nbp2 01830  (Novus Biologicals)

Journal: Cells

Article Title: DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies

doi: 10.3390/cells12071100

Figure Lengend Snippet: DPP8-dependent antineoplastic effect of high-dose 1G244. ( A ) 1.0 × 10 5 of hematological cancer cell lines (MM.1S, KARPAS299, THP-1, KG1, Daudi, or NAMALWA) were cultured with DPP8/9 inhibitors (1G244 or talabostat) at doses of 0–100 µM for 72 h. Cell number was estimated by a colorimetric assay using WST-1 reagent (n = 6). ( B ) Knockdown studies of DPP8 and DPP9 in MM.1S or KARPAS299 cells. NT, no treatment; GFP, control vector; KD, knockdown. Expression level of DPP8 or DPP9 was estimated by Western blot analysis. ( C ) 1.0 × 10 5 of MM.1S and KARPAS299 cells and their transfectants were cultured with DPP8/9 inhibitors (1G244 or talabostat) at doses of 0–100 µM for 6 h. Cytotoxicity was estimated by a LDH release assay (n = 6).

Article Snippet: This was followed by transfer to an Immobilon-P membrane (Millipore, Burlington, MA, USA) and hybridization with an anti-DPP8 antibody (OTI1D2), monoclonal, mouse, NBP2-01830 (Novus Biologicals, Englewood, CO, USA), an anti-DPP9 antibody (OTI2E3), monoclonal, mouse, NBP2-01521 (Novus Biologicals), an anti-GSDMD antibody, monoclonal, rabbit, ab210070 (Abcam, Cambridge, UK), an anti-Cleaved Caspase-3 antibody (Asp175, 5A1E), monoclonal, rabbit, #9664S (Cell Signaling Technology, Danvers, MA, USA), an anti-DFNA5/GSDME antibody (EPR19859, N-terminal), monoclonal, rabbit, ab215191 (Abcam), an anti-Caspase-1 antibody, polyclonal, rabbit, #2225S (Cell Signaling Technology), an anti-CARD8 antibody (2108C2a), monoclonal, mouse, sc-81213 (Santa Cruz Biotechnology, Dallas, TX, USA), an anti-Caspase-3 antibody, polyclonal, rabbit, #9662S (Cell Signaling Technology), an anti-HCK antibody (E1I7F), monoclonal, rabbit, #14643 (Cell Signaling Technology), an anti-LCK antibody (3A5), monoclonal, mouse, sc-433 (Santa Cruz Biotechnology), an anti-Fyn antibody (15), monoclonal, mouse, sc-434 (Santa Cruz Biotechnology), an anti-c-Fgr antibody (B-8), monoclonal, mouse, sc-166079 (Santa Cruz Biotechnology), an anti-Lyn antibody (H-6), monoclonal, mouse, sc-7274 (Santa Cruz Biotechnology), an anti-Blk antibody (9D10D1), monoclonal, mouse, sc-65980 (Santa Cruz Biotechnology), an anti-β-actin antibody (8H10D10), monoclonal, mouse, #3700S (Cell Signaling Technology), an anti-AK2 antibody, polyclonal, rabbit, ab37594 (Abcam), an anti-FADD antibody (A66-2), monoclonal, mouse, 556402 (BD Biosciences, Franklin Lakes, NJ, USA), an anti-phospho-FADD (Ser194) antibody, polyclonal, rabbit, #2781S (Cell Signaling Technology), or an anti-DUSP26 antibody, polyclonal, rabbit, GTX109283 (GeneTex, Zeeland, MI, USA).

Techniques: Cell Culture, Colorimetric Assay, Knockdown, Control, Plasmid Preparation, Expressing, Western Blot, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies

doi: 10.3390/cells12071100

Figure Lengend Snippet: Caspase-3-mediated apoptosis as anticancer effect by high-dose 1G244. ( A ) 1 × 10 6 MM.1S cells were cultured with DPP8/9 inhibitors (1G244 or talabostat) at doses of 0–100 µM for 3–48 h. The cleaved form (CL) of gasdermin-D (GSDMD), caspase-3, or gasdermin-E (GSDME) was detected by Western blot analysis. The upper caspase-3 CL: 19 kDa; and the lower caspase-3 CL: 17 kDa. ( B , C ) 1.0 × 10 5 of MM.1S cells were cultured with DPP8/9 inhibitors (1G244 or talabostat) at doses of 0–100 µM for 6 h with disulfiram (DSF), Z-DEVD, Z-VAD, necrostatin-1 (Nec), or necrosulfonamide (NSA). Cytotoxicity was estimated by a LDH release assay (n = 6).

Article Snippet: This was followed by transfer to an Immobilon-P membrane (Millipore, Burlington, MA, USA) and hybridization with an anti-DPP8 antibody (OTI1D2), monoclonal, mouse, NBP2-01830 (Novus Biologicals, Englewood, CO, USA), an anti-DPP9 antibody (OTI2E3), monoclonal, mouse, NBP2-01521 (Novus Biologicals), an anti-GSDMD antibody, monoclonal, rabbit, ab210070 (Abcam, Cambridge, UK), an anti-Cleaved Caspase-3 antibody (Asp175, 5A1E), monoclonal, rabbit, #9664S (Cell Signaling Technology, Danvers, MA, USA), an anti-DFNA5/GSDME antibody (EPR19859, N-terminal), monoclonal, rabbit, ab215191 (Abcam), an anti-Caspase-1 antibody, polyclonal, rabbit, #2225S (Cell Signaling Technology), an anti-CARD8 antibody (2108C2a), monoclonal, mouse, sc-81213 (Santa Cruz Biotechnology, Dallas, TX, USA), an anti-Caspase-3 antibody, polyclonal, rabbit, #9662S (Cell Signaling Technology), an anti-HCK antibody (E1I7F), monoclonal, rabbit, #14643 (Cell Signaling Technology), an anti-LCK antibody (3A5), monoclonal, mouse, sc-433 (Santa Cruz Biotechnology), an anti-Fyn antibody (15), monoclonal, mouse, sc-434 (Santa Cruz Biotechnology), an anti-c-Fgr antibody (B-8), monoclonal, mouse, sc-166079 (Santa Cruz Biotechnology), an anti-Lyn antibody (H-6), monoclonal, mouse, sc-7274 (Santa Cruz Biotechnology), an anti-Blk antibody (9D10D1), monoclonal, mouse, sc-65980 (Santa Cruz Biotechnology), an anti-β-actin antibody (8H10D10), monoclonal, mouse, #3700S (Cell Signaling Technology), an anti-AK2 antibody, polyclonal, rabbit, ab37594 (Abcam), an anti-FADD antibody (A66-2), monoclonal, mouse, 556402 (BD Biosciences, Franklin Lakes, NJ, USA), an anti-phospho-FADD (Ser194) antibody, polyclonal, rabbit, #2781S (Cell Signaling Technology), or an anti-DUSP26 antibody, polyclonal, rabbit, GTX109283 (GeneTex, Zeeland, MI, USA).

Techniques: Cell Culture, Western Blot, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies

doi: 10.3390/cells12071100

Figure Lengend Snippet: Dependence on HCK for DPP8/9 inhibitor-induced pyroptosis. ( A ) Expression level of DPP8, DPP9, caspase-1, CARD8, GSDMD, or caspase-3 in THP-1, MM.1S, KARPAS299, KG1, NAMALWA, or Daudi cells was estimated by Western blot analysis. ( B ) Gene expression of THP-1, MM.1S, KARPAS299, KG1, NAMALWA, or Daudi cells was analyzed by microarray method using 3D-Gene. ( C ) Expression level of HCK, LCK, Fyn, c-Fgr, Lyn, or Blk in THP-1, MM.1S, KARPAS299, KG1, NAMALWA, or Daudi cells was estimated by Western blot analysis. ( D ) 1.0 × 10 5 of hematological cancer cell lines (Jurkat, K562, MOLM-13, NOMO-1, RPMI8226, Raji, or SKM-1) were cultured with 1G244 at doses of 0–10 µM for 72 h. Cell number was estimated by a colorimetric assay using WST-1 reagent (n = 6). ( E ) Expression level of HCK or CARD8 in Jurkat, K562, MOLM-13, NOMO-1, RPMI8226, Raji, or SKM-1 cells was estimated by Western blot analysis. ( F ) Knockdown studies of HCK in MM.1S cells. NT, no treatment; GFP, control vector; KD, knockdown. Expression level of HCK was estimated by Western blot analysis. ( G ) 1.0 × 10 5 of MM.1S cells and their transfectants were cultured with DPP8/9 inhibitors (1G244 or talabostat) at doses of 0–100 µM for 6 h. Cytotoxicity was estimated by a LDH release assay (n = 6).

Article Snippet: This was followed by transfer to an Immobilon-P membrane (Millipore, Burlington, MA, USA) and hybridization with an anti-DPP8 antibody (OTI1D2), monoclonal, mouse, NBP2-01830 (Novus Biologicals, Englewood, CO, USA), an anti-DPP9 antibody (OTI2E3), monoclonal, mouse, NBP2-01521 (Novus Biologicals), an anti-GSDMD antibody, monoclonal, rabbit, ab210070 (Abcam, Cambridge, UK), an anti-Cleaved Caspase-3 antibody (Asp175, 5A1E), monoclonal, rabbit, #9664S (Cell Signaling Technology, Danvers, MA, USA), an anti-DFNA5/GSDME antibody (EPR19859, N-terminal), monoclonal, rabbit, ab215191 (Abcam), an anti-Caspase-1 antibody, polyclonal, rabbit, #2225S (Cell Signaling Technology), an anti-CARD8 antibody (2108C2a), monoclonal, mouse, sc-81213 (Santa Cruz Biotechnology, Dallas, TX, USA), an anti-Caspase-3 antibody, polyclonal, rabbit, #9662S (Cell Signaling Technology), an anti-HCK antibody (E1I7F), monoclonal, rabbit, #14643 (Cell Signaling Technology), an anti-LCK antibody (3A5), monoclonal, mouse, sc-433 (Santa Cruz Biotechnology), an anti-Fyn antibody (15), monoclonal, mouse, sc-434 (Santa Cruz Biotechnology), an anti-c-Fgr antibody (B-8), monoclonal, mouse, sc-166079 (Santa Cruz Biotechnology), an anti-Lyn antibody (H-6), monoclonal, mouse, sc-7274 (Santa Cruz Biotechnology), an anti-Blk antibody (9D10D1), monoclonal, mouse, sc-65980 (Santa Cruz Biotechnology), an anti-β-actin antibody (8H10D10), monoclonal, mouse, #3700S (Cell Signaling Technology), an anti-AK2 antibody, polyclonal, rabbit, ab37594 (Abcam), an anti-FADD antibody (A66-2), monoclonal, mouse, 556402 (BD Biosciences, Franklin Lakes, NJ, USA), an anti-phospho-FADD (Ser194) antibody, polyclonal, rabbit, #2781S (Cell Signaling Technology), or an anti-DUSP26 antibody, polyclonal, rabbit, GTX109283 (GeneTex, Zeeland, MI, USA).

Techniques: Expressing, Western Blot, Gene Expression, Microarray, Cell Culture, Colorimetric Assay, Knockdown, Control, Plasmid Preparation, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies

doi: 10.3390/cells12071100

Figure Lengend Snippet: Antitumor effects of tominostat. ( A ) 1.0 × 10 5 of MM.1S, KARPAS299, or Daudi cells were cultured with DPP8/9 inhibitors (1G244 or tominostat) at doses of 0–100 µM for 6 h. Cytotoxicity was estimated by a LDH release assay (n = 6). ( B , C ) 5 × 10 6 of MM.1S ( B ) or Daudi ( C ) cells were subcutaneously inoculated into NSG mice (n = 6). Three days after inoculation, 1G244 or tominostat was administered subcutaneously once a week. Tumor volume was assessed at the same time.

Article Snippet: This was followed by transfer to an Immobilon-P membrane (Millipore, Burlington, MA, USA) and hybridization with an anti-DPP8 antibody (OTI1D2), monoclonal, mouse, NBP2-01830 (Novus Biologicals, Englewood, CO, USA), an anti-DPP9 antibody (OTI2E3), monoclonal, mouse, NBP2-01521 (Novus Biologicals), an anti-GSDMD antibody, monoclonal, rabbit, ab210070 (Abcam, Cambridge, UK), an anti-Cleaved Caspase-3 antibody (Asp175, 5A1E), monoclonal, rabbit, #9664S (Cell Signaling Technology, Danvers, MA, USA), an anti-DFNA5/GSDME antibody (EPR19859, N-terminal), monoclonal, rabbit, ab215191 (Abcam), an anti-Caspase-1 antibody, polyclonal, rabbit, #2225S (Cell Signaling Technology), an anti-CARD8 antibody (2108C2a), monoclonal, mouse, sc-81213 (Santa Cruz Biotechnology, Dallas, TX, USA), an anti-Caspase-3 antibody, polyclonal, rabbit, #9662S (Cell Signaling Technology), an anti-HCK antibody (E1I7F), monoclonal, rabbit, #14643 (Cell Signaling Technology), an anti-LCK antibody (3A5), monoclonal, mouse, sc-433 (Santa Cruz Biotechnology), an anti-Fyn antibody (15), monoclonal, mouse, sc-434 (Santa Cruz Biotechnology), an anti-c-Fgr antibody (B-8), monoclonal, mouse, sc-166079 (Santa Cruz Biotechnology), an anti-Lyn antibody (H-6), monoclonal, mouse, sc-7274 (Santa Cruz Biotechnology), an anti-Blk antibody (9D10D1), monoclonal, mouse, sc-65980 (Santa Cruz Biotechnology), an anti-β-actin antibody (8H10D10), monoclonal, mouse, #3700S (Cell Signaling Technology), an anti-AK2 antibody, polyclonal, rabbit, ab37594 (Abcam), an anti-FADD antibody (A66-2), monoclonal, mouse, 556402 (BD Biosciences, Franklin Lakes, NJ, USA), an anti-phospho-FADD (Ser194) antibody, polyclonal, rabbit, #2781S (Cell Signaling Technology), or an anti-DUSP26 antibody, polyclonal, rabbit, GTX109283 (GeneTex, Zeeland, MI, USA).

Techniques: Cell Culture, Lactate Dehydrogenase Assay

Journal: Virchows Archiv : an international journal of pathology

Article Title: Expression and clinical role of the dipeptidyl peptidases DPP8 and DPP9 in ovarian carcinoma.

doi: 10.1007/s00428-018-2487-x

Figure Lengend Snippet: Fig. 2 DPP8 and DPP9 protein expression in HGSC effusions. Cytoplasmic expression of DPP8 (a, b) and DPP9 (c, d) in tumor cells

Article Snippet: The DPP8 antibody was a mouse monoclonal antibody purchased from Novus Biologicals (cat no. NBP2-01830, clone OTI1D2; Littleton, CO), applied at a 1:200 dilution.

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes

doi: 10.3389/fphys.2018.01622

Figure Lengend Snippet: Structural analysis of gliptins-DPP4/8/9 binding. (A) A representative Western blot ( n = 3) showing expression of DPP8 and DPP9 in total protein lysate (50 μg) of HL-1 cardiomyocytes and mouse LV. (B) 3D structural alignment of saxagliptin bound to DPP4 (PDB code: 3bjm) and sitagliptin (PDB code: 1×70) aligned with the 3D structure of DPP8 (PDB code: 6eoo) using pymol. The 3D structure of DPP4 and DPP8 are shown as cartoon representation and colored in orange and gray, respectively. The DPP4-bound forms of saxagliptin and sitagliptin are shown in sticks and colored in magenta and green, respectively. The DPP4 and DPP8 amino-acids present in the gliptin-binding surface of DPP4 are shown in sticks and colored in orange and gray, respectively, if conserved in the structural model or, in red and cyan, if not. (C) 3D structural alignment as described in (B) but using DPP9 (PDB code: 6eoq, colored in yellow) in place of DPP8. Representative Western blots ( n = 6) for (D) pCaMKII (T286), (E) pPLB (T17), and (F) CaMKII and PLB expression in HL-1 cardiomyocytes treated with TC-E 5007 (2 μM) for the indicated time points. GAPDH was used as a loading control. “n” represents the number of experiments.

Article Snippet: The following primary antibodies (diluted in 5% [w/v] BSA-TBST) were used: phospho-CaMKII (pCaMKII, T286, 1:1000, Abcam, ab32678), phospho-PLB (pPLB, T17, 1:5000, Badrilla, A010-13), DPP8 (1:500, Santa Cruz, sc-376399), DPP9 (1:500, Santa Cruz, sc-271634), CaMKII (1:500, Santa Cruz, sc-9035), and PLB (1:2000, Thermo Fisher Scientific, MA3-922).

Techniques: Binding Assay, Western Blot, Expressing, Control

Journal: Frontiers in Physiology

Article Title: Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes

doi: 10.3389/fphys.2018.01622

Figure Lengend Snippet: Saxagliptin inhibits the CaMKII-PLB axis via DPP9 inhibition. (A) mRNA and (B) protein expression of DPP8 and DPP9 in HL-1 cardiomyocytes transfected with si-scr, si-DPP8 or si-DPP9. GAPDH was used as a house keeping gene/protein. A representative Western blot showing (C) pCaMKII (T286), and (D) pPLB (T17) expression in HL-1 cardiomyocytes transfected with si-DPP8 or si-DPP9, and/or treated with saxagliptin or sitagliptin (2 μM each) as indicated for (C) 5 min and (D) 30 min. All values are expressed as mean ± SEM ( n = 6). ∗ p < 0.05 vs. si-scr DPP8 and # p < 0.05 vs. si-scr DPP9 by one-way ANOVA followed by Tukey’s post hoc test. “n” represents the number of experiments.

Article Snippet: The following primary antibodies (diluted in 5% [w/v] BSA-TBST) were used: phospho-CaMKII (pCaMKII, T286, 1:1000, Abcam, ab32678), phospho-PLB (pPLB, T17, 1:5000, Badrilla, A010-13), DPP8 (1:500, Santa Cruz, sc-376399), DPP9 (1:500, Santa Cruz, sc-271634), CaMKII (1:500, Santa Cruz, sc-9035), and PLB (1:2000, Thermo Fisher Scientific, MA3-922).

Techniques: Inhibition, Expressing, Transfection, Western Blot

Journal: Frontiers in Physiology

Article Title: Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes

doi: 10.3389/fphys.2018.01622

Figure Lengend Snippet: Saxagliptin inhibits PKC via DPP9 inhibition. Quantification of active PKC levels in HL-1 cardiomyocytes treated with saxagliptin or sitagliptin in a (A) time-dependent (2 μM) or (B) concentration-dependent manner (10 min), and (C) TC-E 5007 (2 μM) for indicated time periods. (D) HL-1 cardiomyocytes were transfected with si-DPP8 or si-DPP9, and/or treated with saxagliptin or sitagliptin (2 μM) for 10 min as indicated to follow measurements of active PKC. All values are expressed as mean ± SEM ( n = 6). ∗ p < 0.05 vs. (A,C) 0 min, (B) 0 μM, and (D) si-scr by one-way ANOVA followed by Tukey’s post hoc test. “n” represents the number of experiments.

Article Snippet: The following primary antibodies (diluted in 5% [w/v] BSA-TBST) were used: phospho-CaMKII (pCaMKII, T286, 1:1000, Abcam, ab32678), phospho-PLB (pPLB, T17, 1:5000, Badrilla, A010-13), DPP8 (1:500, Santa Cruz, sc-376399), DPP9 (1:500, Santa Cruz, sc-271634), CaMKII (1:500, Santa Cruz, sc-9035), and PLB (1:2000, Thermo Fisher Scientific, MA3-922).

Techniques: Inhibition, Concentration Assay, Transfection