Sonopuls gm 3100

The samples of NPs were added to filtered seawater to obtain the working suspensions with a concentration of 1000 mg/L. Before each series of bioassays, the working suspensions of NPs were sonicated with ultrasound homogenizer Bandelin Sonopuls GM 3100 (Bandelin Electronic GmbH & Co. KG, Berlin, Germany) using maximal intensity for 30 min.
The exposition of microalgae cells to the suspensions of NPs was carried out in 24-well plates. Each well was filled with 2 mL of microalgae cell aliquot and the corresponding volume of the working suspension to obtain the final concentrations 1, 10, and 100 mg/L. The filtered seawater without NPs was added to the control group. The exposure of each used concentration and control group was performed in four biological replicates.

Microalgal cultures were provided by The Resource Collection Marine Biobank of the National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences (NSCMB FEB RAS). The bioassays were conducted on two marine diatoms (Bacillariophyta), namely A. ussuriensis (Stonik, Orlova et Crawford, 2006) and C. muelleri (Lemmermann, 1896) (Figure S1).
Microalgae cultivation and toxicity tests were conducted according to the guidance of OECD No. 201 [42 ] with minor modifications, as described previously [43 (link)]. For microalgae bioassays, we used 24-well plates with VEPs at the concentrations of 1, 10, and 100 mg/L. The working suspensions of VEPs were sonicated with an ultrasound homogenizer (Bandelin Sonopuls GM 3100, Bandelin Electronic GmbH & Co. KG, Berlin, Germany) with a high-frequency power of 100 W for 30 min before each series of bioassays. The sonication was performed to prevent initial particle agglomeration, according to the protocols of particle suspension testing [44 (link)] and previous work [27 (link),45 (link)]. The wells with only the f/2 medium [46 (link)] were taken as a control group. All the bioassays were performed in quadruplicate. The volume of microalgae aliquots in each well was 2 mL.

His-mATGL and its point mutants were recombinantly expressed in Expi293F™ cells (Thermo Fisher Scientific, Waltham, USA). The cells were cultivated in Expi Expression Medium (Thermo Fisher Scientific, Waltham, USA) at standard conditions (37°C, 95% humidified atmosphere, 7% CO₂). Cell density was determined in a CASY Cell Counter and Analyzer System (OMNI Life Science, Bremen, Germany). Cells in a total culture volume of 10 ml were transfected with 10 μg of plasmid DNA using the ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Two days after transfection, cells were harvested by centrifugation at 500 g and 4°C for 5 min. The cell pellet was washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). Cells were disrupted immediately after harvesting by sonication (Sonopuls GM3100 equipped with a MS-72 tip; Bandelin, Berlin, Germany) in sucrose solution (250 mM sucrose, 1 mM EDTA, 1 mM DTT, 20 μg/ml leupeptin, 2 μg/ml antipain, and 1 μg/ml pepstatin) on ice. The homogenate was centrifuged at 1,000 g and 4°C for 20 min. The postnuclear fraction was collected and protein concentration was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, USA) using (BSA) as standard.