Journal: Viruses

Article Title: Unveiling the Role of TMPRSS2 in the Proteolytic Activation of Pandemic and Zoonotic Influenza Viruses and Coronaviruses in Human Airway Cells

doi: 10.3390/v16111798

Figure Lengend Snippet: Activation of SARS-CoV and MERS-CoV S by TMPRSS2 and further host proteases. ( A , B ) HeLa cells were co-transfected with plasmids coding for MERS-CoV S ( A ) or SARS-CoV ( B ) with a C-terminal FLAG-tag and plasmids coding for human TMPRSS2 or HAT or murine TMPRSS4 or TMPRSS13. Cell lysates were analysed via SDS-PAGE at 48 h p.t. Western blot analysis was performed using an antibody targeting the C-terminal FLAG-tag. Tubulin was used as a loading control. The data shown are representative of three individual experiments. EV = empty vector. ( C , E ) HeLa cells co-expressing MERS-CoV ( C ) or SARS-CoV ( E ) S, as well as the respective receptors DPP4 or ACE2, were incubated for 48 h. After fixation, the cells were stained with a primary antibody targeting the C-terminal FLAG-tag of S and a fluorescence-coupled secondary antibody. DAPI was used to stain the nuclei. Representative images of three independent experiments are shown. The scale bar represents 100 µM. ( D , F ) Quantification of MERS-CoV S ( D ) or SARS-CoV ( F ) mediated cell–cell fusion. For each condition, ten randomly taken images were analysed by counting the nuclei per syncytium (with at least three nuclei). The data shown are the mean values + SEM of three independent experiments. Statistical significance was determined with a one-way ANOVA followed by Šídák’s multiple comparisons test. ns = not significant, ** = p < 0.01, **** = p < 0.0001.

Article Snippet: The expression vectors coding for SARS- or MERS-CoV S with a C-terminal FLAG-tag, pCMV3-MERS-Spike-cFLAG (VG40069-CF) and pCMV3-SARS-Spike-cFLAG (VG40150-CF), as well as a vector encoding human dipeptidyl peptidase 4 (DPP4) with a C-terminal HA-tag, pCMV3-hDPP4-cHA (HG10688-CY), were purchased from Sino Biological, Houston, TX, USA.

Techniques: Activation Assay, Transfection, FLAG-tag, SDS Page, Western Blot, Control, Plasmid Preparation, Expressing, Incubation, Staining, Fluorescence

Journal: Cell Reports

Article Title: Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells

doi: 10.1016/j.celrep.2020.107894

Figure Lengend Snippet: Activation and Killing by T Cells Is Selective for iPSC-β (A) Experimental design: PBMCs co-cultured with autologous iPSC-derived cells. (B, C, and F) CD25 or CD69 expression shown as MFI. (B) PBMCs (gated on CD3 + , CD4 + , and CD8 + populations) co-cultured for 48 h with autologous iPSC-β (purple) or iPSC-α (red). (n = 3 T1D and n = 1 ND donor, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (C) Donor-matched PBMCs (CD3 + gated) (n = 1 T1D, n = 3 differentiation batches per donor line) co-cultured for 48 h with autologous iPSC-β (purple) or iPSC-cardiomyocytes (orange). (D) Percentage of live iPSC-β (C-peptide + /glucagon − ) or iPSC-α (C-peptide − /glucagon + ) from iPSC-β or iPSC-α differentiations, respectively (n = 3 T1D donors, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (F) Representative flow cytometry histograms after 48 h of co-culture. Dashed histogram represents the control (untreated target cells). (E) Percentage of apoptotic (apopxin + ) iPSC-β (CD49a + /CD26 − ) or iPSC-α (CD49a − /CD26 + ) from iPSC-β or iPSC-α differentiations (n = 1 T1D iPSC donor, n = 3 differentiation batches per donor line). (G) Unmatched PBMCs (CD3 + gated cells) co-cultured for 48 h with iPSC-α (n = 3 T1D donors, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (H) Donor-matched PBMCs (gated on CD3 + , CD4 + , and CD8 + populations) co-cultured for 48 h with autologous enriched iPSC-β or iPSC-α. Data are means ± SEMs, 2-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001; ns, non-significant. See also Figure S4 .

Article Snippet: For the apoptosis assay, clusters were dissociated, and single cells were stained at room temperature for 30 min using a 1:100 dilution of recently reported stem cell derived β-cell marker, anti-human CD49a ( ) PE-conjugated (BD Biosciences, 559596), α-cell marker anti-human CD26 APC-conjugated (Miltenyi Biotech, 130-120-769), and Apopxin green indicator (Abcam, ab176750).

Techniques: Activation Assay, Cell Culture, Derivative Assay, Expressing, Flow Cytometry, Co-Culture Assay, Control

Journal: Cell Reports

Article Title: Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells

doi: 10.1016/j.celrep.2020.107894

Figure Lengend Snippet:

Article Snippet: For the apoptosis assay, clusters were dissociated, and single cells were stained at room temperature for 30 min using a 1:100 dilution of recently reported stem cell derived β-cell marker, anti-human CD49a ( ) PE-conjugated (BD Biosciences, 559596), α-cell marker anti-human CD26 APC-conjugated (Miltenyi Biotech, 130-120-769), and Apopxin green indicator (Abcam, ab176750).

Techniques: Virus, Recombinant, Software, Real-time Polymerase Chain Reaction

Journal: Molecular Cancer Therapeutics

Article Title: PhAc-ALGP-Dox, a Novel Anticancer Prodrug with Targeted Activation and Improved Therapeutic Index

doi: 10.1158/1535-7163.mct-21-0518

Figure Lengend Snippet: Figure 1. PhAc-ALGP-Dox is activated sequentially by extracellular THOP1 and cytoplasmic DPP4/FAPa in the tumor microenvironment. A–C, Generation of PhAc-ALGP-Dox cleavage products in the presence of recombinant enzymes (THOP1, 0.1 mg/mL; FAPa, 0.04 mg/mL; or DPP4, 0.04 mg/mL). Data are represented as mean SD of triplicate analysis, LoQ, 3 nmol/L. D, Quantification of Dox autofluorescence following exposure to 20 mmol/L PhAc-ALGP-Dox for 5 hours. Nuclear/cytoplasmic fluorescence was assessed in HUVEC cells in the presence of recombinant THOP1 (0.1 mg/mL) alone or together with either recombinant FAPa (0.04 mg/mL) or PT-100 (300 nmol/L), an inhibitor of FAPa/DPP4. Bars represent mean þ SD of n ¼ 8–12. Asterisks indicate significant differences versus no enzyme or as specified by brackets. , P < 0.001, , P < 0.0001 as defined by one-way ANOVA corrected for multiple comparisons (Holm–Sidak). E–L, Representative images of HUVEC cells stimulated with PhAc-ALGP-Dox alone (E, F), together with THOP1 (G, H), THOP1 and FAPa (I–J) or THOP1, and PT-100 (K, L). Top panels are merged images of cytoplasmic (Agglutinin, green), nuclear (DAPI, blue), or Dox (autofluorescence, red). Bottom panels are black-white images of Dox autofluorescence. Scale bar, 20 mm. M, Quantification of ELISA data of human THOP1 protein levels in cell lysates and conditioned media of cancer cells (MDA-MB-231 and LS 174T) compared with normal (HME-1) cells. Bars are mean þ SD of three independent ELISAs run in duplicate. Asterisks indicate significant differences versus no enzyme or as specified by brackets. , P < 0.001, , P < 0.0001 as defined by one-way ANOVA corrected for multiple comparisons (Holm–Sidak). N, Kinetic activation of Dnp(k)-ALGP-CouAla by extracellular peptidases in normal (HME-1, blue) or tumor (LS 174T, purple) conditioned medium. Dashed purple line represents the signal in tumor conditioned media, preincubated with a THOP1 inhibitor (THOPi, 10 mmol/L). O, Quantification of Dnp(k)-ALGP-CouAla conversion after 180 minutes. Bars are mean þ SD, pooled from three independent experiments (n ¼ 6). , P < 0.01 as defined by one-way ANOVA.

Article Snippet: Following inhibitors were used to block activity of endogenous peptidases: THOP1 was specifically inhibited with Cpp-AAF-pAB (100 mmol/L Bachem), THOP1 and CD10 were blocked with JMV-390 (10 mmol/L, Tocris #2575), DPP4 and FAPa was constrained using talabostat (1⁄4PT-100, 10 mmol/L, Tocris #3719), whereas FAPi (MedChemExpress, HY100684) was used to specifically inhibit FAPa.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Journal of Virological Methods

Article Title: A high-throughput inhibition assay to study MERS-CoV antibody interactions using image cytometry

doi: 10.1016/j.jviromet.2018.11.009

Figure Lengend Snippet: Exogenously expressed DPP4 in various cell lines. Fluorescence images (A) and fluorescence intensity FlowJo analysis histograms (B) comparing untransfected A549, BHK-21, or Vero E6 cells (left panels) to cells expressing optimal amounts of DPP4, depicted by red staining (right panels).

Article Snippet: Cell nuclei were stained with DAPI, and DPP4 receptors were stained with DPP4 PE-conjugated mouse antibody (Sino Biological, Wayne, PA) or unconjugated DPP4 rabbit polyclonal anitbodies (Abcam, Cambridge, MA) followed by anti-rabbit antibody labeled with Alexa Flour® 488 (AL488) (Abcam).

Techniques: Fluorescence, Expressing, Staining

Journal: Journal of Virological Methods

Article Title: A high-throughput inhibition assay to study MERS-CoV antibody interactions using image cytometry

doi: 10.1016/j.jviromet.2018.11.009

Figure Lengend Snippet: MERS-CoV S1 and S proteins binding to DPP4-expressing cells. Whole well fluorescence images of MERS-CoV S1 (A) and MERS-CoV S (B) proteins binding DPP4-expressing BHK-21 cells. Both S1 monomer and S trimer proteins were stained with anti-MERS-CoV S polyclonal AL488, demonstrating binding to DPP4-expressing cells in green. FlowJo analysis of the image-based fluorescence intensity data for MERS-CoV S1 monomer (C) and S trimer (D) showed peak shifts resulting from binding.

Article Snippet: Cell nuclei were stained with DAPI, and DPP4 receptors were stained with DPP4 PE-conjugated mouse antibody (Sino Biological, Wayne, PA) or unconjugated DPP4 rabbit polyclonal anitbodies (Abcam, Cambridge, MA) followed by anti-rabbit antibody labeled with Alexa Flour® 488 (AL488) (Abcam).

Techniques: Binding Assay, Expressing, Fluorescence, Staining

Journal: Journal of Virological Methods

Article Title: A high-throughput inhibition assay to study MERS-CoV antibody interactions using image cytometry

doi: 10.1016/j.jviromet.2018.11.009

Figure Lengend Snippet: Ability of mAbs to inhibit MERS-CoV S protein binding to DPP4-expressing cells . Inhibition of MERS-CoV S binding to DPP4 by D12 mAb was measured by image cytometry (A) and fluorescence intensity was analyzed by FlowJo histograms (B). As a negative control, a non-relevant isotype mAb was used. Dose response curves of Log mAb concentration versus percent inhibition were generated for D12 (C), G2 (D), and G4 (E) mAbs. (C–E) Each dot represents duplicates. Error bars represent SEM.

Article Snippet: Cell nuclei were stained with DAPI, and DPP4 receptors were stained with DPP4 PE-conjugated mouse antibody (Sino Biological, Wayne, PA) or unconjugated DPP4 rabbit polyclonal anitbodies (Abcam, Cambridge, MA) followed by anti-rabbit antibody labeled with Alexa Flour® 488 (AL488) (Abcam).

Techniques: Protein Binding, Expressing, Inhibition, Binding Assay, Cytometry, Fluorescence, Negative Control, Concentration Assay, Generated

Journal: Journal of Virological Methods

Article Title: A high-throughput inhibition assay to study MERS-CoV antibody interactions using image cytometry

doi: 10.1016/j.jviromet.2018.11.009

Figure Lengend Snippet: Ability of mAbs to neutralize MERS-CoV in cells exogenously-expressing DPP4. Neutralization of MERS-CoV pseudovirus by D12 (red), G2 (blue), and G4 (black) in DPP4-expressing BHK-21 cells. 50% neutralization titers (IC 50 ) were calculated based on sigmoidal curves. Each dot represents duplicates. Error bars represents SEM.

Article Snippet: Cell nuclei were stained with DAPI, and DPP4 receptors were stained with DPP4 PE-conjugated mouse antibody (Sino Biological, Wayne, PA) or unconjugated DPP4 rabbit polyclonal anitbodies (Abcam, Cambridge, MA) followed by anti-rabbit antibody labeled with Alexa Flour® 488 (AL488) (Abcam).

Techniques: Expressing, Neutralization

Journal: Pharmaceutical Biology

Article Title: Ethanol extract of the mushroom Coprinus comatus exhibits antidiabetic and antioxidant activities in streptozotocin-induced diabetic rats

doi: 10.1080/13880209.2022.2074054

Figure Lengend Snippet: Dipeptidyl peptidase (DPP)-4 levels after extract treatment. Histograms with the same letter are not significantly different, values are expressed as mean ± SD ( n : 24) p < 0.05. HC: Healthy control; NC: negative control (45 mg STZ); PC: positive control (45 mg metformin); T1: 250 mg ethanol extract of C. comatus ; T2: 500 mg ethanol extract of C. comatus ; T3: 750 mg ethanol extract of C. comatus .

Article Snippet: Rat-specific DPP-4 (Cat.No E0226Ra), insulin (Cat.No E0707Ra), and GLP-1 (Cat.No E0719Ra) ELISA kits (BT Laboratories, Shanghai, China) were used for the measurement of the corresponding parameters (BT Laboratory ).

Techniques: Control, Negative Control, Positive Control