Total protein kit

Protein concentrations were assayed using a total protein kit (Sigma, St. Louis, MO, USA) based on the method of Lowry [69 (link)] and modified by Peterson [70 (link)]. Standards were prepared by diluting 400 μg/mL of BSA in water. Sodium chloride (final concentration of 0.1 M) was added in order to reduce ampholyte interference. Proteins were precipitated by adding 0.1% trichloroacetic acid in the presence of 0.15% deoxycholate. Color was measured at 500 nm in a Wallac 1420 VICTOR3TM Multilabel counter spectrophotometer (PerkinElmer, Wellesley, MA, USA).

For enzymatic assays, 30 mL of bioreactor culture was harvested by centrifugation (5 min, 7745 ×g) after 17 h of cultivation in the presence of IPTG. CFE were made according to the Y-PER Yeast Protein Extraction Reagent kit instructions (Thermo Scientific). Protein concentrations were determined by using the Total Protein Kit, Micro Lowry, Onishi and Barr Modification (Sigma–Aldrich).
The activity of CadA and Irg1 was measured with cis- and trans-aconitate according to Vuoristo et al. (2015) (link). Besides, the Irg1 activity was measured by using a method adapted from Michelucci et al. (2013) (link) with the following modifications: CFE’s were incubated with 200 mM of cis-aconitate in 25 mM HEPES buffer (pH 7.1) supplied with proteinase inhibitor (cOmplete Protease Inhibitor Cocktail Tablets, Roche) for 50 min at 30°C.
For whole cell assays, 20 mL of bioreactor culture was harvested by centrifugation (5 min, 7745 × g) after 17 h of cultivation in the presence of IPTG. Cells were washed with M9 salts in 100 mM MOPS (pH 7.1) and resuspended in 10 ml of the same medium. Twenty-millimeter of substrate (cis- or trans-aconitate) was added to 2 mL of the cell suspension and incubated at 30°C and 100 rpm. 1 M HCl was added to terminate the reaction, after which the supernatants were analyzed for itaconate formation by HPLC.

Total cell proteins were isolated and purified using the Total Protein Kit (Sigma) according to the manufacturer’s protocol. Extraction and isolation of nuclear and cytoplasmic proteins were performed according to the manufacturer’s instructions (Yeasen, China). Briefly, after treatment, cells were washed twice with PBS, scraped and collected by centrifugation at 1500×g for 5 min. Cell pellets were resuspended in 250 μl extraction buffer A and incubated for 10 min on ice. Afterwards, extraction buffer B was added, and samples were vortexed for 30 s at 4 °C. After centrifugation at 12000×g for 5 min at 4 °C, supernatants, which contained the cytosolic fractions, were removed out. The rest pellets, which contained the nuclei, were resuspended in 50 μl of nuclear extraction buffer, and nuclear proteins were extracted by shaking the samples for 30 min at 4 °C. Afterwards, samples were centrifuged at 12000×g for 5 min at 4 °C and the supernatants were removed for analyzing. The protein concentration was determined using the BCA assay method (Pierce, USA).

The examination of cells morphology on TCP was performed by optical microscopy. The cell response was evaluated by quantification of alkaline phosphatase (ALP) and total protein (TP) in the cell populations after exposure to an optimum electric treatment selected after performing several experiments and analysing cells morphology. ALP activity is the most widely recognized biochemical marker for early osteoblast differentiation. ALP enzyme activity (Sigma Aldrich, P7998) was assessed after 1 and 3 days of cell culture. Further on, the TP is an indication of cells proliferation. The intracellular quantity for TP synthesis was assessed after 1 and 3 days of cell culture by Total Protein Kit (Sigma TP0400). An Infinite F200PRO UV/visible Spectrometer has been used to detect the ALP and the TP levels at 405 and 600 nm wavelengths, respectively. Experimental data were collected from three separate experiments and were expressed as mean values 6SD. They were analysed using SPSS 14.0 software (SPSS, USA). The level of significance was calculated using Student's test for single comparisons and ANOVA for multiple comparisons. Statistical significance was assumed at the 95% confidence limit or greater (p<0.05).

Brain tissue was dissected from 4 cortical areas: parahippocampal cortex (Brodmann area 36), cingulate cortex (Brodmann area 24), frontal cortex (Brodmann area 8) and posterior thalamus (pulvinar). Brain tissue (200 mg) samples were sequentially extracted in 1% NP-40 buffer (140 mM NaCl, 3 mM KCl, 25 mM Tris ph 7.4, 5 mM EDTA, 2 mM 1,10-phenanthroline, 0.1 M PMSF, 1.7 mg/ml aprotinin, NP-40 detergent, 100 ml dH₂O) (soluble extract) in a Precellys 24 homogeniser (Stretton Scientific, Derbyshire, UK) with 2.3 mm ceramic beads (Biospec, Glasgow, UK). The homogenates were spun at 13000 g for 15 min at 4°C and the supernatant was removed and stored at −80°C. Insoluble material was solubilized by vigorous agitation in 6 M GuHCl, re-homogenized and left for 4 h at room temperature (RT) before storage at −80°C.Total protein concentrations were determined using the Total Protein Kit (Sigma Aldrich, Dorset, UK) following the manufacturer’s guidelines.

A total of 3–4 colonies of A. hydrophila were transferred into 10 ml TSB and incubated at 28°C overnight. After incubation, cells were collected by centrifugation at 8,000 rpm for 10 min and adjusted to the concentration of 1 × 10⁹ CFU/ml (PRJNA808687) in 10 ml of phosphate-buffered solution (PBS). Then, SMEO at the concentration of 1 × MIC was added to the abovementioned solutions and treated at 28°C for 4, 8, and 12 h. Finally, the supernatants were separated from bacterial cells by centrifugation (8,000 rpm) and filtration (with 0.22 μm filter, Sigma-Aldrich, United States), which will be used for the nucleic acid and protein assay as described. At the same time, SMEO treatment for 0 h was used as the zeroing sample for UV spectroscopic detection.
For the nucleic acid assay, the absorbance of the supernatant samples was measured by a spectrophotometer (UV759S, INESA Analytical Instrument Co., Ltd., China) at the optical density of 260 nm.
For the protein assay, a Total Protein Kit, Micro (Sigma-Aldrich, United States) was used to measure the concentration of protein. According to the operating instructions, the protein assay solution was mixed with the supernatant samples. After approximately 2 min, the absorbance of the sample was also measured by a spectrophotometer at the optical density of 595 nm (UV759S, INESA Analytical Instrument Co., Ltd., China).