Journal: Heliyon

Article Title: N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine induces endothelium-dependent hyperpolarization-mediated vasorelaxation via store-operated calcium entry mechanism in healthy and intestinal inflammatory mice

doi: 10.1016/j.heliyon.2024.e33994

Figure Lengend Snippet: TPEN induced vasorelaxation of the second-order branch of mice mesenteric arteries via EDH mechanisms predominantly. (A) Summarized data shows TPEN-induced CRC, R max , AUC and EC 50 of the second-order branch of mice mesenteric arteries pre-constricted with NE (5 μM, n = 6) or KCl (80 mM, n = 6). (B) Summarized data shows TPEN-induced CRC, R max , AUC and EC 50 of the second-order branch of mice mesenteric arteries with 100 μM L-NNA plus 10 μM INDO (n = 6), L-NNA + INDO (&) + 0.3 μM SKA-31(n = 6), or L-NNA + INDO (&) + 3 μM apamin (Apa) + 30 μM TRAM-34 (TRAM, n = 6). (C) Summarized data shows TPEN-induced CRC, R max , AUC and EC 50 of the second-order branch of mice mesenteric arteries with L-NNA + INDO (n = 6), or L-NNA + INDO (&) + 10 μM SN-6 (n = 6), or L-NNA + INDO (&) + 100 μM ouabain (n = 6). Data were expressed as a percentage of NE-induced vasoconstriction and shown as means ± SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns: no significance.

Article Snippet: TPEN, TRAM-34, apamin, SKA-31, SN-6, ouabain, GSK-7975 A, SKF96365, ML-9, and Brefeldin A (BFA) were supplied by MedChemExpress (MCE), headquartered in New Jersey, USA.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Functional expression of K Ca 3.1 in THP-1-derived M 2 macrophages. ( A , B ): Simultaneous measurements of changes in the membrane potential ( A ) and [Ca 2+ ] i ( B ) in the three different cells [a (black), b (blue), and c (red)], following the application of a selective K Ca 3.1 activator, SKA-121 (1 μM), and/or the K Ca 3.1 inhibitor, TRAM-34 (1 μM) using DiBAC 4 (3) and Fura 2, respectively. The relative time-course changes in fluorescence intensities (1.0 at 0 s) from three different THP-1-derived M 2 macrophages are shown. ( C ): Real-time PCR examination of K Ca 3.1 expression in THP-1-derived M 0 macrophages (‘M 0 ’) and M 2 macrophages (‘M 2 ’). Expression levels are shown as a ratio to ACTB ( p > 0.05, n = 4 for each). ( D , E ): Protein expression levels of K Ca 3.1 in the ‘M 0 ’ and ‘M 2 ’ groups were determined by Western blot. Specific band signals for K Ca 3.1 were observed at approximately 50 kDa ( D , upper panel). After compensation with the optical density of the ACTB signal (43 kDa) ( D , lower panel), the expression level in the ‘M 0 ’ group was expressed as 1.0 ( p > 0.05, n = 4 for each) ( E ). ( F ): SKA-121 (1 μM)-induced relative hyperpolarizing responses in the ‘M 0 ’ and ‘M 2 ’ groups ( p > 0.05, n = 37 and 29, respectively).

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Functional Assay, Expressing, Derivative Assay, Membrane, Fluorescence, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Whole-cell patch-clamp recordings of SKA-121-activated K + currents in THP-1-derived M 2 macrophages. ( A ): Typical current density and voltage-relationships after addition of vehicle (black), 1 μM SKA-121 (red), and 1 μM SKA-121 plus 1 μM TRAM-34 (blue). Currents were elicited by ramp depolarization from −120 to +40 mV from a holding potential of −80 mV every 10 s. ( B ): Typical current density and voltage-relationship of SKA-121-sensitive component. ( C ): Summarized results of current densities (pA/pF) at +40 mV in three groups (n = 9 for each). *: p < 0.05 vs. vehicle control (−/−); ## p < 0.01 vs. SKA-121 alone (+/−).

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Patch Clamp, Derivative Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Effects of treatment with SKA-121 on IL-10, IL-8, VEGF-A, and TGF-β1 expression and on IL-10 and IL-8 secretion in THP-1-derived M 2 macrophages. A–D: Real-time PCR examination of IL-10 ( A ), IL-8 ( B ), VEGF-A ( C ), and TGF-β1 ( D ) expression in THP-1-derived M 2 macrophages treated (+) or untreated (−) with 1 μM SKA-121 and 10 μM TRAM-34 for 24 h. Relative mRNA expression in the vehicle control (‘−’ for SKA and ‘−’ for TRAM) is expressed as 1.0 (n = 4 for each). ( E , F ): Quantitative detection of IL-10 ( E ) and IL-8 ( F ) secretion by an ELISA assay in THP-1-derived M 2 macrophages treated and untreated with SKA-121 and TRAM-34. Relative cytokine secretion in the vehicle control (−/−) is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control (−/−), ## : p < 0.01 vs. SKA-121 alone (+/−).

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Effects of treatment with SKA-121 on high [K + ] e -enhanced IL-10 and IL-8 expression and secretion in THP-1-derived M 2 macrophages. A , B : Real-time PCR examination of IL-10 ( A ) and IL-8 ( B ) expression in normal [K + ] e (5 mM)- and high [K + ] e (25 mM)-treated THP-1-derived M 2 macrophages for 24 h in the presence (+) or absence (−) of SKA-121 (1, 10 μM). Relative mRNA expression in normal [K + ] e is expressed as 1.0 (n = 4 for each). ( C , D ): Quantitative detection of IL-10 ( C ) and IL-8 ( D ) secretion by an ELISA assay in THP-1-derived M 2 macrophages treated and untreated with SKA-121. Relative cytokine secretion in normal [K + ] e is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. normal [K + ] e , ## : p < 0.01 vs. the vehicle control (−/−) of high [K + ] e .

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Protein expression levels of phosphorylated ERK1/2 (P-ERK1/2) and P-JNK in THP-1-derived M 2 macrophages following the SKA-121 treatment and high [K + ] e exposure. A – D : Western blot showing P-ERK1/2, total ERK1/2 (ERK1/2) ( A,B ), P-JNK, and total JNK (JNK) ( C , D ) in 1 μM SKA-121-treated ( A , C ) and high [K + ] e -exposed ( B , D ) THP-1-derived M 2 macrophages. Specific band signals were observed at 42 (P-ERK2), 42 (ERK2), 43/50 (P-JNK), and 43/50 (JNK) kDa. E – H : Summarized results of the relative protein expression of P-ERK2/ERK2 ( E , F ) and P-JNK/JNK ( G , H ) in 1 μM SKA-121-treated ( E , G ) and high [K + ] e -exposed ( F , H ) THP-1-derived M 2 macrophages. After compensation with the optical density of the ACTB signal (43 kDa), the expression level in the vehicle control or 5 mM K + is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control and 5 mM K + .

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Expressing, Derivative Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Protein expression levels of phosphorylated c-Jun (P-c-Jun) following the SKA-121 treatment and high [K + ] e exposure in THP-1-derived M 2 macrophages. ( A , B ): Western blot showing P-c-Jun, and unphosphorylated c-Jun (c-Jun) in 1 μM SKA-121-treated ( A ) and high [K + ] e -exposed ( B ) THP-1-derived M 2 macrophages. Specific band signals were observed at 42-46 (P-c-Jun) and 40 (c-Jun) kDa. ( C , D ): Summarized results of the relative protein expression of P-c-Jun/c-Jun in 1 μM SKA-121-treated ( C ) and high [K + ] e -exposed ( D ) THP-1-derived M 2 macrophages. After compensation with the optical density of the ACTB signal (43 kDa), the expression level in the vehicle control is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control and 5 mM K + .

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Expressing, Derivative Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Effects of treatment with a CREB inhibitor on expression levels of IL-10, IL-8, VEGF-A, and TGF-β1 transcripts and on phosphorylated P-CREB protein levels following the SKA-121 treatment and high [K + ] e exposure in THP-1-derived M 2 macrophages. ( A – D ): Real-time PCR examination of IL-10 ( A ), IL-8 ( B ), VEGF-A ( C ), and TGF-β1 ( D ) expression in THP-1-derived M 2 macrophages treated with vehicle and 1 μM 666-15 for 24 h. Relative mRNA expression in the vehicle control is expressed as 1.0 (n = 4 for each). E-F: Western blot showing P-CREB and total CREB (CREB) in 1 μM SKA-121-treated ( E ) and high [K + ] e -exposed ( F ) THP-1-derived M 2 macrophages. Specific band signals for P-CREB and CREB were observed at approximately 40 kDa. G, H: Summarized results of the relative protein expression of P-CREB/CREB in 1 μM SKA-121-treated ( G ) and high [K + ] e -exposed ( H ) THP-1-derived M 2 macrophages. After compensation with the optical density of the ACTB signal, the expression level in the vehicle control or 5 mM K + is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control or 5 mM K + .

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by the Activation of Ca 2+ -Activated K + Channel K Ca 3.1 in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms23158603

Figure Lengend Snippet: Effects of treatment with SKA-121 on expression levels of IL-10, IL-8, VEGF-A, and TGF-β1 transcripts and IL-10 and IL-8 secretion in human prostate cancer PC-3 cell-cultured medium-treated THP-1-derived M 2 macrophages. ( A – D ): Real-time PCR examination of IL-10 ( A ), IL-8 ( B ), VEGF-A ( C ), and TGF-β1 ( D ) expression in PC-3 media-treated THP-1-derived M 2 macrophages treated (+) or untreated (−) with 1 or 10 μM SKA-121 for 24 h. Relative mRNA expression in the group untreated with PC-3 medium is expressed as 1.0 (n = 4 for each). ( E , F ): Quantitative detection of IL-10 ( E ) and IL-8 ( F ) secretion by an ELISA assay in THP-1-derived M 2 macrophages treated or untreated with SKA-121. Relative secretion in the group untreated with PC-3 medium is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control (−/−).

Article Snippet: TRAM-34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SKA-121 [ ] (MedChemExpress), Luna Universal qPCR master Mix (New England Biolabs Japan, Tokyo, Japan), the IL-10/IL-8/IL-1β Human Uncoated ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA), AZD5363, LY364947, everolimus, SCH772984, Bay11-7082 (Cayman Chemical, Ann Arbor, MI, USA), LY294002 (Chem Scene, Monmouth Junction, NJ, USA), ciclosporin A (FUJIFILM Wako Pure Chemicals), T-5224 (APExBIO, Boston, MA, USA), ML385, sulforaphane, 666-15 (Selleckchem), DiBAC 4 (3), Fura 2-acetoxymethyl ester, WST-1, 1-methoxy PMS (Dojindo, Kumamoto, Japan), SP600125 (LC Laboratories, Woburn, MA, USA), and phorbol 12-myristrate 13-acetate (PMA) (AdipoGen, San Diego, CA, USA) were also purchased form the indicated sources.

Techniques: Expressing, Cell Culture, Derivative Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control