Journal: Nano letters

Article Title: Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

doi: 10.1021/acs.nanolett.5b00490

Figure Lengend Snippet: Figure 2. Involvement of SCARA in PPMO uptake. (A) Differentiated C2C12 cells (1 × 105) were pretreated SR ligands and control (fucoidin sulfate, dextran sulfate, and chondroitin sulfate) at 50 μg/mL for 1 h then treated with PPMO (500 nM) for 4 h in Opti-MEM before changing the medium to differentiation medium and incubation for 20 h. The products of nested reverse transcription-PCR (RT-PCR) were examined by electrophoresis on a 2% agarose gel. The top band indicates full-length transcript and the bottom band represents exon-skipped transcript (B) C2C12 myoblasts differentiated for 1 d, then treated with either siRNA cocktail targeting SACRA1,2,4 and 5 (25 nM each) using Lipofectamine RNAiMax or scrambled control siRNA (Cntrl. siRNA, 100 nM). After 24 h, medium was changed and cells treated with PPMO (500 nm) as stated above. The products of nested RT-PCR were examined by gel electrophoresis and the percent of exon skipping was calculated using densitometry. (C) Quantitative PCR (qPCR) analysis of dystrophin exon 23-skipping in tibialis anterior muscle (TA), diaphragm and heart 1 week following intravenous injection of 10 mg/kg PPMO (Pi6a-PMO) in adult SCARA1−/−and wild-type (WT) C57 BL/6 mice (n = 4). The percentage of exon 23-skipping of the DMD transcripts was determined by normalizing exon 23−24 amplification levels to exon 20−21 levels. *P < 0.05; Student’s t- test; error bars represent mean ± SEM.

Article Snippet: Subsequently, His-tagged recombinant mouse SR-AI (SCARA1) (R&D Systems, U.S.A.) receptor was immobilized on a CM5 chip to give <420 RU.

Techniques: Control, Incubation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Injection

Journal: Nano letters

Article Title: Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

doi: 10.1021/acs.nanolett.5b00490

Figure Lengend Snippet: Figure 4. Interaction with SCARA1. (A) Representative images of the cellular colocalization of Cy5-PPMO or FITC-2′OMePS and FITC-tcDNA with rat antimouse SCARA1 antibody in differentiated C2C12 myotubes at 4 h as measured by fluorescence microscopy (Cy5- PPMO, FITC- tcDNA and FITC- 2′OMePS were used at 200, 500, and 500 nM, respectively). Scale bar, 20 μm. (B) Binding experiments were performed using a Biacore 3000 system. His-tagged SCARA1 receptor was immobilized on the chip using an antihis-tag antibody to give <420 RU. Different ASOs in PBS were injected at 10 μL/min at 25 °C. Data traces were zeroed in the x- and y-axis after subtraction of nonspecific binding.

Article Snippet: Subsequently, His-tagged recombinant mouse SR-AI (SCARA1) (R&D Systems, U.S.A.) receptor was immobilized on a CM5 chip to give <420 RU.

Techniques: Microscopy, Binding Assay, Injection

Journal: Frontiers in cell and developmental biology

Article Title: Effect of AMH on primordial follicle populations in mouse ovaries and human pre-pubertal ovarian xenografts during doxorubicin treatment.

doi: 10.3389/fcell.2024.1449156

Figure Lengend Snippet: FIGURE 2 Treatment regimen for administration of anti-Müllerian hormone (AMH) and/or doxorubicin (DXR) to SCID mice (severe combined immunodeficient) containing human ovarian transplants. Ten days after human ovarian tissue was xenotransplanted into SCID mice, animals were intraperitoneally (IP) injected daily with PBS or rhAMH for 8 days with an additional single IP injection of PBS or DXR on the second day.

Article Snippet: Recombinant human AMH (rhAMH) was purchased from R&D Systems (1737-MS-010/CF, United Kingdom).

Techniques: Injection

Journal: Frontiers in cell and developmental biology

Article Title: Effect of AMH on primordial follicle populations in mouse ovaries and human pre-pubertal ovarian xenografts during doxorubicin treatment.

doi: 10.3389/fcell.2024.1449156

Figure Lengend Snippet: FIGURE 3 Analysis of mouse ovarian follicles in cultured pre-pubertal ovaries treated with DXR and AMH stained with H and E. (A) Representative images of morphologically healthy follicles (scale bar = 25 μm): i: Primordial follicles. ii-iv: Growing follicles (ii: Transitional; iii: Primary; iv: Secondary). (B) Unhealthy follicles (scale bar = 100 μm): black arrows denote pyknotic granulosa cells, red arrows denote atretic oocytes. (C) Total number of morphologically healthy primordial follicles in mouse ovaries treated with different concentrations of DXR. Values are presented as Mean ± S.E.M. Control n = 3; 50 ng/mL DXR n = 3; 100 ng/mL DXR n = 5; 200 ng/mL DXR n = 3. (D) Number of morphologically healthy primordial follicles in mouse ovaries cultured with different concentrations of rhAMH and with or without exposure to DXR. Values represent Mean ± S.E.M ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

Article Snippet: Recombinant human AMH (rhAMH) was purchased from R&D Systems (1737-MS-010/CF, United Kingdom).

Techniques: Cell Culture, Staining, Control

Journal: JCI insight

Article Title: Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity.

doi: 10.1172/jci.insight.186268

Figure Lengend Snippet: Figure 1. Alanine scanning of JZQ-B4 VHH using a yeast surface display system. (A) The schematic of the cell surface display of the VHH variants (created with BioRender.com). Different VHH variants were subcloned into the vector within NheI-BamHI restriction sites. (B) The schematic illustrating the topology of the fusion proteins on the yeast cell surface (created with BioRender.com). Aga2p is disulfide bonded to Aga1p, forming α-agglutinin. The HA tag at the N-terminus was used for the detection of displayed fusion proteins while Alexa Fluor 647–labeled human MSLN was used to measure the affinity of VHHs. (C) The bar graph shows relative binding affinities of VHH variants with alanine substitutions in CDRs, normalized to the level of the parental JZQ-B4 (n = 1 culture per variant). (D) Dot plots are shown for yeast cells expressing the parental JZQ-B4 and CDR3 VHH variants after staining with anti-HA antibody and Alexa Fluor 647–labeled human MSLN.

Article Snippet: The relative binding affinities of JZQ-B4 VHH variants displayed on the yeast surface were determined by staining cells with 300 nM of monomeric human MSLN protein (R&D Systems, Bio-Techne, catalog 3265-MS-050), conjugated in-house with Alexa Fluor 647 using a microscale labeling kit (Thermo Fisher Scientific, catalog A30009).

Techniques: Plasmid Preparation, Labeling, Binding Assay, Variant Assay, Expressing, Staining

Journal: JCI insight

Article Title: Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity.

doi: 10.1172/jci.insight.186268

Figure Lengend Snippet: Figure 2. Binding affinities of MSLN CAR variants. (A) Schematic representation of the lentiviral vectors containing MSLN CAR variants. (B) Relative binding of MSLN CAR variants was estimated by staining CAR-expressing Jurkat T cells with 25 nM of Alexa Fluor 647–conjugated monomeric human and mouse MSLN, using NT cells as control. (C) Determination of the equilibrium binding affinities of CAR variants using a saturation binding assay with Alexa Fluor 647–conjugated human and mouse MSLN (n = 1).

Article Snippet: The relative binding affinities of JZQ-B4 VHH variants displayed on the yeast surface were determined by staining cells with 300 nM of monomeric human MSLN protein (R&D Systems, Bio-Techne, catalog 3265-MS-050), conjugated in-house with Alexa Fluor 647 using a microscale labeling kit (Thermo Fisher Scientific, catalog A30009).

Techniques: Binding Assay, Staining, Expressing, Control, Saturation Assay

Journal: JCI insight

Article Title: Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity.

doi: 10.1172/jci.insight.186268

Figure Lengend Snippet: Figure 3. Activity of CAR T cells expressing VHH variants. (A) Flow cytometric histograms showing the cell surface expression of JZQ-B4 VHH CAR vari- ants in primary CAR T cells. (B) Surface expression of human MSLN in tumor cell lines. NCI-H226/KO represents NCI-H226 with human MSLN knockout using a CRISPR/Cas9 system. MSTO-211H/hMSLN is MSTO-211H cell line that was transduced for human MSLN overexpression. (C) Bioluminescence-based cytotoxicity assay using NT cells as control. CAR T cell samples were normalized to contain the same percentage of CAR-expressing cells (50% CAR+) using donor-matched NT cells. Target cells were cocultured with T cells at an effector-to-target ratio of 2.5:1, and the percentages of target cell lysis were normalized to target cell–only group at 24 hours after coculture. Data represent mean ± SD of 4 technical replicates from 1 independent experiment.

Article Snippet: The relative binding affinities of JZQ-B4 VHH variants displayed on the yeast surface were determined by staining cells with 300 nM of monomeric human MSLN protein (R&D Systems, Bio-Techne, catalog 3265-MS-050), conjugated in-house with Alexa Fluor 647 using a microscale labeling kit (Thermo Fisher Scientific, catalog A30009).

Techniques: Activity Assay, Expressing, Knock-Out, CRISPR, Over Expression, Cytotoxicity Assay, Control, Lysis

Journal: Oncogene

Article Title: MST1R Kinase Accelerates Pancreatic Cancer Progression Via Effects on both Epithelial Cells and Macrophages

doi: 10.1038/s41388-019-0811-9

Figure Lengend Snippet: Bone marrow derived myeloid cells were isolated and treated with either IL-4 or IFN-g to induce M1 or M2 macrophage polarization, respectively. Cells were treated with either Mst1, LMP conditioned media, or a combination of both Mst1 and LMP conditioned media. Cytokine expression was assayed by qRT-PCR (A) Mst1 attenuated M1 macrophage polarization, shown by decreased INFγ, IL-12b and iNOS. (B) Mst1 did not independently induce M2 polarization, as it failed to induce expression of Arginase-I or TGFβ. LMP-conditioned media was capable of inducing M2 macrophage differentiation. Data reflect mean fold change +/− SEM.

Article Snippet: Treatment groups included DMEM Complete alone or DMEM Complete with 100ng/ml recombinant murine Mst1 (R&D 6244-MS).

Techniques: Derivative Assay, Isolation, Expressing, Quantitative RT-PCR

Journal: Oncogene

Article Title: MST1R Kinase Accelerates Pancreatic Cancer Progression Via Effects on both Epithelial Cells and Macrophages

doi: 10.1038/s41388-019-0811-9

Figure Lengend Snippet: (A) KRC and CK epithelial cell lines derived from KC and KRC pancreata were screened for their relative expression of Mst1r and Mst1 by qRT-PCR. High levels of Mst1r expression correlated with similarly high levels of Mst1 expression in 3 out of 4 KRC epithelial cell lines. (B&C) Luciferase reporter assay results from murine pancreatic cell lines PanIN 4313 and 4964 LM. Cells were stimulated with 250ng/ml of Mst1 for 24–48h. A regulatory response element was located between −335bp and −485bp within the Mst1r promoter (p<0.0001, p =0.002). (D) Bone marrow-derived macrophages were exposed to 100 ng/mL Mst1 or vehicle. Q-RT-PCR demonstrated a significant increase in Mst1r transcript after Mst1 (p< 0.05). (E) Immunostaining in 9m pancreata demonstrate increased Mst1 expression in high grade PanIN lesions in KRC versus KC mice.

Article Snippet: Treatment groups included DMEM Complete alone or DMEM Complete with 100ng/ml recombinant murine Mst1 (R&D 6244-MS).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Immunostaining

Journal: Oncogene

Article Title: MST1R Kinase Accelerates Pancreatic Cancer Progression Via Effects on both Epithelial Cells and Macrophages

doi: 10.1038/s41388-019-0811-9

Figure Lengend Snippet: (A) shRNA knockdown resulted in ~90% reduction in Mst1 expression in LMP cells compared to control. B) Mst1 KD tumors weighed significantly less than control tumors (p<0.001). (C) Flow cytometry was performed on single cell suspensions derived from LMP control or Mst1 knockdown tumors. Flow sorting demonstrated a reduction in MRC1 + /CD11b + Grl − cells derived from Mst1 knockdown tumors, p=0.03. (D) Immunostaining for CD3 in LMP and LMP Mst1 KD tumors reveal an increase in CD3+ cells in the LMP Mst1 KD compared to the control LMP-derived tumors. (E) Multiplex ELISA assay of conditioned media from LMP control versus LMP Mst1 knockdown cell lines revealed an 8 fold reduction in Ccl2. (F) Q-RT-PCR for Ccl2 in 3 and 6 month old KRC and KTK −/− C pancreata demonstrated greater than 2 fold reduction in expression in the absence of functional Mst1r kinase.

Article Snippet: Treatment groups included DMEM Complete alone or DMEM Complete with 100ng/ml recombinant murine Mst1 (R&D 6244-MS).

Techniques: shRNA, Knockdown, Expressing, Control, Flow Cytometry, Derivative Assay, Immunostaining, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Functional Assay

Journal: Oncogene

Article Title: MST1R Kinase Accelerates Pancreatic Cancer Progression Via Effects on both Epithelial Cells and Macrophages

doi: 10.1038/s41388-019-0811-9

Figure Lengend Snippet: (A) Model suggesting that Mst1r overexpression results in increased Mst1 and increased Ccl2 within the tumor microenvironment, contributing to the attenuation of M1 differentiation and skewing macrophages towards an alternative tumor-promoting M2 state. (B&C) Analysis of the TCGA’s pancreatic cancer cohort revealed the association between high MST1R expression and both poor overall (p = 0.0009) and disease-free survival (p < 0.0001).

Article Snippet: Treatment groups included DMEM Complete alone or DMEM Complete with 100ng/ml recombinant murine Mst1 (R&D 6244-MS).

Techniques: Over Expression, Expressing

Journal: JCI Insight

Article Title: Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity

doi: 10.1172/jci.insight.186268

Figure Lengend Snippet: ( A ) Schematic representation of the lentiviral vectors containing MSLN CAR variants. ( B ) Relative binding of MSLN CAR variants was estimated by staining CAR-expressing Jurkat T cells with 25 nM of Alexa Fluor 647–conjugated monomeric human and mouse MSLN, using NT cells as control. ( C ) Determination of the equilibrium binding affinities of CAR variants using a saturation binding assay with Alexa Fluor 647–conjugated human and mouse MSLN ( n = 1).

Article Snippet: Two alpacas (obtained from local alpaca farms by Tufts University) were immunized with 5 subcutaneous injections of the C-terminal domains of both recombinant human MSLN (aa 296–580, R&D Systems, Bio-Techne, catalog 3265-MS) and mouse MSLN (aa 298–600, R&D Systems, Bio-Techne, catalog 8604-MS).

Techniques: Binding Assay, Staining, Expressing, Control, Saturation Assay