Journal: Cell Reports

Article Title: Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells

doi: 10.1016/j.celrep.2019.12.057

Figure Lengend Snippet:

Article Snippet: RNA-seq libraries were then prepared from 200 ng of RNA using the NEBNext Ultra II Directional RNA-seq kit (NEB).

Techniques: Plasmid Preparation, Recombinant, Capture-C, Hybridization, Multiplex Assay, Ligation, Software

Journal: Gastroenterology

Article Title: Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

doi: 10.1053/j.gastro.2019.05.065

Figure Lengend Snippet: Figure 4. Reduction of neuronal PLXND1 expression decreases intratumoral nerve density and metastasis in murine model of PDA. (A) PLXND1f/f mice and PLXND1 Adv-Creþ/ mice underwent orthotopic injection of KPC cells into the pancreas. Nerves per whole tumor area were quantified. Data are means ± SEM from n ¼ 14 PLXNDf/f and n ¼ 19 PLXND1 Adv-Creþ/ mice in each group, unpaired, 2-tailed Student t test. (B) Orthotopic tumors grown from PDA tumor cells were collected and measured in WT and PLXND1 Adv-Creþ/ mice 28 days later. Data are means ± SEM from at least 10 mice, unpaired, 2-tailed Student t test. (C) Representative H&E staining of the diaphragm from WT and PLXND1 Adv-Creþ/ mice indicating metastasis in the WT tissue (metastasis indicated with arrows). Scale bars ¼ 50 mm. (D) Micrometastasis formation was assessed by H&E staining of the liver, gut, and diaphragm tissue collected 28 days after injection (Fisher’s exact test). (E) Number of metastatic organ sites/ mouse in liver, gut and diaphragm was analyzed in organs collected on autopsy. PLXND1f/f n ¼ 17, PLXND1 Adv-Creþ/ n ¼ 18 mice. Note: both WT and PL mice were used as control. n.s., not significant, * P < .05.

Article Snippet: One-hour primary incubation with rabbit antibodies against PLXND1 (1:200 Novus) or SEMA3D (1:200 Abcam) was followed by 2 washes and kit-supplied secondary amplifier antibody.

Techniques: Expressing, Injection, Staining, Control

Journal: Gastroenterology

Article Title: Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

doi: 10.1053/j.gastro.2019.05.065

Figure Lengend Snippet: Figure 5. High SEMA3D and PLXND1 expression and shorter distance between SEMA3D/PLXND1-high tumor cells and nerves in human PDAs is associated with PNI. (A) Multiplex IHC was performed on a single slide of mouse PDA tissue for hematoxylin, TUJ1, SEMA3D, and PLXND1. An intratumoral nerve bundle is shown and arrows indicate single nerve cells stained with respective markers. (B) Multiplex IHC was used to identify PNI-positive and -negative human PDA tissue with epithelial marker, EpCam, staining tumor cells and nerve marker, TUJ1, staining neural cells. Representative images are shown, scale bar ¼ 100 mm. (C) Banked human paraffin-embedded resected PDA tissue with or without PNI was stained for H&E, SEMA3D, and PLXND1. Arrows indicate nerves and arrowheads indicate tumor cells. Images representative of n ¼ 16 with PNI and n ¼ 8 without PNI. (D) Colocalization of SEMA3D and PLXND1 on tumor cells and nerves in human PDA was analyzed using multiplex IHC. Respective images are from PNI-positive and -negative samples. Tumor cells and nerves are from different areas of the same PNI-positive or -negative slide. EpCam identifies tumor cells, TUJ1identifies nerves, and the merge image displays SEMA3D and PLXND1 colocalization. Scale bar ¼ 300 mm for tumor cells and ¼ 200 mm for nerve images. (E) Halo image analysis software was used to measure the amount of SEMA3D or (F) PLXND1 positively stained cells per mm2 within 100 to 500 mm of nerves in human PDA tissue with and without PNI, unpaired 2-tailed Student t test. *P < .05.

Article Snippet: One-hour primary incubation with rabbit antibodies against PLXND1 (1:200 Novus) or SEMA3D (1:200 Abcam) was followed by 2 washes and kit-supplied secondary amplifier antibody.

Techniques: Expressing, Multiplex Assay, Staining, Marker, Software

Journal: Gastroenterology

Article Title: Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

doi: 10.1053/j.gastro.2019.05.065

Figure Lengend Snippet: Figure 6. Decrease in both tumoral SEMA3D and neural PLXND1 shows further reduction in tumor invasion and nerve density. (A) ShSEMA3D or shCTRL KPC cells were plated on the top chamber and DRG cells were plated on the bottom with control goat antibody or neutralizing PLXND1 antibody, invasion was measured after 24 hours. Data are means ± SEM from 4 technical replicates, unpaired, 2-tailed Student t test. (B) Orthotopic tumors were grown from shSEMA3D or shCTRL PDA cells in PLXND1f/f and PLXND1 Adv-Creþ/ mice. The number of nerves per whole tumor area from mice pancreas H&E slides were quantified. Note PL and PLAC shCTRL data from Figure 4A is combined for comparison. Data are means ± SEM from at least 6 mice in each group, unpaired, 2-tailed Student t test. *P < .05, **P < .01, ***P < .0001. (C) Model presenting the proposed paracrine signaling mechanism. ANXA2 (yellow block) controls the extracellular release of SEMA3D (purple circle) allowing interaction with its receptor PLXND1 (blue) on neurons aiding in increased tumor cell invasion and metastatic capabilities. The black circles and orange receptors indicate other axon guidance molecules potentially contributing to this signaling pathway.

Article Snippet: One-hour primary incubation with rabbit antibodies against PLXND1 (1:200 Novus) or SEMA3D (1:200 Abcam) was followed by 2 washes and kit-supplied secondary amplifier antibody.

Techniques: Control, Comparison, Blocking Assay

Journal: Cell Transplantation

Article Title: Therapeutic Efficacy of Human Mesenchymal Stem Cells With Different Delivery Route and Dosages in Rat Models of Spinal Cord Injury

doi: 10.1177/09636897221139734

Figure Lengend Snippet: Histopathological and immunofluorescence analyses of the spinal cord in each group. The histopathological and immunofluorescence quantitative analysis of spinal cord tissues with ImageJ software in each group, including SO-CON, SCI control, hUC-MSCs treatment, and methylprednisolone treatment. The arithmetical mean of each spinal section and mean of quantified immunofluorescence were compared with the SCI control group through one-way analysis of variance and post hoc Dunnett’s test (100%). (N = 5, mean ± SEM). (A) Morphometry measurement of the spinal cord lesion of spared white and gray matter in the lesion center by H&E staining. (B-E) Expression of NF-200 (B), the synapse markers SYP (C), pan-Cadherin (D), and ICAM-1 (E) in spinal cord tissue by immunofluorescence staining. * P < 0.05, vs SCI-CON; *** P < 0.001, vs SCI-CON; **** P < 0.0001, vs SCI-CON. SO: sham operation; SO-CON: sham operation control; SCI: spinal cord injury; hUC-MSCs: human umbilical cord-derived mesenchymal stem/stromal cells; H&E: hematoxylin and eosin; NF-200: neurofilament-H; SYP: synaptophysin; ICAM-1: intercellular adhesion molecule-1; SEM: standard error of the mean; SCI - CON: spinal cord injury rat without treatment; SCI-ITH-L/M/H: spinal cord injury rat with low, medium or high dose of hUC-MSCs intrathecal injection; SCI - i.v.: spinal cord injury rat with UC-MSCs intravenous injection; SCI - PC: spinal cord injury rat with methylprednisolone treatment.

Article Snippet: Anti-NF-200 , CST , 55453s , Rabbit , 1:100 , Goat anti-rabbit IgG (Alexa Fluor® 488) , CST , 4412s , 1:2000.

Techniques: Immunofluorescence, Software, Staining, Expressing, Derivative Assay, Injection

Journal: Cell Transplantation

Article Title: Therapeutic Efficacy of Human Mesenchymal Stem Cells With Different Delivery Route and Dosages in Rat Models of Spinal Cord Injury

doi: 10.1177/09636897221139734

Figure Lengend Snippet: Histopathologic and immunofluorescence staining of the spinal cord in each group. H&E and immunofluorescence staining of spinal cord tissues in each group, including SO-CON, SCI control, hUC-MSCs treatment, and methylprednisolone treatment. For immunofluorescence staining, the paraffin sections of spinal cord tissue were labeled with Neurofilament-H antibody (NF-200, green), Synaptophysin antibody (SYP, red), pan-Cadherin antibody (CDH, green), and intercellular adhesion molecule-1antibody (ICAM-1 red). H&E: hematoxylin and eosin; SO-CON: sham operation control; SCI: spinal cord injury; hUC-MSCs: human umbilical cord-derived mesenchymal stem/stromal cells; NF-200: neurofilament-H; SYP: synaptophysin; CDH: pan-Cadherin; ICAM-1: intercellular adhesion molecule-1; SCI - CON: spinal cord injury rat without treatment; SCI-ITH-L/M/H: spinal cord injury rat with low, medium, or high dose of hUC-MSCs intrathecal injection; SCI - i.v.: spinal cord injury rat with UC-MSCs intravenous injection; SCI - PC: spinal cord injury rat with methylprednisolone treatment.

Article Snippet: Anti-NF-200 , CST , 55453s , Rabbit , 1:100 , Goat anti-rabbit IgG (Alexa Fluor® 488) , CST , 4412s , 1:2000.

Techniques: Immunofluorescence, Staining, Labeling, Derivative Assay, Injection

Journal: Cell

Article Title: Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis

doi: 10.1016/j.cell.2018.01.031

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Article Snippet: Mouse anti-BrdU , Rockland Antibodies and Assays , #200-301-H50.

Techniques: Recombinant, Activation Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Sandwich ELISA, Software

Journal: Scientific Reports

Article Title: Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells

doi: 10.1038/s41598-021-85534-6

Figure Lengend Snippet: Characterization of EVs produced by immortalized human airway basal cells in culture. ( A ), Representative size distribution histogram of EVs isolated from conditioned media of BCi-NS1.1 cells grown in culture. The NanoSight NS500 machine with Nanoparticle Tracking Analysis software was used to calculate the quantity of particles as well as their size distribution (mean, mode, etc.). A representative image of the particles is shown in the upper right. ( B ), Immunoblots (cropped for clarity and conciseness) of BCi-NS1.1 cell lysates and EVs produced from these cells with antibodies against proteins found in exosomes—CD63, HSP90, Hsc70, and TSG101—as well as Calnexin, which is not typically seen in exosomes. Equal amounts of total protein were loaded into the cell-lysate and EV lanes. As a negative control, BEGM without BPE was incubated for 48 h in flasks without cells and then processed by means of the same procedures used to isolate EVs. A volume equal to that of the EV preparation was loaded onto the lane marked “(—)”. ( C ), Representative image of BCi-NS1.1 EVs (marked by arrows) taken using electron microscopy.

Article Snippet: The following primary antibodies were used for immunoblotting of EV preparations and cells: mouse monoclonal anti-human CD63 (NK1/C3) (sc-59286; 1/1000; Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-human HSP90 (SMC-107A/B; 1/833.3; StressMarq Biosciences, Victoria, British Columbia, Canada), mouse monoclonal anti-human Hsc70 (B-6) (sc-7298; 1/200; Santa Cruz Biotechnology), mouse monoclonal anti-human TSG101 (#612,697; 1/200; BD Biosciences, San Jose, CA, USA), rabbit monoclonal anti-human/monkey Calnexin (C5C9) (#2679; 1/1000; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-human Alix (1A12) (sc-53540; 1/200; Santa Cruz Biotechnology), and rabbit anti-human β-actin (ab822750; 1/5000; Abcam, Cambridge, MA, USA).

Techniques: Produced, Isolation, Software, Western Blot, Negative Control, Incubation, Electron Microscopy