Varioskan

SIRT1 activity modulation was assessed by using a Direct Fluorescent Screening Assay Kit (SIRT1 Direct Fluorescent Screening Assay Kit, Cat # KA1366, ABNOVA, Taiwan) following the instructions of the provider (Figures S1 and S2, Supplementary material). The formation of the final fluorescent product was detected using a Varioskan (Varioskan, Thermo Scientific, MA, USA) with an excitation wavelength of 360 nm and an emission wavelength of 465 nm.
For each EPH fraction, the % inhibition/activation was calculated following the Eq. (4), and the fold activation was calculated following the instructions of the provider by using the Eq. (5): $$\%Inhibition/Activation=\left[\frac{InitialActivityfluorescenc{e}_{\mathit{control}}-Samplefluorescence}{InitialActivityfluorescenc{e}_{\mathit{control}}}\right]\times 100$$ $$FoldActivation=\frac{Samplefluorescence}{InitialActivityfluorescenc{e}_{\mathit{control}}}$$ where “Initial Activity fluorescence _control” is the fluorescence obtained in wells with SIRT1 dissolved in assay buffer and solvent;
“Sample fluorescence” is the fluorescence from wells with SIRT1 dissolved in buffer assay and solvent plus samples.
To perform the Pearson´s correlation test and the principal component analysis, negative values of SIRT1 activity were scaled and normalized into a range of 0 and 1, according to Teknomo⁷⁵ .

The antioxidant capacities of the samples were determined with the ABTS method. ABTS+ (ABTS radical cation) method was used with Trolox as a standard antioxidant for the antioxidant analysis. As stated in the literature, 7 mM aqueous ABTS solution was mixed with 2.45 mM potassium persulfate solution to form ABTS+. The prepared mixture was then left in the dark at ambient temperature for 12–16 h to complete the reaction. The ABTS+ solution was diluted with ethanol to obtain 0.7 A (±0.02) at 734 nm. Each sample was analyzed 3 times. Antioxidant capacities of the samples were calculated as Trolox equivalent. Sample (10 µL) and ABTS solution (200 µL) were mixed and waited in the dark for 30 min. Then absorbance values were recorded at 734 nm with a spectrophotometer (Varioskan, Thermo Fischer, Waltham, MA, USA).
Total phenolic content was determined with the Folin-Ciocalteu method using gallic acid as a standard. Samples were prepared as 20 μL of the diluted sample solution and 100 μL Folin-Ciocalteu reagent previously diluted with a 1:10 ratio in a 96-well plate. Then 80 µL 7.5% Na₂CO₃ solution was added to the mixture and waited in the dark for an hour. Then absorbance values were recorded at 725 nm with a spectrophotometer (Varioskan, Thermo Fischer, USA).

Cells were washed in phosphate buffered saline and harvested with passive lysis buffer (Promega) for luciferase and protein measurements. Extracts were distributed into the wells of a 96 plate and Luciferase Assay Reagent II (Promega) was injected onto the samples in a Varioskan (Thermo Scientific) plate reader. The integration time was set at 1000ms for each measurement. The light emission values were normalized against the protein content of the samples measured by BCA protein assay (Thermo Scientific): 200 µl working reagent (50 part BCA ‘A’ +1 part BCA ‘B’) was added to 25 µl of each sample and after 30 minutes incubation in the dark the absorbance of the samples were measured in a Varioskan plate reader at 562 nm.

BMCs stimulated by 10 ng/mL of M-CSF and 2.5 ng/mL IFN-γ were exposed to 100 μM nicotine or PBS for 4 days followed by an activation period of 6 or 18 hours by 10 ng/mL of LPS (Sigma Aldrich), with or without 100 μM nicotine. Supernatant was harvested by centrifugation at ~300 x g for 10 minutes and conserved at -80°C. The presence of the following cytokines in the supernatants was evaluated by ELISA using Peprotech’s (Rocky Hill, NJ, USA) ELISA Development kit: TNF-α, IL-1β, IL-12 and IL-10, following the manufacturer’s instructions. Fluorescence was quantified by a Varioskan (Thermo Electron Corporation, Marietta, OH, USA) and the results were analyzed by the SkanIt^™ software by Thermo Scientific.

Two fluorescent dyes were used to assess cytotoxicity in PLHC-1 cells: Alamar Blue (AB), which estimates the metabolic activity, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which monitor membrane impairment. PLHC-1 cells were seeded in 96-well plates at a density of 7.5 × 10⁴ cells per well (200 µL) and were allowed to attach for 24 h. After 24 h exposure of the cells to sediment extracts, the exposure medium was removed and 100 µL of a solution containing 5% AB and 4 µM CFDA-AM, was added to each well and the plate incubated for 1 h. Results were obtained as relative fluorescent units (RFUs) at the excitation/emission wavelengths pairs of 530/590 nm for AB, and 485/530 nm for CFDA-AM in a fluorescent plate reader (Varioskan, Thermo Electron Corporation). At least six replicates in three different plates were read to measure the cytotoxic effect of the extract at a given concentration by comparison to the fluorescence read in non-exposed cells. Metabolic impairment (AB) and disruption of membrane integrity (CFDA-AM) were determined in PLHC-1 cells after 24 h exposure to organic sediment extracts at 120 mg eQsed·mL⁻¹.

The assay was performed to test whether disulfide cross-linking using the cysteine pair P221C/M394C affects the chaperone activity of full-length ClpB. 0.2 μm α-glucosidase from Bacillus stearothermophilus was denatured for 8 min at 75 °C in reaction buffer containing 50 mm MOPS, pH 7.5, 150 mm KCl, 10 mm MgCl₂, and 5 mm ATP. Chaperones were added prior to refolding at 55 °C. The total chaperone concentrations were c(ClpB) = 1.0 μm, c(DnaK) = 1.6 μm, c(DnaJ) = 0.5 μm, and c(GrpE) = 0.2 μm. The co-chaperones DnaK, DnaJ, and GrpE from T. thermophilus were expressed and purified as described previously (32 (link)). Samples were taken after 30, 60, and 120 min and diluted 1:10 into assay buffer containing 50 mm KP_i, pH 6.8, 2 mmpara-nitrophenyl-α-d-glucopyranoside, 0.1 mg/ml BSA. The α-glucosidase activity was measured at 40 °C using a microplate spectrophotometer (Varioskan, Thermo Electron, Vantaa, Finland). The average rate of absorption increase at 405 nm was monitored and normalized against a positive control containing α-glucosidase that was not heat-aggregated. Importantly, reducing agents were strictly excluded from all buffers to maintain the intermolecular disulfide bonds of covalently linked ClpB dimers.