flow cytometry. Left panels show representative histograms of ( A, C ) CCR3 or ( B, D ) CCR10 expression on the surface of neutrophils (gated on live, CD45 + CD11b + Ly6G + cells) from ( A, B ) the gut (blue), blood (red), and bone marrow (BM; black) or ( C, D ) BAL (blue), blood (red), and bone marrow (BM; black). Right panels show the percentage of ( A, C ) CCR3 + or ( B, D ) CCR10 + neutrophils obtained from ( A, B ) gut, blood, and BM or ( C, D ) BAL, blood, and BM. Data are from six independent experiments. ( E–H ) Uninfected bone marrow neutrophils were unstimulated or treated with the indicated stimuli for 4 hr. Surface expression of ( E, G ) CCR3 and ( F, H ) CCR10 on neutrophils was determined by flow cytometry. Left panels show representative histograms of ( E, G ) CCR3 or ( F, H ) CCR10 surface expression after stimulation with: ( E, F ) cytokines IFNγ + TNFɑ + GM-CSF (blue); fMLP (magenta); phorbol 12-myristate 13-acetate (PMA) (purple); lipopolysaccharide (LPS) (red); ( G, H ) cytokines IFNγ + TNFɑ + Granulocyte-macrophage colony stimulating factor (GM-CSF, blue); beads alone (magenta); cytokines plus beads (red). Right panels show the percentage of ( E, G ) CCR3 + or ( F, H ) CCR10 + neutrophils following stimulation with the indicated stimuli. US = unstimulated. Data shown are pooled from two independent experiments. ( I, J ) Bone marrow cells enriched for neutrophils were infected with opsonized STm at a multiplicity of infection (MOI) = 10 for 1 hr with (violet) or without (red) pretreatment with cytochalasin D for 30 min before infection. Surface expression of ( I ) CCR3 or ( J ) CCR10 was determined by flow cytometry. Data are from two independent experiments. Left panels show representative histograms of surface receptor staining on neutrophils, and right panels show the percentages. ( A–J , right panels) Bars represent the mean ± standard deviation (SD). ( A–D ) Data were analyzed by one-way analysis of variance (ANOVA) for paired samples (non-parametric Friedman test), assuming non-normal distribution and non-equal SD given the differences in the variance among the groups, followed by Dunn’s multiple comparisons test. ( E–J ) Data were analyzed by one-way ANOVA for paired samples, applying the Greenhouse–Geisser correction given the differences in variance among the groups; Bonferroni’s multiple comparison test was performed to compare between relevant stimulation conditions. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant.

Article Snippet: Antibody , Anti-mouse CCR10 (Rat monoclonal; unconjugated) , R&D Systems , Clone: 248918; Cat#MAB2815; RRID: AB_2074258 , In vitro signaling blockade (5 µg/100 µl).

Techniques: Expressing, Flow Cytometry, Infection, Staining, Standard Deviation, Comparison

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Surface expression of CCR3 and CCR10 on neutrophils obtained from the gut of WT mice ( n = 19, pooled from six independent experiments) infected with STm for 3 days, analyzed by flow cytometry. ( B ) Percentage of CCR3 + and CCR10 + neutrophils obtained from the gut, blood, and bone marrow of Ccl28 +/+ ( n = 19) and Ccl28 −/− mice ( n = 14) infected with STm for 3 days, analyzed by flow cytometry. ( C ) Surface expression of CCR3 and CCR10 on neutrophils obtained from the bronchoalveolar lavage (BAL) of WT mice ( n = 8, pooled from two independent experiments) infected with Ab for 1 day, analyzed by flow cytometry. ( D ) Percentage of CCR3 + neutrophils (WT n = 9; Ccl28 −/− n = 8) and CCR10 + neutrophils (WT n = 4; Ccl28 −/− n = 4) obtained from the BAL, lung, blood, and bone marrow of WT and Ccl28 −/− littermates infected with Ab for 1 day, analyzed by flow cytometry. ( A, C ) Left panels show representative contour plots, and right panels show the percentages of neutrophils expressing the indicated receptor on their surface. Symbols represent data from individual mice, bars represent the geometric means.

Article Snippet: Antibody , Anti-mouse CCR10 (Rat monoclonal; unconjugated) , R&D Systems , Clone: 248918; Cat#MAB2815; RRID: AB_2074258 , In vitro signaling blockade (5 µg/100 µl).

Techniques: Expressing, Infection, Flow Cytometry

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Neutrophils enriched from wild-type mouse bone marrow were infected at multiplicity of infection (MOI) = for 5 min to 4 hr with ( A, B ) opsonized Salmonella enterica serovar Typhimurium (STm) or ( C, D ) Acinetobacter baumannii (Ab). ( A, C ) Surface CCR10 or ( B, D ) intracellular CCR10 staining was detected by flow cytometry. Neutrophils were obtained from ( E ) the gut, blood, and bone marrow 3 dpi with STm or ( F ) bronchoalveolar lavage (BAL), blood, and bone marrow 1 dpi with Ab. Surface (clear histograms) and intracellular (filled histograms) CCR10 expression was analyzed by flow cytometry. ( A–F ) Left panels show representative histograms, and right panels show the percentage of neutrophils expressing CCR10 on their surface (clear bars) or intracellularly (filled bars). Bars represent the mean. Data were analyzed by paired t test ( A–D ) or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test ( E, F ) on log-transformed data. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, not significant.

Article Snippet: Antibody , Anti-mouse CCR10 (Rat monoclonal; unconjugated) , R&D Systems , Clone: 248918; Cat#MAB2815; RRID: AB_2074258 , In vitro signaling blockade (5 µg/100 µl).

Techniques: Infection, Staining, Flow Cytometry, Expressing, Comparison, Transformation Assay

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Murine bone marrow neutrophils were stimulated with IFNγ + TNFɑ + GM-CSF for 4 hr before adding 1 × 10 6 cells to the upper compartment of a transwell chamber for chemotaxis assays. Each of the chemokines (CCL28, CCL11, or CXCL1), or no chemokine (NC), was placed in separate lower compartments. The transwell plate was incubated for 2 hr at 37°C. Cells that migrated to the lower compartment were enumerated by flow cytometry. Neutrophil chemotaxis index was calculated by taking the number of cells that migrated in response to a chemokine and dividing it by the number of cells that migrated in the absence of a chemokine. Data are from four independent experiments. ( B, C ) Infection of bone marrow neutrophils. ( B ) Opsonized STm (1 × 10 7 CFU) or ( C ) opsonized Ab (1 × 10 7 CFU) were cultured alone, or added to bone marrow neutrophils (1 × 10 6 cells) stimulated with CCL28, CCL11, or no chemokine, for 2.5 hr (STm) or 4.5 hr (Ab) at 37°C. Neutrophils were lysed with 1% Triton-X and surviving bacteria were enumerated by plating serial dilutions. Percentage of bacterial survival was calculated for each condition by taking the CFU from bacteria incubated with neutrophils and dividing it by the CFU from bacteria incubated without neutrophils, multiplied by 100. Data shown for each infection comprise three independent experiments. Bars represent the mean ± standard deviation (SD). ( D ) The effect of the CCR3 antagonist SB328437 on neutrophil-mediated STm killing was evaluated by performing the experiment as described in panel ( B ), with or without the antagonist. Data shown comprise three independent experiments. ( E–G ) Reactive oxygen species (ROS) production (2′,7′-dichlorodihydrofluorescein diacetate [H 2 DCFDA] conversion to fluorescent DCF) detected by flow cytometry in bone marrow neutrophils infected with STm as described in panel ( B ). In ( F, G ), cells were stimulated with CCL28 in the presence of an anti-CCR3 antibody, an anti-CCR10 antibody, or isotype controls. Left panels show representative histograms, and right panels show the percentage of ROS + neutrophils in the indicated treatment groups. ( H, I ) Neutrophil extracellular trap (NET) formation detected by fluorescence microscopy using Helix dye in human neutrophils activated with platelets. Cells were unstimulated (no chemokine, NC), stimulated with CCL28 alone, or with CCL28 and the CCR3 agonist SB328737 and/or the CCR10 agonist BI-6901, as indicated. ( H ) Representative images of fluorescence microscopy with DAPI (blue) and Helix (green). ( I ) Quantification of NETs represented as percentage of cells forming NETs based on observed morphology. Connected circles represent NET abundance in cell populations from the same donor following different indicated treatments. ( A–E ) Bars represent the mean ± SD. ( A–C ) Data were analyzed by non-parametric analysis of variance (ANOVA) (Kruskal–Wallis’s test), assuming non-equal SD given the differences in the variance among the groups, followed by Dunn’s multiple comparisons test. ( D, I ) Data were analyzed by ratio paired t test. ( E–G ) Log-transformed data were analyzed by one-way ANOVA for paired samples. Greenhouse–Geisser correction was applied in F and G given the differences in variance among the groups. Tukey’s multiple comparison test was performed to compare all conditions to each other. ( I ) Ratio paired t tests were used to compare NET levels in samples from the same donor. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant.

Article Snippet: Antibody , Anti-mouse CCR10 (Rat monoclonal; unconjugated) , R&D Systems , Clone: 248918; Cat#MAB2815; RRID: AB_2074258 , In vitro signaling blockade (5 µg/100 µl).

Techniques: Chemotaxis Assay, Incubation, Flow Cytometry, Infection, Cell Culture, Bacteria, Standard Deviation, Fluorescence, Microscopy, Transformation Assay, Comparison

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: As indicated, cells were unstimulated (NC), stimulated with CCL28 alone, or with CCL28 and the CCR3 antagonist SB328437 and/or the CCR10 antagonist BI-6901 (as in ). ( A ) Representative contour plots, and ( B ) percentage of Helix + MPO + neutrophils in the indicated treatment groups. Connected circles represent NET abundance in cell populations from the same donor following different indicated treatments. Ratio paired t tests were used to compare NET levels in samples from the same donor. Significant changes are indicated by *p ≤ 0.05; ns, not significant.

Article Snippet: Antibody , Anti-mouse CCR10 (Rat monoclonal; unconjugated) , R&D Systems , Clone: 248918; Cat#MAB2815; RRID: AB_2074258 , In vitro signaling blockade (5 µg/100 µl).

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse CCR10 (Rat monoclonal; unconjugated) , R&D Systems , Clone: 248918; Cat#MAB2815; RRID: AB_2074258 , In vitro signaling blockade (5 µg/100 µl).

Techniques: Sequencing, Generated, CRISPR, Isolation, Blocking Assay, In Vitro, Control, Recombinant, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Incubation, Selection, Software, Infection, RNA Extraction, Staining, Immunofluorescence, Bacteria, Protease Inhibitor, Inhibition, Preserving, Cell Isolation, Extraction

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Primary HKs were transduced with pLXSN HPV38 E6/E7 or pLSXN. mRNA levels were measured by RT-qPCR and normalized to GAPDH . Error bars represent standard deviations from 3 biological replicates of 2 different donors ( n = 6). ***, p <0.001; ****, p <0.0001; ns, not significant. (B) Total RNA levels of HKs expressing or not expressing HPV38 E6 and E7 were analyzed by TaqMan PCR. Commercial probes for ITGA1 and GAPDH were used. Results were normalized to GAPDH . Data shown are the means of 3 independent experiments for 2 different donors ( n = 6). ****, p <0.0001. (C) Proteins extracts from HKs expressing or not expressing HPV38 E6 and E7 were analyzed by immunoblotting (IB) with the indicated antibodies. (D) The TaqMan assay was also performed as previously described in primary HKs previously retrovirally transduced with the hTERT gene and expressing E6 and/or E7 from HPV38 ( n = 3). Results were normalized to GAPDH . ****, p <0.0001. (E) Proteins extracts from hTERT pLXSN or hTERT HPV38 E6/E7 cells were analyzed by IB with the indicated antibodies. Images shown are representative examples of 2 different experiments. (F) hTERT pLXSN or hTERT HPV38 E6/E7 cells were plated on coverslips and after 24 h were probed for ITGA1 using anti-ITGA1 antibody followed by secondary Alexa Fluor 488-conjugated antibody. Nuclei were stained with DAPI (pseudocoloured red), and cells were analyzed under a microscope. Images were merged using ImageJ software.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, TaqMan Assay, Staining, Microscopy, Software

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Electromobility shift assay performed with 38HK nuclear protein extracts and biotinylated probes containing p53RE WT or mutated sequences. Probes were incubated and cross-linked with protein extracts. Unlabeled WT or mutant p53RE2 probes were used as a control. Images shown are representative examples of 2 different experiments. (B) 38HK were cross-linked and chromatin was processed for ChIP using p53 antibody. Results were analyzed by qPCR with primers spanning p53RE1, p53RE2, p53RE3, or the intergenic region of chromosome 22 as a negative control (nc). Error bars represent standard deviations of 3 independent experiments performed in triplicate. **, p <0.01. (C) HKs or 38HK were cross-linked and chromatin was processed for ChIP using p53 or IgG antibodies. Results were analyzed by qPCR using primers spanning for p53 REs of the ITGA1 promoter and normalized to IgG enrichment (negative control). Error bars represent the standard deviation of 2 independent experiments performed in 2 different HKs donors. *, p <0.05, ns, not significant. (D) Cell lysate was incubated with WT biotinylated probe containing p53 REs of the ITGA1 promoter. Incubation without a probe was used as a control. DNA-associated proteins were recovered by precipitation with streptavidin beads and analyzed by IB. Images shown are representative examples of 3 independent experiments. Signals of 3 different IBs were quantified with Image Lab software (right panel). Data shown are the means of 3 independent experiments. *, p <0.05. (E) Chromatin from 38HK was processed for ChIP experiments using p53 or DNMT1 antibodies. Results were obtained by qPCR with primers spanning p53RE2 or the intergenic region of chromosome 22 (nc). Error bars indicate standard deviations from 3 independent experiments performed in duplicate. **, p <0.01; ***, p <0.001. (F) 38HK were cultured in medium containing cyclic pifithrin-α hydrobromide or DMSO as a control. Chromatin was processed for ChIP using p53 or DNMT1 antibodies. Results were obtained by qPCR using primers spanning p53RE2. Data shown are the means of 2 independent experiments performed in triplicate. *, p <0.05, **, p <0.01. (G) Chromatin was processed for a ChIP-reChIP assay in which p53-immunoprecipitated DNA was re-immunoprecipitated by DNMT1. Enrichment of p53RE2 or the intergenic region of chromosome 22 (nc) was obtained by qPCR. Data shown are the means of 3 independent experiments performed in triplicate. **, p <0.01. (H) Nuclear protein extracts from 38HK were processed for IP. Agarose beads were conjugated with IgG or p53 antibodies. Conjugated beads were incubated with protein lysate overnight. IgG was used as a control. Results were obtained by IB using the indicated antibodies. (I) 38HK were transfected with DNMT1 siRNA or control siRNA (Scramble). After 72 h, a ChIP assay was performed with p53 or DNMT1 antibodies. Results were obtained by qPCR using p53RE2 primers. Error bars represent standard deviations from 3 independent experiments. *, p <0.05; **, p <0.01. DNMT1 protein levels in different cells were determined by IB with the indicated antibodies (right panel).

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Electro Mobility Shift Assay, Incubation, Mutagenesis, Control, Negative Control, Standard Deviation, Software, Cell Culture, Immunoprecipitation, Transfection

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) mRNA levels of 38HK treated with cyclic pifithrin-α hydrobromide or DMSO for 6 h were analyzed by RT-qPCR and normalized to GAPDH . Histograms represent the mean of at least 3 independent experiments. ****, p <0.0001. (B and C) ITGA1 and p53 mRNA and protein levels from 38HK expressing WT p53 (Scramble) or with CRISPR/Cas9-mediated p53 deletion (CRISPR-p53) were measured by RT-qPCR (B) and IB (C). (B) Gene expression was normalized to GAPDH . (C) Protein quantification was normalized to β-actin. Signals of 4 different IBs were quantified with Image Lab software (right panel). Data shown are the means of 4 independent experiments. *, p <0.05. (D and E) 38HK N-HA-p53 or p53-C-HA cells were generated by retroviral transduction with WT p53 tagged at the N- or C-terminus. As a control, 38HK were transduced with the corresponding empty plasmid. Protein extracts and total mRNA levels were processed for IB and RT-qPCR analysis, respectively. (D) IB images shown are representative examples of 3 independent experiments. (E) ITGA1 mRNA levels were normalized to GAPDH . Error bars indicate standard deviations of 4 independent experiments. ***, p <0.001. (F) 38HK were transfected with control siRNA (Scramble) or with DNMT1 siRNA (siDNMT1). After 72 h, cells were collected for RNA extraction and RT-qPCR analysis. Error bars indicate standard deviations of 3 independent experiments. *, p <0.05; ***, p <0.001. (G) ITGA1 expression was evaluated by RT-qPCR after 24 h of treatment with 5-aza-2′- deoxycytidine (Aza) or DMSO at 30 μM final concentration. Error bars represent standard deviations of 3 independent experiments. **, p <0.01. (H) H3K9ac at the ITGA1 promoter was evaluated by ChIP assay after treatment with Aza or DMSO as previously described ( n = 4). Results were obtained by qPCR using primers for p53RE2. *, p <0.05.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, CRISPR, Gene Expression, Software, Generated, Retroviral, Transduction, Control, Plasmid Preparation, Transfection, RNA Extraction, Concentration Assay

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Protein extracts from 38HK were processed for oligonucleotide pulldown as previously described. Images shown are representative examples of 3 independent experiments. (B) HK and 38HK were processed for protein extraction and IB with the indicated antibodies. After incubation with p446PKR antibody, the membrane was stripped and incubated with total PKR antibody. (C) Proteins extracts from U2OS cells retrovirally transduced with E6 and/or E7 from HPV38 were analyzed by IB with the indicated antibodies. (D) 38HK were treated with PKR inhibitor, 2AP, or PBS:glacial acetic acid (200:1) as a control for 4 h at 10 mM final concentration. p-p53 S392 and p53 band intensities were quantified and normalized to total p53 (central panel) or β-actin (right panel). Membranes were first incubated with p446PKR, then stripped and incubated with total PKR. Data shown are the means of 3 independent experiments. *, p <0.05, **, p <0.01. (E and F) 38HK were treated with 2AP, and ITGA1 mRNA (D) and protein levels (E) were determined by RT-PCR and IB, respectively. (D) Data shown are the means of 3 independent experiments (**, p <0.01). (E) Images shown are representative examples of 3 independent experiments. (G and H) ChIP assay using p53 or DNMT1 antibodies was performed in 38HK treated with or PBS:glacial acetic acid (200:1) as a control (CTR) or 2AP. Data shown are the means of 2 independent experiments performed in duplicate by qPCR using p53RE2 primers. *, p <0.05.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Protein Extraction, Incubation, Membrane, Transduction, Control, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) 38HK were transfected with ITGA1 cDNA (ITGA1) or empty plasmid control (pcDNA). After zeocin selection, 38HK were fixed with crystal violet (left panel) and total colony number per well was counted (right panel). Data shown are the means of 3 independent experiments. ****, p <0.0001. (B) 38HK transfected with ITGA1 cDNA (ITGA1) or empty plasmid control (pcDNA) were fixed and stained with propidium iodide for flow cytometry analysis. The histograms (right) represent the means of the sub-G0 population of 3 independent experiments. **, p <0.01. (C and D) Total protein and mRNA extracts from transfected 38HK were analyzed by IB and RT-qPCR. (C) Protein band intensity was quantified and normalized to β-actin. (D) ITGA1 , EGFR , and CCND1 mRNA levels were normalized to GAPDH . For (C) and (D), data shown are the means of 4 independent experiments *, p <0.05; **, p <0.01; ***, p <0.001; ns, not significant.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Control, Selection, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Skin keratinocytes were isolated from WT animals ( n = 4) and K14 HPV38 E6/E7 transgenic mice ( n = 3). After 30 weeks of UV irradiation, cSCC samples (cSCC1–3) were isolated from HPV38 E/E7 transgenic mice. Total RNA extraction was performed and Itga1 mRNA levels were determined by quantitative RT-PCR by normalizing to Gapdh . Whole-exome sequencing of the same mice was also performed. Mutational analysis of the Trp53 , Itga1 , and Egfr genes was performed as described in Materials and Methods. (B) Genomic position of the exonic mutations and the corresponding amino acid change are represented for the Itga1 and Egfr genes. WT exons are represented as blue boxes, and mutated exons are represented in red boxes. Text boxes describe the cSCC sample, the genomic position of the nucleotide change on the GRCm38/mm10 mouse reference genome, the type of mutation, and the corresponding amino acid change.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Isolation, Transgenic Assay, Irradiation, RNA Extraction, Quantitative RT-PCR, Sequencing, Mutagenesis

Journal: Cell stem cell

Article Title: Integrated molecular-phenotypic profiling reveals metabolic control of morphological variation in a stem-cell-based embryo model.

doi: 10.1016/j.stem.2025.03.012

Figure Lengend Snippet: Figure 5. Nodal and Wnt signaling activation can partially rescue T/BRA expression in glycolysis-inhibited human gastruloids (A–D) Immunofluorescence images of micropatterned ESI07 human gastruloids at 48 hpdi, with control BMP treatment from 0 to 48 hpdi (A), combined with 2-DG treatment from 24 to 48 hpdi (B), 2-DG + Act-A from 24 to 48 hpdi (C), and 2-DG + CHIR from 24 to 48 hpdi (D). Panels have the same contrast limits, but because T/BRA intensity in control (A) is very high compared with other conditions, we show a non-saturated image in the pie above the white dotted line. Scale bars: 50 mm. All colonies are 700 mm in diameter. (E–G) Spatial distribution of T/BRA (E) and SOX2 (F) log(intensity), and nuclear-cytoplasmic intensity ratio of Smad2/3 (Act-A/Nodal signaling) (G), averaged over n = 3 colonies. Dark lines represent the mean over colonies, colored bands represent the standard deviation.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Ndiff Takara Cat# 40002 2-Deoxyglucose Sigma Cat# D8375 NaN3 Sigma Cat# S8032 Activin-A (mouse cells) R&D Systems Cat# 338-AC Activin-A (human cells) R&D Systems Cat# AFL338 CHIR99021 (mouse cells) Merck Cat# SML1046 CHIR99021 (human cells) Tocris Cat# 4423 BMP4 R&D Systems Cat# 314BP/CF Fgf8 Tebubio Cat# AF-100-25 Glucose Agilent Cat# 103577-100 Critical commercial assays HCR RNA-FISH Bundle Molecular Instruments https://store.molecularinstruments.com/ Seahorse XF Real-Time ATP Rate Assay Kit Agilent Cat# 103592-100 L-Lactate Assay Kit abcam Cat# ab65330 NAD/NADH-Glo Assay Promega Cat# G9071 Deposited data Single gastruloid RNA sequencing data This paper BioStudies: E-MTAB-13543 Single-cell RNA sequencing data This paper GEO: GSE282050 Experimental models: Cell lines E14 mESCs T/Bra::GFP Laboratory of Alfonso Martinez Arias originating from Fehling et al.87 E14 mESCs T/Bra::GFP, CAG::SUC2CD8HA, PGK::mcherry This paper N/A H9 hESCs WiCell hPSCReg #WAe009-A ESI07 hESCs ESIBIO hPSCReg #SSCCe003-A Oligonucleotides HCR probe: Aldh1a2 Molecular Instruments Acc# NM_009022 HCR probe: Eomes Molecular Instruments Acc# NM_010136.3 HCR probe: Gata6 Molecular Instruments Acc# NM_010258 HCR probe: Meox1 Molecular Instruments Acc# NM_010791 HCR probe: Nanog Molecular Instruments Acc# NM_001289828.1 HCR probe: Sox1 Molecular Instruments Acc# NM_009233.3 HCR probe: Sox17 Molecular Instruments Acc# NM_011441.5 HCR probe: Sox2 Molecular Instruments Acc# NM_011443.4 HCR probe: T/Bra Molecular Instruments Acc# NM_009309.2 Antibodies rabbit anti-SOX2 (1:200) Cell Signaling Technology Cat# 3579S; RRID: AB_2864411 goat anti-TBXT/BRA (1:300) R&D Systems Cat# AF2085; RRID: AB_2200235 mouse anti-Smad2/3 (1:100) BD Biosciences Cat# 610843; RRID: AB_398162 donkey anti-mouse IgG, Alexa Fluor 488 (1:500) Thermo Fisher Scientific Cat# A21202; RRID: AB_141607 donkey anti-rabbit IgG, Alexa Fluor 555 (1:500) Thermo Fisher Scientific Cat# A31572; RRID: AB_162543 donkey anti-goat IgG, Alexa Fluor 647 (1:500) Thermo Fisher Scientific Cat# A21447 (Continued on next page) e1 Cell Stem Cell 32, 1–15.e1–e7, May 1, 2025

Techniques: Activation Assay, Expressing, Control, Standard Deviation

Journal: Cell stem cell

Article Title: Integrated molecular-phenotypic profiling reveals metabolic control of morphological variation in a stem-cell-based embryo model.

doi: 10.1016/j.stem.2025.03.012

Figure Lengend Snippet: Figure 6. scRNA-seq reveals cell-type-specific rescue by Nodal and Wnt signaling activation (A) UMAP plot showing the combined cell populations identified in control, glycolysis-inhibited (2-DG), and rescued (2-DG + Act-A, 2-DG + CHIR) gastruloids at 96 hpa. Cells are colored and numbered by their cluster annotation. (B) UMAP plots visualizing the abundance of different cell types in gastruloids of each condition. (C) Stacked bar plot representation of cell populations from (A) shown as percentage of total analyzed cell numbers and separated by condition. Colors correspond to clusters specified in (A). Cell types contributing >3% are numbered according to their cluster. All percentages and cell numbers are listed in Figure S9B. Total analyzed cell number: n = 8,792 (control), n = 9,596 (2-DG), n = 10,713 (2-DG + Act-A), n = 9,334 (2-DG + CHIR). (D) Dot plot of the Top5 differentially expressed genes of each cluster (see Table S1). (E) Feature plots with the expression of key marker genes in the integrated UMAP. See also Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Ndiff Takara Cat# 40002 2-Deoxyglucose Sigma Cat# D8375 NaN3 Sigma Cat# S8032 Activin-A (mouse cells) R&D Systems Cat# 338-AC Activin-A (human cells) R&D Systems Cat# AFL338 CHIR99021 (mouse cells) Merck Cat# SML1046 CHIR99021 (human cells) Tocris Cat# 4423 BMP4 R&D Systems Cat# 314BP/CF Fgf8 Tebubio Cat# AF-100-25 Glucose Agilent Cat# 103577-100 Critical commercial assays HCR RNA-FISH Bundle Molecular Instruments https://store.molecularinstruments.com/ Seahorse XF Real-Time ATP Rate Assay Kit Agilent Cat# 103592-100 L-Lactate Assay Kit abcam Cat# ab65330 NAD/NADH-Glo Assay Promega Cat# G9071 Deposited data Single gastruloid RNA sequencing data This paper BioStudies: E-MTAB-13543 Single-cell RNA sequencing data This paper GEO: GSE282050 Experimental models: Cell lines E14 mESCs T/Bra::GFP Laboratory of Alfonso Martinez Arias originating from Fehling et al.87 E14 mESCs T/Bra::GFP, CAG::SUC2CD8HA, PGK::mcherry This paper N/A H9 hESCs WiCell hPSCReg #WAe009-A ESI07 hESCs ESIBIO hPSCReg #SSCCe003-A Oligonucleotides HCR probe: Aldh1a2 Molecular Instruments Acc# NM_009022 HCR probe: Eomes Molecular Instruments Acc# NM_010136.3 HCR probe: Gata6 Molecular Instruments Acc# NM_010258 HCR probe: Meox1 Molecular Instruments Acc# NM_010791 HCR probe: Nanog Molecular Instruments Acc# NM_001289828.1 HCR probe: Sox1 Molecular Instruments Acc# NM_009233.3 HCR probe: Sox17 Molecular Instruments Acc# NM_011441.5 HCR probe: Sox2 Molecular Instruments Acc# NM_011443.4 HCR probe: T/Bra Molecular Instruments Acc# NM_009309.2 Antibodies rabbit anti-SOX2 (1:200) Cell Signaling Technology Cat# 3579S; RRID: AB_2864411 goat anti-TBXT/BRA (1:300) R&D Systems Cat# AF2085; RRID: AB_2200235 mouse anti-Smad2/3 (1:100) BD Biosciences Cat# 610843; RRID: AB_398162 donkey anti-mouse IgG, Alexa Fluor 488 (1:500) Thermo Fisher Scientific Cat# A21202; RRID: AB_141607 donkey anti-rabbit IgG, Alexa Fluor 555 (1:500) Thermo Fisher Scientific Cat# A31572; RRID: AB_162543 donkey anti-goat IgG, Alexa Fluor 647 (1:500) Thermo Fisher Scientific Cat# A21447 (Continued on next page) e1 Cell Stem Cell 32, 1–15.e1–e7, May 1, 2025

Techniques: Activation Assay, Control, Expressing, Marker

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Sequences of primers used in this study

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques:

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modifies the expression of the CXCL12 signaling axis in MCF-7 cells. (A) Characterization of the control and COUP clones. An immunofluorescence cytochemistry assay was used to detect the relative expression of HA/COUP-TFI or COUP-TFI proteins in the control (Cont.) and COUP clones. The cells were fixed and processed for immunofluorescence as described in Methods; the nuclei were stained with DAPI. (B) CXCL12 , CXCR4 , and CXCR7 mRNAs were quantified by a real-time PCR analysis from two independent MCF-7 control and COUP clones. The results were normalized to GAPDH mRNA used as an internal control. The results were expressed as the relative mRNA expression level of CXCL12 , CXCR4 , or CXCR7 . Data are the mean values ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (C) The amount of intracellular HA/COUP-TFI, COUP-TFI, CXCL12, CXCR4, and CXCR7 protein was determined from whole-cell extracts of the different MCF-7 clones and compared to total ERK. A representative western blot is shown. (D) The control and COUP clones were fixed, and an immunofluorescence cytochemistry assay was used to detect the relative expression of CXCL12, CXCR4, and CXCR7 proteins. Staining with DAPI is also presented to visualize the nucleus of the cells.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Control, Clone Assay, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modulates the chromatin structure of the CXCL12 and CXCR4 gene promoters. The FAIRE assay was performed as described in Methods. Real-time PCR was performed to monitor the enrichment of DNA corresponding to the proximal promoter of the CXCL12 (A) , the CXCR4 (B) and the CXCR7 (C) genes relative to the input chromatin from the control (Cont.) and COUP clones. The data are from triplicate samples and are representative of three separate experiments. The asterisk indicates significant differences ( p < 0.05) between the control and COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Real-time Polymerase Chain Reaction, Control, Clone Assay

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Estrogenic regulation of CXCL12 and CXCR4 in control and COUP clones. Control (Cont.) and COUP clones were treated with ethanol (EtOH) as the vehicle or E2 10 −8 M and ICI 10 −6 M alone or both together for 48 h. The CXCL12 (A) and CXCR4 (B) relative mRNA levels were monitored by a real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Control, Clone Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: The effect of COUP-TFI on the CXCL12/CXCR4 axis is mediated by EGF/EGFR activation. (A) The relative expression of EGF and EGFR mRNA was monitored by a real-time PCR analysis using MCF-7 control (Cont.) and COUP clones. The results were normalized against GAPDH as the internal control and are expressed as the mean EGF or EGFR mRNA/GAPDH mRNA ratio ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (B) ERK activation was examined in the MCF-7 control (Cont.) and COUP clones after a 5- or 10-min stimulation with EGF (10 −9 M) or CXCL12 (200 ng/mL). Western blots were performed using antibodies against phospho-ERK (P-ERK) and total ERK (ERK1/2); a representative western blot is presented. The importance of EGFR-specific signaling and general ERK signaling on CXCL12 (C) and CXCR4 (D) regulation was assayed by treating the cells with EGF (10 −9 M), AG1478 (25 μM), or U0126 (25 μM) for 48 h. The CXCL12 and CXCR4 relative mRNA levels were monitored by the real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Clone Assay, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI overexpression influences cellular responses to CXCL12. (A) The relative growth of the control (Cont.) and COUP clones was assayed with or without CXCL12 treatment for 7 days. The basal and CXCL12-induced cell growth were evaluated by MTT assays (n = 6) and determined in three independent experiments. The results are expressed as the relative cell number obtained when the control cells were treated with the vehicle control. Significant differences between the unstimulated control cells and the other conditions ( p < 0.05) are indicated with an asterisk. Significant differences between stimulated control cells and stimulated COUP clones ( p < 0.05 ) are indicated with a pound sign. (B) The migratory capacity of control (Cont.) and COUP clones was analyzed. The cells were maintained in phenol red-free DMEM/2.5% dsFBS for 48 h and then seeded in phenol red-free DMEM/0.5% dsFBS in the upper chamber of a PET 8-μm pore insert. The cells were allowed to migrate for 24 h toward the phenol red-free DMEM/2.5% dsFBS medium complemented or not with CXCL12 (200 ng/mL) and AMD3100 (50 μM). (C) CXCL12 was also added to the culture medium in the upper chamber prior to migration. The results are expressed as the mean ± SEM of the relative number of migratory cells compared to the basal migration of the control cells measured in three independent experiments. The asterisks indicate significant differences ( p < 0.05) from the basal migration of the control clones. The pound sign indicates significant differences ( p < 0.05) between two conditions linked by black lines.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Over Expression, Control, Clone Assay, Migration

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Box plots of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA expression in breast cancer and normal tissue. CXCR4 (A) , CXCR7 (B) , CXCL12 (C) , and COUP-TFI (D) mRNA expression was measured by real-time PCR in 23 normal breast tissue samples (NT), in 20 SBR grades 1 and 2, and in 19 SBR grades 3. The expression level was normalized by 18S RNA expression and analyzed with IQ5 software (Bio-Rad). The data are presented as whisker plots in which the horizontal bar represents the median, the grey boxes are the 25 th and 75 th percentiles, the vertical bar is the standard deviation, and the plus signs are the extreme points. All the Mann-Whitney tests were performed with Minitab 16 software, and the p value is indicated on the different graphs (ns denotes non-significant).

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Software, Whisker Assay, Standard Deviation, MANN-WHITNEY

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) Western blots of siCTL–, siRTN4–, and siCLIMP-63–transfected HT-1080 cells were probed with anti-RTN4, anti-CLIMP-63, and anti-β-actin as a loading control. (B) Representative images of ER tubules in HT-1080 cells transfected with ERmoxGFP and siCTL, siRTN4, or siCLIMP-63. Arrowheads indicate the tubules with increased blob length. Scale bar, 2 μm. (C) The quantification of maxima length, variation of maxima length (SD), and maxima-minima intensity differentials of ER tubules in HT-1080 cells transfected with siRTN4, siCLIMP-63, or siCTL. Bar graphs show mean ± SEM and scatter dot plots median with interquartile range. Significance assessed by Student t test from three independent experiments (20–40 line scans/each repeat). ** P < 0.01; *** P < 0.001. Numerical values that underlie the graphs and plots are shown in . CLIMP-63, cytoskeleton-linking membrane protein 63; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4, reticulon4; SD, standard deviation; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRTN4, siRNA to RTN4.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Western Blot, Transfection, Standard Deviation

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) STED images of ERmoxGFP in HT-1080 cells transfected with ERmoxGFP or cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1). Peripheral ER regions (white boxes) are shown as zooms; line scans of selected tubules in these regions (yellow boxes) are shown with ERmoxGFP in green and ER-shaping proteins in red. Scale bar, 5 μm; zooms, 2 μm. (B) Peripheral ER tubule maxima length, variation of maxima length (SD), and maxima-to-minima intensity differential are shown for cells transfected with ERmoxGFP alone (CTL) or cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1). Significance assessed by one-way ANOVA from three independent experiments (40 line scans/each repeat). Bar graphs show mean ± SEM and scatter dot plots median with interquartile range. * P < 0.05; ** P < 0.01; *** P < 0.001. Numerical values that underlie the graphs and plots are shown in . (C) Based on line scan analysis of peripheral ER tubules of HT-1080 cells cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1), percent localization of CLIMP-63, RTN4a, and ATL1 puncta to minima or maxima of lumenal ERmoxGFP-labeled tubules was quantified. Significance assessed by one-way ANOVA from four independent experiments (40 line scans/each repeat). Bar graphs show mean ± SEM. * P < 0.05; *** P < 0.001. Numerical values that underlie the graphs are shown in . ATL, atlastin; CLIMP-63, cytoskeleton-linking membrane protein 63; CTL, control ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4a, reticulon4a; SD, standard deviation; STED, stimulated emission depletion.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Transfection, Labeling, Standard Deviation

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) Representative merged images of single peripheral ER tubules expressing ERmoxGFP or Sec61βGFP labeled for calnexin or derlin-1. The dashed line indicates the site of line scan analysis along tubule. Fluorescence intensities of ER reporter (green) and protein (red) from line scans are presented as graphs. Scale bar, 0.5 μm. (B) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin and derlin-1 puncta to ERmoxGFP or Sec61βGFP maxima and minima was quantified. Values plotted are mean ± SEM from three independent experiments (40 tubules per repeat) with one-way ANOVA for significance. *** P < 0.001. Numerical values that underlie the graphs are shown in . (C) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin and derlin-1 puncta to ERmoxGFP maxima and minima was quantified in cells transfected with siCTL, siCLIMP-63, or siRTN4. Significance was assessed by χ 2 test from three independent experiments (20–40 tubules per repeat). *** P < 0.001. Numerical values that underlie the graphs are shown in . (D) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin puncta to ERmoxGFP maxima and minima was quantified in HT-1080 cells cotransfected with mCherry-CLIMP-63, mCherry-RTN4a, or mCherry-ATL1 compared with CTL. Significance assessed by χ 2 test from three independent experiments (40 tubules per repeat). *** P < 0.001. Numerical values that underlie the graphs are shown in . ATL, atlastin; CLIMP-63, cytoskeleton-linking membrane protein 63; CTL, control; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4a, reticulon4a; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRTN4, siRNA to RTN4.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Expressing, Labeling, Fluorescence, Transfection

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: STED live cell imaging (40 ms/frame over 4 seconds) of isolated ROIs of peripheral ERmoxGFP-labeled tubules (a) was performed for COS-7 cells cotransfected with mCherry-CLIMP-63 or mCherry-RTN4a (A) or transfected with siCLIMP-63, siRTN4, or siCTL (B). Kymograms show the distribution of ERmoxGFP at specific sites along ER tubules over time (b). From plots of normalized average intensity over time (c), we determined the CoV along the tubule length as a measure of localized distribution of ERmoxGFP to distinct domains along peripheral ER tubules (d). Scatter dot plots show median with interquartile range from three independent experiments (20–50 tubules per condition) with one-way ANOVA for significance. * P < 0.05; *** P < 0.001. Numerical values that underlie the plots are shown in . CLIMP-63, cytoskeleton-linking membrane protein 63; CoV, coefficient of variation; CTL, control; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; ROI, region of interest; RTN4a, reticulon4a; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRNA, small interfering RNA; siRTN4, siRNA to RTN4; STED, stimulated emission depletion.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Live Cell Imaging, Isolation, Labeling, Transfection, Small Interfering RNA

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) Volcano plots showing statistically enriched proteins in DCC-IP and Nrp1-IP samples identified by permutation-based FDR-corrected t-test based on three biological replicates. The LFQ intensity of the DCC or Nrp1 pulldowns over IgG pulldowns are plotted against the - log10 p-value. FDR <0.05; S0 = 2. (B) Gene enrichment analysis of statistically enriched proteins in the DCC and Nrp1 pulldown samples. (C-F) Western blot validation of RP co-immunoprecipitation with DCC, Nrp1 and Robo2 but not with EphB2. (G-J) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns shows enrichment of rRNA in DCC, Nrp1, and Robo2 but not EphB2 pulldowns (unpaired two-tailed t-test; three biological replicates). Error bars indicate standard deviation; * p<0.05.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Western Blot, Immunoprecipitation, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A-C) Western blot validation of RP co-immunoprecipitation with DCC, Nrp1 and Robo2 in SH-SY5Y cells. (D-E) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldowns or receptor pulldowns shows enrichment of rRNA in DCC and Nrp1 IPs in SH-SY5Y cells (unpaired two-tailed t-test; three biological replicates). (F) iBAQ-based relative quantification of 60S or (G) 40S subunit RPs between DCC and Nrp1 (unpaired two-tailed t-test; three biological replicates). Error bars indicate standard deviation. *p<0.05.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Western Blot, Immunoprecipitation, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns with or without EDTA or RNase A/T1 treatments (two-way ANOVA with Bonferroni’s multiple comparisons test; three biological replicates; Error bars indicate standard deviation; ***p<0.0001). (B) Western blot analysis and quantification of ribosomal proteins after DCC and (C) Nrp1 pulldowns. (two-way ANOVA with Bonferroni’s multiple comparisons test; three biological replicates; Error bars indicate standard deviation; **p<0.01; ***p<0.0001). (D, E) Volcano plots indicating both DCC and Nrp1 bind significantly to different RBPs (orange dots). FDR <0.05; S0 = 2. (F) DCC and Nrp1 each bind to differentially to the RBPs Staufen1 and hnRNPA2B1. FDR <0.05. (G) Distance matrix showing a high correlation between replicates and a distinct signature between samples. (H) Volcano plot showing differential expression analysis for DCC and Nrp1 pulldowns. (I) Enrichment analysis plot of known RBP targets detected in RNA-sequencing data after DCC and Nrp1 pulldown.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Standard Deviation, Western Blot, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) Expansion imaging shows partial colocalization of DCC and (B) Nrp1 with ribosomal proteins (Scale bars, 5 μm). (C) Representative proximity ligation assay signal in axonal growth cones between DCC and RPL5/uL18, RPS4X/eS4 or IgG control (Scale bars, 5 μm). (D) Representative proximity ligation assay signal in axonal growth cones between Nrp1 and RPS3A/eS1, RPS23/uS12 or IgG control (Scale bars, 5 μm). (E) EphB2 and RPL5/uL18 show a significantly lower amount of PLA signal in axonal growth cones compared to DCC-RPL5/uL18 or Nrp1-RPS23/uS12 (Mann-Whitney test; ***p<0.0001; Scale bars, 5 μm). (F, G) Quantification of PLA signal in cue-stimulated axonal growth cones relative to control (unpaired two-tailed t-test; error bars indicate SEM; ***p<0.0001; *p = 0.0423). (H) Relative PLA quantification of DCC and RPL5/uL18 compared to control after Netrin-1, EphrinA1, or co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; error bars indicate SEM; **p = 0.0058). For all PLA experiments, numbers in bars indicate amount of growth cones quantified collected from at least three independent experiments.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Imaging, Proximity Ligation Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) PLA images showing DCC and RPL10A/uL1 are in close proximity in axonal growth cones, whereas DCC and IgG control generates little to no PLA signal. Scale bars, 5 μm. (B) Sema3A stimulation at protein-synthesis independent concentration does not decrease PLA signal between Nrp1 and RPS3A/eS1 (Mann-Whitney test; error bars indicate SEM; p = 0.2555). or (C) puromycin levels in axonal growth cones (Mann-Whitney test; error bars indicate SEM; p = 0.2487). (D) Relative PLA quantification of Nrp1 and RPS23/uS12 compared to control after Sema3A, EphrinA1, or co-stimulation with Sema3A and EphrinA1 (one-way ANOVA with Bonferroni’s multiple comparisons test; Error bars indicate SEM; *p=0.032078; **p<0.018577; *** p<0.001). For all PLA and QIF experiments, numbers in bars indicate amount of growth cones quantified collected from at least three independent experiments.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Concentration Assay, MANN-WHITNEY

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Phf6 loss decreases the leukemogenic potential of cells in vivo and triggers a change in disease presentation. ( A ) PHF6 is a lineage-specific regulator of tumor growth in B-ALL and T-cell acute lymphoblastic leukemia (T-ALL). ( B ). Kaplan-Meier survival analysis of mice injected with either 10 3 (dotted) or 10 6 (solid) Phf6 WT (blue) and Phf6 KO (red) B-ALL cells. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6 WT cells. P- values are shown for the comparisons. ( C ) Representative hematoxylin and eosin (H&E) ( top ) and immunohistochemistry ( bottom ) staining of serial sections from lymph nodes (LNs) and lymphoma (mass) of recipient mice injected with Phf6 WT , shPhf6, and Phf6 KO cells. mCherry immunochemistry demarcates tumor cells. Bars, 600 µm. ( D , top ) Size comparison of representative LNs from Phf6 WT ( left ) and Phf6 KO ( right ) recipient mice. ( Bottom ) Quantification of combined LN weight of Phf6 WT (blue; n = 5) and Phf6 KO (red; n = 5) recipients. ( E ) Tumor burden in the blood of Phf6 WT (blue; n = 7) and Phf6 KO (red; n = 8) recipient mice. mCherry demarcates tumor cells. ( F ) Bar graphs showing the percentage of the CD4 + fraction among mCherry + cells isolated from Phf6 WT (blue; n = 9) and Phf6 KO (red; n = 5) tumors in bone marrow ( left ) and LNs ( right ). Data represent the mean ± standard deviation (SD) in D – F . Statistics were calculated with two-sided Student's t -test. (***) P < 0.001; (****) P < 0.0001.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: In Vivo, Injection, Comparison, Immunohistochemistry, Staining, Isolation, Standard Deviation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: The absence of Phf6 promotes an altered gene expression program in B-cell leukemia. ( A ) Heat map showing differentially expressed genes (fold change >4, false discovery rate [FDR] <0.05) in pairwise comparisons between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Each column represents a replicate sample. The scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. ( B ) The top gene ontology (GO) and PANTHER terms found to be enriched in Phf6 KO cells. The P -value for each term is plotted as −log 10 ( P -value). ( C ) GSEA plot depicting significant ( P < 0.001) changes in pre-B lymphocyte signature genes upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( D ) Quantitative PCR (qPCR) analysis of Phf6 WT (blue) and Phf6 KO (red) cells transduced with empty vector (EV; solid) or a vector expressing Phf6 cDNA (cDNA; dotted). Relative mRNA levels for B-cell-associated genes are shown: Phf6 , Cd22 , Cd74 , Il4ra , Lyn , Ly86 , and Blk . ( E ) Schematic representation of ICA used to identify differential expression signatures (independent components [ICs]) in the integrated RNA-seq data set comprised of Phf6 WT , shPhf6, and Phf6 KO cells. Hinton diagram representation of ICA-derived signatures. Columns denote signatures, and rows denote samples. Colors denote relative directionality of gene expression ([red] up-regulation; [green] down-regulation), and the size of each square represents the magnitude of the contribution of each sample to the respective IC. Each signature is two-sided. Vertical boxes denote statistically significant ( P = 0.01, Mann-Whitney test) independent components. IC2 identified a Phf6 KO -specific gene signature. ( F ) GSEA plot depicting ( P = 0.08) enrichment in T-cell signal transduction signature upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. Data represent the mean ± SD. Statistics for these data were calculated with two-sided Student's t -test. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001; (n.s.) not significant.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Gene Expression, RNA Sequencing, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Quantitative Proteomics, Derivative Assay, MANN-WHITNEY

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: PHF6 exerts transcriptional regulation by interacting with histones rather than binding sequence-specific DNA sites. ( A ) Pie chart showing the distribution of 77,749 PHF6-binding sites across genomic regions in B-ALL cells. (TTS) Transcription termination site; (UTR) untranslated region. ( B ) De novo DNA sequence motifs identified in PHF6-bound regions at promoters of differentially expressed genes with their associated P -values. Shown are sequence logos of de novo position-weight matrices found by the MEME motif discovery tool ( left ) or those of known transcription factors whose motifs are found to be most similar to the de novo motif discovery results by Tomtom software ( right ). ( C ) Endogenous coimmunoprecipitation (co-IP) assay of TCF12, NF-κB, and PHF6 in the absence ( top ) or presence ( bottom ) of ethidium bromide (EtBr). Input is 7% of immunoprecipitation lysate. ( D , left ) Metagene tracks of PHF6 ChIP-seq signal averaged over all promoter–TSS tracks grouped by relative expression levels. (Red) High; (green) genomic; (purple) low. ( Right ) Metagene track of H3K27ac ChIP-seq signal averaged over all promoter–TSS regions. Shaded regions around average tracks denote estimates of 95% confidence interval (CI) of the metagene average signals based on resampling. ( E ) Metagene tracks of PHF6 ChIP-seq signal and correlation with histone marks: H3K27ac (blue), H3K4me3 (yellow; GSE66234), H3K27me3 (green). Pearson correlation of PHF6 and histone ChIP-seq signals across 10-kb regions spanning the TSS. Differentially expressed genes (solid line) and genome-wide genes (dotted line) are shown. ( F ) Endogenous co-IP assay of histone H3 and PHF6 in the absence ( top ) or presence ( bottom ) of EtBr. Input is 7% of immunoprecipitation lysate.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Binding Assay, Sequencing, Software, Co-Immunoprecipitation Assay, Immunoprecipitation, ChIP-sequencing, Expressing, Genome Wide

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: PHF6 has distinct binding patterns within lineage-specific genes that result in drastic changes in nucleosome occupancy upon genetic deletion. ( A ) Heat maps comparing gene expression of curated CD19 + B-cell ( left ) and CD4 + T-cell ( right ) gene sets between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. Each column represents a replicate sample. ( B ) Motif enrichment analysis for DNA regions that undergo significant changes in chromatin accessibility in Phf6 KO cells. ( Top ) DNA motifs with decreased chromatin accessibility upon loss of Phf6 . ( Bottom ) DNA motifs with increased chromatin accessibility upon loss of Phf6 . ( C ) Metagene analysis of nucleosome positions ( top ) and fold enrichment of PHF6 binding ( bottom ) plotted for global analysis ( left ), the CD19 + B-cell gene set ( middle ), and the CD4 + T-cell gene set ( right ) assessed at TSSs ±1-kb genomic regions. ( Top ) Nucleosome positions are shown for Phf6 WT (solid line) and Phf6 KO (dotted line) cells, called by the NucleoATAC algorithm and normalized for batch effects using the chromVAR package. ( Bottom ) Metagene tracks of PHF6 ChIP-seq signal averaged over the TSSs ±1 kb in Phf6 WT cells. (−1/+1) +1/−1 nucleosomes flanking the TSS; (NFR) nucleosome-free region. Gray bars indicate major changes in nucleosome positioning that correspond to enriched PHF6 binding. Shaded regions around average tracks denote estimates of 95% CI of the metagene average signals based on resampling.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Transformation Assay, Expressing, ChIP-sequencing

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Chromatin instability allows for aberrant T-cell transcription factor signaling. ( A ) Cell proliferation assay comparing Phf6 WT (blue; up arrow), Phf6 KO (red; up arrow), Phf6 WT + control vector (blue; circle-dotted), Phf6 KO + control vector (red; circle-dotted), Phf6 WT + NICD cDNA (blue; down triangle), and Phf6 KO + NICD cDNA (red; down triangle) cells. n = 3. ( B ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes infected with control vector or activated NICD vector. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6 WT + control vector cells. P -values are shown for the comparisons. ( C ) Quantification of the combined thymus weight of Phf6 WT + control vector (blue; n = 4), Phf6 KO + control vector (red unfilled; n = 6), and Phf6 KO + NICD cDNA (red patterned; n = 7) recipients. ( D ) Bar graphs showing the percentage of the CD4 + fraction among mCherry + cells isolated from Phf6 WT + control vector (blue; n = 5), Phf6 KO + control vector (red unfilled; n = 4), and Phf6 KO + NICD cDNA vector (red patterned; n = 6) tumors from LNs. Data represent the mean ± SD in C and D . Statistics were calculated with two-sided Student's t -test. (*) P < 0.05; (**) P < 0.01; (****) P < 0.0001.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Proliferation Assay, Control, Plasmid Preparation, Injection, Infection, Comparison, Isolation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Loss of Phf6 results in decreased dependence on the driving oncogene BCR–ABL1 in vivo. ( A ) GSEA plots depicting significant changes in the targets of the BCR–ABL1 fusion signature ( P = 0.0129) and dasatinib resistance signature ( P = 0.0056) upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( B ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes with subsequent mock or ponatinib treatment (30 mg/kg daily for four consecutive days). The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P -value is shown for the comparison. ( C ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes with mock or 10 mg/kg doxorubicin (single immunoprecipitation dose) treatment. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P -value is shown for the comparison.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: In Vivo, Injection, Comparison, Immunoprecipitation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Model of PHF6 as a chromatin state regulator, permitting transcription factor binding through chromatin accessibility. ( A ) In wild-type cells, PHF6 binds to the ±1 nucleosome flanking the open TSSs of genes, allowing B-cell-specific transcription factors to bind, drive gene expression, and maintain B-cell identity. Conversely, PHF6 binds nucleosomes surrounding the TSSs of T-cell-specific genes, coordinating chromatin compaction and thus blocking the binding of T-cell-specific transcription factors. ( B ) In the absence of PHF6, chromatin is no longer maintained in an open state, B-cell transcription factors cannot bind, and expression of B-cell identity genes is down-regulated. However, T-cell identity genes are no longer inaccessible, allowing T-cell-specific transcription factors to bind and activate aberrant transcriptional programs.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Binding Assay, Gene Expression, Blocking Assay, Expressing