Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Cytokinetic phenotype in Kif5b knockdown ATDC5 cells. a Western blot of protein extracts from single cell clones with stable expression of sh-ctl or sh-kif5b constructs. b Immunofluorescence of KIF5B (green) in sh-Kif5b clone #4. Scale bar: 10 μm. c sh-ctl and sh-Kif5b cells stained with α-tubulin (red) and DAPI (blue). Yellow asterisks denote typical binucleated cells. Scale bar: 20 μm. d Quantification of the bi- and multi-nucleation rate in control cell clones ( n = 3, sh-ctl clone #1–3) and Kif5b-knockdown cell clones ( n = 5, sh-Kif5b clone #4–8). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Re-introduced GFP tagged full length KIF5B reduced the bi- and multi-nucleation rate in Kif5b knockdown ATDC5 cells clone #4 ( n = 10 independent experiments) and #5 ( n = 5 independent experiments). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. f Time-lapse images of sh-ctl and sh-Kif5b cells in mitosis. Scale bar: 10 μm. g Quantification of duration of cytokinesis in sh-ctl cells ( n = 84 cells from sh-ctl clone #1–3) and sh-Kif5b cells ( n = 68 cells from sh-Kif5b clone #4, #5 and #8). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 5% to 95% percentile (whiskers). Duration of cytokinesis was calculated from the furrow ingression to the final separation of the two daughter cells. Cells fusing back were not included in this analysis

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Knockdown, Western Blot, Clone Assay, Expressing, Construct, Immunofluorescence, Staining, Control, Two Tailed Test, MANN-WHITNEY, Whisker Assay

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Midbody microtubule organization is impaired in Kif5b deficient cells. a Immunofluorescence of α-tubulin in primary chondrocytes in late cytokinesis. Scale bar: 10 μm. b Quantification of tubulin intensity in midbody in primary chondrocytes ( Kif5b fl/ + cells: n = 24; Col2cre ; Kif5b fl/ − cells: n = 27). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 10% to 90% percentile (whiskers). c Electron micrographs of midbody regions from sh-ctl and sh-Kif5b ATDC5 cells. Red lines denote the region of Flemming body. Yellow arrows and boxes denote the broken regions in Flemming body. Scale bar: 0.5 μm. d Quantification of the length of Flemming body in sh-ctl and sh-Kif5b cells (sh-ctl cells: n = 12; sh-Kif5b cells: n = 9). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Live imaging of sh-ctl and sh-Kif5b cells expressing GFP-tubulin in cytokinesis. Scale bar: 10 μm. f Quantification of the bi- and multi-nucleation rate in sh-Kif5b cells transiently expressing GFP, GFP-Kif5b and GFP-Kif5bΔMT ( n = 5 independent experiments). *** P < 0.0001; NS, P = 0.4039; unpaired two-tailed t-test. Data are mean ± S.D. g A model showing the function of KIF5B in cytokinesis. In late cytokinesis, KIF5B alone or together with other unknown molecules cross-links microtubules in the midbody, and therefore the structure of midbody can be stably maintained

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Imaging, Expressing, Stable Transfection

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: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: ( A ) Images from representative time lapse movies of chromosomes (marked with H2B-mScarlet) and meiotic spindles (marked with MAP4 microtubule-binding domain (mNeonGreen-MAP4-MTBD)) in wild-type and REC8-FKBP12 F36V -mClover3 oocytes. ( B ) Representative metaphase II chromosome spreads with centromeres labeled by ACA staining in wild-type and REC8-FKBP12 F36V -mClover3 oocytes. ( C ) The percentage of aneuploid eggs matured in vitro from wild-type or REC8-FKBP12 F36V -mClover3 oocytes. ( D ) Representative images of metaphase II-arrested eggs matured in vitro from REC8-FKBP12 F36V -mClover3 oocytes treated with DMSO or dTAG-13 for 1 h and stained with ACA and Hoechst. ( E ) Quantification of the percentage of eggs with prematurely separated sister chromatids following 1-h dTAG-13 treatment during metaphase I followed by washout and progression into meiosis II. ( F ) Representative images of embryos at various stages of preimplantation development following fertilization of DMSO- or dTAG-13-treated REC8-FKBP12 F36V -mClover3 eggs. ( G ) Quantification of developmental progression from the zygote to blastocyst stage in embryos generated by fertilization of DMSO- or dTAG-13-treated REC8-FKBP12 F36V -mClover3 eggs. Data are from three independent experiments. Statistical significance was assessed using Student’s t-test. The number of analyzed cells or embryos is indicated in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Binding Assay, Labeling, Staining, In Vitro, Generated

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: a , Representative confocal images of REC8 (GFP antibody) and chromosomes (Hoechst) in metaphase I-stage REC8−FKBP12 F36V −mClover3 oocytes treated with DMSO (0 minutes) or dTAG-13 for 30 minutes, 60 minutes or 90 minutes. b , Quantification of normalized REC8 fluorescence intensity in metaphase I oocytes treated with dTAG-13 for the indicated durations, fixed immediately after treatment. Fluorescence was measured on chromosomes and normalized to DMSO control. c , Representative confocal images of metaphase II chromosomes (Hoechst) and centromeres (ACA) in oocytes treated with dTAG-13 for the indicated durations during metaphase I, followed by washout and progression into meiosis II. d , Quantification of the number of prematurely separated sister chromatids per metaphase II egg after the treatment and washout scheme in c . e , Integrated analysis of the data from b and d showing the relationship between REC8 fluorescence intensity and the number of prematurely separated sister chromatids per egg. REC8 levels were measured in metaphase I oocytes immediately after treatment, and corresponding chromatid separation was quantified in metaphase II eggs after washout and meiotic progression. Chromatid separation remained minimal until REC8 levels dropped below approximately 50% of control, beyond which separation increased sharply, supporting a threshold-based model of cohesion loss. Data are from three independent experiments. Statistical comparisons were performed using Student’s t -test ( b , d ). The number of analyzed oocytes is indicated in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Fluorescence, Control

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: ( A ) Representative confocal images of metaphase II-arrested REC8-FKBP12 F36V -mClover3 eggs treated with DMSO or dTAG-13 and immunostained with ACA (centromere marker) and HEC1 (kinetochore protein). Insets show magnified views of kinetochores. ( B ) Quantification of the proportion of oocytes exhibiting fragmented kinetochores after treatment. No significant increase in kinetochore fragmentation was observed in dTAG-13–treated REC8-FKBP12 F36V -mClover3 eggs compared to controls. ( C ) Representative images of γH2AX immunostaining in REC8-FKBP12 F36V -mClover3 metaphase II eggs treated with DMSO or dTAG-13, showing comparable levels of DNA damage marker signal. ( D ) Quantification of normalized γH2AX fluorescence intensity. No significant increase in γH2AX signal was detected in dTAG-13-treated REC8-FKBP12 F36V -mClover3 eggs. ( E ) Positive control: Representative γH2AX staining of wild-type metaphase II eggs treated with etoposide, a topoisomerase II inhibitor known to induce DNA damage, confirming assay sensitivity. Data are from three independent experiments. Statistical comparisons were performed using Student’s t-test. The number of analyzed oocytes is indicated in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Marker, Immunostaining, Fluorescence, Positive Control, Staining

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: a , Images from representative time-lapse movies of sister chromatids (H2B−mScarlet) in DMSO-treated and IgG TRIM-Away, DMSO-treated and CENP-A TRIM-Away, dTAG-13-treated and IgG TRIM-Away or dTAG-13-treated and CENP-A TRIM-Away eggs from REC8−FKBP12 F36V −mClover3 mice. t = 0 minutes denotes the start of live imaging experiment. b , Representative maximum intensity projected immunofluorescence images of CENP-A and chromosomes in metaphase II chromosomal spreads of DMSO-treated and IgG TRIM-Away, DMSO-treated and CENP-A TRIM-Away, dTAG-13-treated and IgG TRIM-Away or dTAG-13-treated and CENP-A TRIM-Away eggs from REC8−FKBP12 F36V −mClover3 mice. Boxes mark regions of interest that are 4.8× magnified. c , Quantification of CENP-A fluorescence intensities in metaphase II chromosomal spreads of DMSO-treated and IgG TRIM-Away (295 centromeres), DMSO-treated and CENP-A TRIM-Away (535 centromeres), dTAG-13-treated and IgG TRIM-Away (347 centromeres) and dTAG-13-treated and CENP-A TRIM-Away (465 centromeres) eggs from REC8−FKBP12 F36V −mClover3 mice. d , Measurements of chromatid scattering volumes from time-lapse movies of DMSO-treated and IgG TRIM-Away, DMSO-treated and CENP-A TRIM-Away, dTAG-13-treated and IgG TRIM-Away or dTAG-13-treated and CENP-A TRIM-Away eggs from REC8−FKBP12 F36V −mClover3 mice. Lines represent mean values, and error bars indicate s.e.m. Data are from three independent experiments. Statistical significance was assessed using two-way ANOVA ( c , d ). The number of analyzed oocytes is provided in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Imaging, Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation

doi: 10.3390/ijms23010182

Figure Lengend Snippet: ERp57 cKO mice show an accelerated development of osteoarthritis during aging. ( A ) Histological analysis and OARSI scoring of toluidine blue-stained knee sections of 18-month-old mice reveals an increase in cartilage degradation, the occurrence of hypertrophic chondrocytes (*), and osteophyte formation (scattered box) in ERp57 cKO mice, but not in WT animals ( p = 0.0039). No significant difference was detected between younger WT and ERp57 cKO mice at the age of 9 months ( p = 0.7924) and 12 months ( p = 0.2503). Scale Bars = 100 µm. ( B ) TUNEL staining indicates a higher percentage of apoptotic cells in the articular cartilage of ERp57 cKO mice at 9 ( p = 0.0271), 12 ( p = 0.0490) and 18 months ( p = 0,000000028) compared to WT animals. Scale bars = 50 µm ( C ) Immunofluorescence of 18-month-old ERp57 cKO cartilage reveals increased staining intensities of matrix degrading proteases MMP3 ( p = 0.0004), MMP9 ( p = 0.0097), ADAMTS4 ( p = 0.0428), and ADAMTS5 ( p = 0.0288) compared to WT controls. M = months. n ≥ 4. *** p < 0.005, ** p < 0.01, * p < 0.05.

Article Snippet: Then the Vectastain ® Elite ABC Universal Kit (VEC-PK-7200, BIOZOL, Eching, Germany) and the Vector ® DAB Peroxidase Substrate (VEC-SK-4100, BIOZOL, Eching, Germany) were used according to the manufacturer’s instructions with polyclonal antibodies (anti-ADAMTS4: bs-4191R, Bioss Antibodies Inc., Woburn, MA, USA, 1:200 and ADAMTS5: bs-3573R, Bioss Antibodies Inc, Woburn, MA, USA, 1:50), which were incubated on the slides overnight at 4 °C.

Techniques: Staining, TUNEL Assay, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation

doi: 10.3390/ijms23010182

Figure Lengend Snippet: ER stress-induced osteoarthritic changes in the knee joint. Knee joints of aged ERp57 cKO animals display severe OA cartilage degeneration with enhanced MMP3, MMP9, ADAMTS4, and ADAMTS5 expression and osteophyte formation. These changes are preceded by reduced compressive stiffness of the cartilage ECM and increased apoptotic cell death. Both effects are likely induced by ER stress that cannot be adequately compensated by the UPR.

Article Snippet: Then the Vectastain ® Elite ABC Universal Kit (VEC-PK-7200, BIOZOL, Eching, Germany) and the Vector ® DAB Peroxidase Substrate (VEC-SK-4100, BIOZOL, Eching, Germany) were used according to the manufacturer’s instructions with polyclonal antibodies (anti-ADAMTS4: bs-4191R, Bioss Antibodies Inc., Woburn, MA, USA, 1:200 and ADAMTS5: bs-3573R, Bioss Antibodies Inc, Woburn, MA, USA, 1:50), which were incubated on the slides overnight at 4 °C.

Techniques: Expressing