Journal: Scientific Reports
Article Title: Mercury and arsenic attenuate canonical and non-canonical NLRP3 inflammasome activation
Figure Lengend Snippet: Effects of heavy metals on NLRP3 inflammasome activation. ( A ) Schematic diagram for inflammasome activation. BMDMs (1 × 10 6 cells per well) were treated with LPS (1 μg/ml) as a 1 st signal for priming, after which cells were replaced with media containing an inflammasome trigger (2 nd signal) with/without heavy metals (HMs) as indicated. Inflammasome activation was assessed by ELISA and/or immunoblotting. ( B ) LPS-primed BMDMs were treated with nigericin (NG) in the presence of media only (Non), cadmium (Cd), mercury (Hg), arsenic (As), or lead (Pb). Release of IL-1β was analyzed by ELISA. ( C ) LPS-primed BMDMs were subjected to activation of NLRP3 inflammasome by NG, monosodium urate crystal (MSU), or aluminum (Alum) treatment in the presence of an increasing dosage of mercury (Hg). The cleaved form of caspase-1 (Casp1, p20) was detected by immunoblotting, and secretion of IL-1β was observed with ELISA. ( D ) LPS-primed BMDMs were treated with NG, MSU, or Alum in the presence of arsenic (As). Release of Casp1 and IL-1β was analyzed with immunoblotting and ELISA. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD with at least two independent experiments.
Article Snippet: To activate NLRP3 inflammasome, cells were treated with RPMI 1640 medium (350 μl/well) without FBS or antibiotics in the presence of nigericin (NG, 40 μM; 4312, Tocris Bioscience, Bristol, UK) for 1 h, monosodium urate crystals (MSU, 400 μg/ml; U2875, Sigma–Aldrich Co.) which were prepared in according to a previous study for 3 h, aluminium potassium sulfate (Alum, 200 μg/ml; 039–4404, Daejung Chemicals & Materials Co., Daejeon, Republic of Korea) for 3 h, or rotenone (160 μM; sc-203242, Santa Cruz Biotechnology, Dallas, TX, USA) for 6 h. For non-canonical inflammasome activation, cells were transfected by LPS (15 μg/ml, Sigma-Aldrich Co) with Lipofectamine 2000 (10 μl/ml, Invitrogen) and incubated for 6 h or infected by Escherichia coli (MOI 10, DH5α, Invitrogen, CA, USA) for 3 h. Heavy metals, cadmium acetate dihydreate (CAS No. 5743-04-04, Cat. No. 2616-4140), mercury(II) chloride (CAS No. 7487-94-7, Cat. No. 5542-4425), arsenic(III) oxide (arsenic trioxide, CAS No. 1327-53-3, Cat. No. 5076-1405), lead(II) acetate trihydrate (CAS No. 6080-56-4, Cat. No. 5075-4405), or lead(II) chloride (CAS No. 7758-95-4, Cat. No. 5076-1405), were purchased from Daejung Chemicals & Materials Co. and co-treated with the above inflammasome triggers.
Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay