Journal: Frontiers in Pharmacology

Article Title: Hematopoietic Cell Kinase (HCK) Is Essential for NLRP3 Inflammasome Activation and Lipopolysaccharide-Induced Inflammatory Response In Vivo

doi: 10.3389/fphar.2020.581011

Figure Lengend Snippet: HCK inhibition attenuated NLRP3 inflammasome-induced inflammatory response in macrophages and microglia. (A) PMs were seeded in a 96-well plate (1 × 10 4 cells/well) plate. After 12 h, the cells were treated with A419259 for 4 h, and cell viability was detected. (B) ELISA for cleaved IL-1 β (P17) in the supernatants of LPS-primed (500 ng, 2 h) PMs treated with JAK3(JANEX-1) or HCK(A419259) inhibitor for 1 h and stimulated with nigericin for 30 min. (C) Immunoblotting analysis of cleaved IL-1 β (P17) in the supernatants from LPS-primed (500 ng, 2 h) PMs treated with JAK3(JANEX-1) or HCK(A419259) inhibitor for 1 h and stimulated with nigericin for 30 min, followed by immunoblotting for pro-IL-1 β or NLRP3 antibody. (D) Immunoblotting analysis of cleaved IL-1 β (P17) and cleaved caspase 1 (P20) in the supernatants of LPS-primed (500 ng, 2 h) PMs treated for 1 h with the HCK inhibitor (A419259) and then stimulated with 1.5 mM ATP for 30 min, followed by immunoblotting analysis with pro-IL-1 β , pro-caspase 1, NLRP3, or β -tubulin antibodies in whole cell lysates. (E, F) Immunoblotting analysis of cleaved IL-1 β or cleaved caspase 1 in the supernatants of LPS-primed (500 ng, 2 h) microglia treated for 1 h with HCK inhibitors (A419259) and then stimulated with 5 μM nigericin (E) or 1.5 mM ATP (F) for 30 min. The cell lysates were immunoblotted with antibodies against pro-IL-1 β , pro-caspase 1, NLRP3, or β - tubulin in cell lysates. Data from (A – F) are representative of at least three independent experiments. Data show means ± SEM. *** P < 0.001, ns, P > 0.05, Student’s t -test.

Article Snippet: Nigericin (HY-127019), JANEX-1 (HY-15508), and A419259 (HY-15764) were from MedChemExpress (Monmouth, NJ, USA).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Oncotarget

Article Title: The farnesoid X receptor negatively regulates osteoclastogenesis in bone remodeling and pathological bone loss

doi: 10.18632/oncotarget.20576

Figure Lengend Snippet: ( A , E ) BMMs from FXR +/+ and FXR −/− mice were cultured with RANKL (200 ng/ml) for 24 h. The mRNA level was analyzed by real-time PCR with IFN-β or JAK3 primer. ( B ) BMMs from FXR +/+ and FXR −/− mice were serum-starved for 16 h and stimulated with RANKL (200 ng/ml) for the indicated time. ( F ) BMMs from FXR +/+ and FXR −/− mice were cultured with RANKL (200 ng/ml) for the indicated time. Cell lysates were then subjected to western blot analysis with anti-p-STAT1 or anti-JAK3 antibody. ( C , D ) BMMs were infected by mock or FXR through a retrovirus packaging system. Infected BMMs were stimulated with RANKL (200 ng/ml) for 4 or 24 h. The mRNA levels of IFN-β and p-STAT1 were analyzed by real-time PCR or western blotting. ( G ) BMMs from FXR +/+ mice were cultured with RANKL (100 ng/ml) in the presence of tofacitinib, a JAK3 inhibitor, for 3 days. TRAP + MNCs were counted as osteoclasts when more than 3 nuclei were present. Scale bar, 200 μm. ( H ) FXR +/+ BMMs were transfected with 40 nM siRNA. The siRNA-transfected FXR +/+ BMMs were cultured with RANKL (200 ng/ml) for 3 days, and then TRAP + osteoclasts were counted. ( I ) The siRNA-transfected FXR +/+ BMMs were serum-starved for 16 h and stimulated with RANKL (200 ng/ml) for 4 h. ( J ) The siRNA-transfected FXR +/+ BMMs were cultured with RANKL (200 ng/ml) for 24 h. Cell lysates were then subjected to western blotting analysis with anti-p-STAT1 or anti-NFATc1 antibody. Data are expressed as mean ± SD from at least three independent experiments. Scale bar, 200 μm. * p < 0.05.

Article Snippet: Tofacitinib (JAK3 inhibitor) was purchased from LC Laboratories (Woburn, MA, USA).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Infection, Transfection