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