Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: List of aptamers used in this study.

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: In Vitro, Inhibition, Activity Assay

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Fluorescence microscopy. ( A ) Direct fluorescence method. FAM labeled anti-PSMA RNA aptamer (A9g) was incubated with either PC3(PSMA+) or PC3(PSMA-) cells. A high salt wash step was performed to remove unbound or surface bound RNA. Internalized RNA was visualized using fluorescence microscopy. A scrambled, non-internalizing aptamer (Scr) was used as a negative control in these experiments. Florescence images were overlaid with DAPI and P/C (phase contrast) channels. Arrows indicate perinuclear localization of internalized A9g aptamer. ( B ) Antibody amplification method. FAM-labeled anti-TrkB RNA aptamer (C4-3) was incubated with either TrkB expressing or non-expressing HEK293 cells at 37 °C. FAM-labeled control aptamer (Scr) was used as a control for specificity. Unbound and surface bound RNA was removed as described above. Internalized RNA signal was amplified by incubation with an anti-FITC antibody and Alexa488 secondary antibody. Vehicle treated cells (No RNA) or cells subjected to incubation with antibodies alone (Ab control) were used as controls.

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, Negative Control, Amplification, Expressing, Control

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Plate-reader assay to assess aptamer binding and internalization. For the binding experiments, cells were fixed to inhibit active transport before incubation with the RNA aptamers. Live cells were used for the internalization experiments. ( A ) Binding ( left ) and internalization ( right ) of A9g into PSMA-expressing prostate cancer cells. ( B ) Binding ( left ) and internalization ( right ) of E1 aptamer into rat HER2-expressing mammary carcinoma cells. Cells only (no RNA) controls were carried out for each condition. Fluorescence was measured using an Analyst HT plate reader. (*, p < 0.001).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Binding Assay, Incubation, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Evaluation of aptamer binding and internalization by flow cytometry. ( A ) Cell-specific binding of a human HER2 aptamer-Qdot conjugate. Cell lines expressing HER2 receptor (N202.1E-hHER2 and SKBR3) and HER2 non-expressing cell line (N202.1E) were incubated for 45min at 37 °C with human HER2 aptamer conjugated to Qdots (605nm). Cell-specific aptamer binding was evaluated by flow cytometry (upper panel). Quantification of specific fluorescence signal is shown in the middle panel bar graph. Cell surface human HER2 receptor expression in N202.1E, N202.1E(hHER2) and SKBR-3 cells (grey for isotype control, colored histograms for anti-HER2 Ab) (lower panel). ( B ) Measurement of A9g cell-internalization. PSMA-positive cells were incubated with FAM-A9g aptamer for 30 min at either 4 °C (left top panel) or 37 °C (right top panel). Cells were then washed with either DPBS or a High Salt (DPBS plus 0.5M NaCl) wash for 5min at 4 °C. The high salt wash step removes any unbound or surface bound aptamer. Bound and/or internalized aptamers were subsequently visualized using flow cytometry. *, internalized aptamer fraction (middle right panel). Fluorescence intensity quantified in the bar graph (**, p < 0.005).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Binding Assay, Flow Cytometry, Expressing, Incubation, Fluorescence, Control

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: Quantitative and ultrasensitive internalization method ( “QUSIM” ). ( A ) 96-well microplate NIR Odyssey imager scan of serial dilutions for binding aptamer-NIR conjugate (A9g-NIR) and mutant, non-binding sequence conjugate (A9g.6-NIR) ( upper panel ) and standard curves with linear regression for RNA aptamer quantification ( lower panel ). ( B ) Quantification of the amount of aptamer-NIR internalized into PC3(PSMA+) cells vs. PC3(PSMA–) cells. ( C ) Time-dependent cell uptake of binding aptamer (A9g-NIR) vs. non-binding (mutant) aptamer (A9g.6-NIR). ( D ) Kinetics of specific A9g internalization using one-phase association curve fit (R 2 = 0.9924, k = 0.542 min −1 , half-time = 1.278 min).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Binding Assay, Mutagenesis, Sequencing

Journal: Pharmaceuticals

Article Title: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

doi: 10.3390/ph6030295

Figure Lengend Snippet: RNA-RIP assay. (A) Schematic of A9g-saporin conjugate internalization and RIP effect leading to cell death. (B) Dose dependent response of A9g-saporin and control, A9g.6-saporin, conjugates in PC3(PSMA+) ( left ) and PC3(PSMA–) cells ( right ).

Article Snippet: Recombinant PSMA was prepared by diluting 2 μg recombinant human PSMA (4234-ZN-010) from R&D Systems (Minneapolis, MN, USA) in 500 μL of 50 mM pH 7.5 Tris buffer.

Techniques: Control

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: Synergistic effects of platelet-rich fibrin and CTLA4Ig gene-transfected porcine skin on accelerating wound healing in a rat model of deep second-degree burns: a mechanistic study

doi: 10.3389/fimmu.2025.1756818

Figure Lengend Snippet: Temporal expression profiles of antioxidant enzymes CAT and SOD1. Immunofluorescence staining of (A) Catalase (CAT, red) and (B) Superoxide Dismutase 1 (SOD1, red) in wound tissues from the Vaseline group, PRF group, Pigskin group, and PRF+pig skin group at days 4, 7, 14, and 21 post-treatment. Cell nuclei are counterstained with DAPI (blue). The PRF+pig skin group shows the most pronounced and sustained enhancement in the expression of both antioxidant enzymes, particularly during the proliferative and remodeling phases (D7–D21), indicating a reinforced antioxidant defense system. Scale bar = 100 μm.

Article Snippet: After deparaffinization and antigen retrieval (similar to IHC), sections were permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and incubated overnight at 4 °C with primary antibodies against CAT (1:200, Proteintech) and SOD1 (1:200, Proteintech).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Impact of adropin on superoxide dismutase expression in pancreatic β‐cells of normoglycemic and diabetic rats. (A) Immunofluorescence labeling with anti‐superoxide dismutase antibody and anti‐insulin antibodies showed expression of superoxide dismutase in pancreatic β‐cells. (B) Quantification of the histological analysis showed a significant (*** p < 0.001) decrease in superoxide dismutase distribution in pancreatic endocrine cells and a significant (**** p < 0.0001) decrease in its localization in β‐cells of rats with diabetes when compared to the normal group. n = 6. Scale bar: 50 μm. Data analysis was done using the ANOVA test.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Expressing, Immunofluorescence, Labeling

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Effect of adropin on superoxide dismutase expression in pancreatic α‐cells of normal and diabetic rats. (A) Immunofluorescence staining using anti‐superoxide dismutase and anti‐glucagon antibodies showed expression of superoxide dismutase in pancreatic α‐cells. (B) Quantification of the histological analysis showed a significant (** p < 0.01) decrease in superoxide dismutase distribution in the pancreatic endocrine cells of diabetic rats compared to the normal group. α‐Cell expression of superoxide dismutase did not change with adropin treatment among all groups. n = 6. Scale bar: 50 μm. Data analysis was done using the ANOVA test.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Effect of adropin on catalase, superoxide dismutase and total glutathione activities in the serum samples of healthy and diabetic rats. (A) Catalase activity was significantly decreased in diabetic rats compared to normal controls. Adropin slightly increased catalase in DMT group. (B) Superoxide dismutase was slightly increased in DMT compared to the diabetic treated with adropin. (C) Total glutathione was significantly raised with adropin treatment in DMT compared to DMUT. n = 4–6. Data analysis was done using the ANOVA test. * p < 0.05.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Activity Assay