Journal: Frontiers in Immunology

Article Title: Making the effect visible – OX40 targeting nanobodies for in vivo imaging of activated T cells

doi: 10.3389/fimmu.2024.1480091

Figure Lengend Snippet: Characterization of cellular binding of hOX40-Nbs. (A) Determination of hOX40-Nb binding to cellular expressed hOX40 by flow cytometry (n=3), exemplary shown for Nb O18 labeled with AlexaFluor647 (AF647; left). The percentage of positively stained U2OS-hOX40 (frequency of parent) was plotted against indicated concentrations of AF647-labeled hOX40-Nbs and K D values shown in table (right) were calculated from a four-parametric sigmoidal model based on the mean ± SD of three replicates. (B) Representative images of U2OS-hOX40 cells (upper panel) and U2OS-WT cells (lower panel) stained with 1000 nM AF647-labeled hOX40-Nbs (left) as well as non-binding AF647-labeled PEP-Nb (Nb Ctrl.) as negative and phycoerythrin (PE)-labeled anti-hOX40 mAb as positive control (right). Shown are individual Nb staining (red), nuclei staining (Hoechst, blue) and merged signals; scale bar: 50 µm.

Article Snippet: Taking advantage of SPAAC (strain-promoted azide-alkyne cycloaddition) click chemistry reaction fluorescent labeling was performed by incubating azide-coupled Nbs with 2-fold molar excess of DBCO-AF647 (Jena Bioscience) for 2 h at room temperature.

Techniques: Binding Assay, Flow Cytometry, Labeling, Staining, Positive Control

Journal: Frontiers in Immunology

Article Title: Making the effect visible – OX40 targeting nanobodies for in vivo imaging of activated T cells

doi: 10.3389/fimmu.2024.1480091

Figure Lengend Snippet: Characterization of binding epitopes of hOX40-Nbs. (A) Domain mapping by immunofluorescence staining with hOX40-Nbs on U2OS cells displaying either surface exposed hOX40 full length (D1-4), or domain deletion mutants as indicated. Shown are representative images of living cells stained with individual AF647-labeled Nbs or anti-hOX40 mAb; scale bar: 50 µm. (B) Schematic overview summarizing the results of domain mapping analysis (crystal structure OX40 PDB: 2HEV). (C) Epitope binning analysis of hOX40-Nbs by BLI. Representative sensograms of combinatorial Nb binding to recombinant hOX40 on sharing/overlapping epitopes or on different epitopes are shown. (D) Graphical summary of epitope binning analysis.

Article Snippet: Taking advantage of SPAAC (strain-promoted azide-alkyne cycloaddition) click chemistry reaction fluorescent labeling was performed by incubating azide-coupled Nbs with 2-fold molar excess of DBCO-AF647 (Jena Bioscience) for 2 h at room temperature.

Techniques: Binding Assay, Immunofluorescence, Staining, Labeling, Recombinant

Journal: Frontiers in Immunology

Article Title: Making the effect visible – OX40 targeting nanobodies for in vivo imaging of activated T cells

doi: 10.3389/fimmu.2024.1480091

Figure Lengend Snippet: Validation of hOX40-Nb binding to activated T cells. (A) Schematic outline of activation of human peripheral blood mononuclear cells (hPBMCs) by phytohaemagglutinin L (PHA-L) and IL-2. (B) Flow cytometry analysis of hOX40-Nbs staining on CD3 + hPBMCs from three different donors (K025, K029 and K034) after 24 h of PHA-L and IL-2 stimulation shown as bar graph. Data are presented as mean ± SD of three replicate stains. (C) Exemplary results of flow cytometry analysis of CD3 + hPBMCs derived from donor K034 stained with AF647-labeled hOX40-Nbs, a non-binding PEP-Nb (Nb. Ctrl.) or a PE-labeled anti-hOX40 mAb before (0 h, lower panel) and after (24 h, upper panel) stimulation. (D) Flow cytometry analysis of hOX40-Nb staining on non Treg CD4 + , CD8 + and regulatory (Treg) T cells from the three same donors after 24 h of PHA-L and IL-2 stimulation. Bar graphs summarizing the percentages of the different T cell subpopulations for each donor (upper left), Nb binding to non Treg CD4 + T cells (upper right), Nb binding to CD8 + T cells (lower left) and Nb binding to Tregs (lower right) in comparison to non-binding PEP-Nb (Nb. Ctrl.) or a PE-labeled anti-hOX40 mAb. Data are presented as mean ± SD of three replicate stains.

Article Snippet: Taking advantage of SPAAC (strain-promoted azide-alkyne cycloaddition) click chemistry reaction fluorescent labeling was performed by incubating azide-coupled Nbs with 2-fold molar excess of DBCO-AF647 (Jena Bioscience) for 2 h at room temperature.

Techniques: Binding Assay, Activation Assay, Flow Cytometry, Staining, Derivative Assay, Labeling, Comparison

Journal: Frontiers in Immunology

Article Title: Making the effect visible – OX40 targeting nanobodies for in vivo imaging of activated T cells

doi: 10.3389/fimmu.2024.1480091

Figure Lengend Snippet: In vivo optical imaging (OI) with O18 AF647 in HT1080-hOX40 and HT1080-WT tumor bearing mice. 5 µg of O18 AF647 were administered intravenously (i.v.) to CD1 nude mice which previously were subcutaneously injected with human HT1080-hOX40 or HT1080-WT cells at the right upper flank for tumor formation. Tumor biodistribution of O18 AF647 was monitored by repetitive OI measurements over the course of 6 h. (A) Acquired images of different measurement time points of one representative O18 AF647 -injected mouse with HT1080-hOX40 tumor (top) or HT1080-WT tumor (bottom, control). Red arrows indicate the tumor localization at the right upper flank. The kidney is marked with a white arrow at the 5 min time point. (B) Quantification of the fluorescence signal from the tumors (n = 3 per group, arithmetic mean of the average radiant efficiency ± SD, unpaired t test, corrected for multiple comparisons using the Holm-Sidak method revealing a significance of p = 0,00004 indicated by ****) determined at indicated time points. (C) Representative ex vivo OI of harvested tumor (left) and organ quantification of O18 AF647 in HT1080-hOX40 and HT1080-WT tumors. After the last imaging time point, tumors were harvested for ex vivo OI, confirming significantly increased accumulation of O18 AF647 in HT1080-hOX40 tumors (n = 3 per group, arithmetic mean ± SD; unpaired t test revealing a significance of p = 0,0017 indicated by **). Data are shown as individual and mean value of three technical replicates. p < 0.05 was considered statistically significant (*) and marked as ** for p < 0.01 and **** for p < 0.0001.

Article Snippet: Taking advantage of SPAAC (strain-promoted azide-alkyne cycloaddition) click chemistry reaction fluorescent labeling was performed by incubating azide-coupled Nbs with 2-fold molar excess of DBCO-AF647 (Jena Bioscience) for 2 h at room temperature.

Techniques: In Vivo, Optical Imaging, Injection, Control, Fluorescence, Ex Vivo, Imaging

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a The flow chart illustrates the process of treating BM-MSCs with GC-CM, and evaluation of the resulting BM-MSC phenotype and function. b , c The ability to educate BM-MSCs was compared between LNM-GCs (SGC-7901 and HGC-27) and primary GCs (AGS and MGC-803). d LNM-GC exosomes and exosome-free CM were prepared as indicated in the flow chart. e , f BM-MSCs were treated with CM, exosomes, or exosome-free CM from LNM-GCs. The educational effect of the three treatments on BM-MSCs was then compared. b , e Immunofluorescence detection of α-SMA in BM-MSCs was conducted to detect their tumor-promoting phenotype (magnification, ×200; scale bars, 50 μm). c , f GC migration and invasiveness analysis, and HLEC tubule formation assay, were performed to analyze the tumor-promoting function of BM-MSCs. The number of migrated and invaded GCs and tubule junctions were counted and represented in column charts. Data are presented as the mean ± SD of three independent experiments. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; * P < 0.05; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Immunofluorescence, Migration, Tube Formation Assay

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a , b Comparison of cellular and exosomal levels of the Wnt5a protein in AGS and SGC-7901 using western blotting. c Detection of Wnt5a protein in BM-MSCs after treatment with CHX (15 μg/ml) alone or with exosomes at the indicated times. d – g AGS stably transfected with pCMV-GFP and pCMV-Wnt5a-GFP were separately established. GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm). Data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed using Student’s t test. *** P < 0.0001; ** P < 0.01.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Comparison, Western Blot, Stable Transfection, Transfection, Incubation, Expressing, Infection, Derivative Assay, In Vivo, Immunohistochemistry

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a – d Characterization of serum exosomes isolated from healthy participants (H), GC patients without LNM (NM), and GC patients with LNM (M). a Representative TEM images of exosomes (magnification, ×60,000; scale bars, 100 μm). b Size distribution of exosomes analyzed using NTA; c Detection of CD81 expression by flow cytometry. d Western blotting analysis of CD63 and CD9 expression. e , f Comparison of BM-MSC-educating capacity among exosomes from the three groups described above. e Detection α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). f Analysis of tumor-promoting properties of BM-MSCs. g , h Wnt5a expression in exosomes isolated from the pooled sera was detected by western blotting. g Representative image. h Fold changes in relative gray values of Wnt5a; i , j Serum concentration of exosomal Wnt5a was detected using ELISA. i Serum concentration of exosomal Wnt5a in clinical serum samples (20 individuals per group). j Wnt5a concentration in serum samples from the LNM model described in Figs. , . Data are presented using means ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; * P < 0.05; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Isolation, Expressing, Flow Cytometry, Western Blot, Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a – d Characterization of AGS- and SGC-7901-exosomes. a Representative transmission electron microscopy (TEM) images (magnification, ×60,000; scale bars, 100 μm). b Size distribution shown by nanoparticle tracking analysis (NTA). c Flow cytometry analysis of CD81 expression. d Western blotting analysis of CD63 and CD9 expression. e – i The ability of AGS- and SGC-7901-exosomes to educate BM-MSCs was evaluated. e α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). f Quantification of migration, invasion, and tubule formation. g – i In vivo analysis of tumor-promoting properties of BM-MSCs. g The draining popliteal lymph nodes (LNs) from each group were harvested and imaged. h Weight of popliteal LNs. i Immunohistochemistry analysis of pan-cytokeratin (AE1/AE3) expression in popliteal LNs. Representative images from each group are shown (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm). Data are expressed as the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Transmission Assay, Electron Microscopy, Flow Cytometry, Expressing, Western Blot, Migration, In Vivo, Immunohistochemistry

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a The flow chart shows removal of exosomal proteins by proteinase K alone or combined with Triton X-100, and purification of these treated exosomes by ultrafiltration centrifugal tubes. b SDS-PAGE and Coomassie brilliant blue stain were used to confirm that proteins were indeed removed from SGC-7901 exosomes. c , d The influence of protein removal on education of BM-MSCs by SGC-7901 exosomes. c Immunofluorescence detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). d Quantification of migrated and invaded GCs, and formed tubule junctions. Data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test. *** P < 0.0001; ** P < 0.01; ns, non significant.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Purification, SDS Page, Staining, Immunofluorescence, Expressing

Journal: Oncogene

Article Title: Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

doi: 10.1038/s41388-021-01722-8

Figure Lengend Snippet: a – d Western blotting analysis of p-YAP s127 and YAP levels in BM-MSCs treated with GC exosomes ( a ), serum exosomes isolated from healthy individuals (H), GC patients without (NM), and those with regional LNM (M) ( b ), exosomes from lentivirus-infected AGS cells ( c ), and exosomes from oligonucleotide-transfected SGC-7901 cells ( d ). e – j BM-MSCs were pretreated with verteporfin before incubation with SGC-7901 exosomes ( e , g , h ) and serum exosomes isolated from GC patients with LNM ( f , i , j ). e , f Western blot analysis of CTGF and CYR61 in BM-MSCs. g , i Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). h , j Numbers of migrated and invaded GCs and formed tubule junctions. Data are presented as the mean ± SD of three independent experiments. Statistical significance was assessed using Student’s t test. *** P < 0.0001; ** P < 0.01.

Article Snippet: GFP and Wnt5a in cells and their exosomes were individually detected by western blot. h Exosome internalization analysis in BM-MSCs after incubation with exosomes from the abovenamed two stably transfected AGS (magnification, ×400; scale bars, 25 μm). i Screening for the most efficient siRNA against Wnt5a (si-Wnt5a) in SGC-7901. j Western blotting analysis of Wnt5a expression in SGC-7901 cells, and analysis of their secreted exosomes after transfection of SGC-7901 with si-Wnt5a and NC. k – o si-Wnt5a- and NC-transfected SGC-7901 exosomes were separately used to treat BM-MSCs in order to compare their effects in education of BM-MSCs. p , q Western blotting analysis of Wnt5a in AGS and in their secreted exosomes after AGS were infected with the pLV-Wnt5a lentivirus. r – v Exosomes derived from pLV-Wnt5a- and pLV-vec-lentivirus-infected AGS were separately used to treat BM-MSCs to observe their BM-MSC-reprogramming abilities. k , r Detection of α-SMA expression in BM-MSCs (magnification, ×200; scale bars, 50 μm). l , s Numbers of migrated and invaded GCs and formed tubule junctions. m – o , t – v In vivo analysis of tumor-promoting properties in BM-MSCs. m , t The draining popliteal LNs harvested from the above groups. n , u Weight of popliteal LNs. o , v Pan-cytokeratin (AE1/AE3) expression in popliteal LNs detected using immunohistochemistry (magnification, ×100, scale bars, 100 μm; magnification, ×400, scale bars, 20 μm).

Techniques: Western Blot, Isolation, Infection, Transfection, Incubation, Expressing

Journal: bioRxiv

Article Title: Visualization of loop extrusion by DNA nanoscale tracing in single human cells

doi: 10.1101/2021.04.12.439407

Figure Lengend Snippet: DNA (Hoechst, grey) and AF647-dUTP replication domain (magenta/green) preservation during different steps of the FISH protocols. ( A ) Overview of linearized single-stranded M13 bacteriophage DNA with biotin-associated docking handles (grey), 64-bp-spaced docking handles (P0, red) and ~600 bp spaced docking handles (P1-P10) in orange to magenta color scale. ( B ) 11-color-DNA-Exchange-PAINT image overview and zoom of indicated regions. Scale 500 nm for image, and 50 nm for inserts. ( C-E ) Comparison of preservation of chromatin structure during individual steps of the FISH protocol rom live to fixed cells (A), from fixed to permeabilized cells (B) and from permeabilization to acid treatment (C). ( F-H ) Comparison of central and apical planes of nucleus (DNA stained with Hoechst) before and after FISH treatment using “tracing FISH” with acid treatment and 86°C denaturation (F), “3D FISH” with acid and 75°C denaturation (G) and non-denaturing FISH (H). Scale bars, 5 µm; RDs in (D), 1 µm. Images representative of >10 (A) or >50 cells (B-F) in two independent experiments. (G) FISH signal with different total BrdU + BrdC concentrations (3:1 ratio). Data from n>50 cells per condition, representative of two independent experiments.

Article Snippet: Afterwards, cells were trypsinized, washed with PBS and resuspended in Resuspension Buffer R (Neon™ Transfection System 10 µL Kit, Cat.# MPK1025, Invitrogen) at a final density of 5.5 × 10 6 cells/ml. dUTP-AF647 (Cat.# NU-803-XX-AF647-L, Jena Biosciences) was added at a final concentration of 60 µM and cells were electroporated with the following pulse parameters: 1 pulse, 1100 v, 20 ms with the Neon transfection system (Invitrogen).

Techniques: Preserving, Staining