Fig 2: ESF tea ameliorated cytotoxicity and attenuated intracellular ROS generation in H2O2-treated HaCaT cells. (A) HaCaT cells were treated with four extract fractions at the indicated concentrations (10, 20, 50, 100 µg/mL) for 24 h. Cell viability was evaluated by a CCK-8 kit. (B) The effect of H2O2 on human immortalized keratinocyte (HaCaT) cell viability was evaluated by CCK-8. (C) HaCaT cells were treated with four extract fractions for 24 h before being exposed to H2O2 (750 µM). The protective effect of tea extract on cell viability in H2O2-treated cells was evaluated by CCK-8 kit. (D) HaCaT were cultured in 35 mm dishes, treated with different extract fractions (50 µg/mL) for 24 h and then exposed to 750 µM H2O2. Intracellular ROS generation was measured by a fluorescence microscope using CM-H2DCFDA probe. (E) Under the same experimental conditions, intracellular ROS intensity was measured with a SpectraMax i3X microplate reader (Molecular Devices, CA, USA) and compared to the control group. Data were represented as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Rough MPEC strains are better activators of inflammatory response in mammary epithelial cells. Using lentivirus vector, mammary epithelial cells EPH4 were transduced with luciferase gene under the control of 5 copies of the NF-kappa-B response element. Transduced EPH4 cells were treated with 1 µgr/mL LPS (blue line A), and the following E. coli strains at MOI = 1; P4 (black line A), P4?GalE (green line A), P4?GalU (red line A), P4-NR (black line B), P4-96 (blue line B), P4-NR?GalE (green line B), P4-NR?GalU (red line B). NF-kappa-B activation was measured via analysis of luciferase activity in the presence of 150 µg/mL d-luciferin (RLU; relative luminescence units) following infection with the above described bacterial strains using SpectraMax i3x multiple detection microplate reader (Molecular Devices, CA, USA). In all experiments, LPS and normal medium were used as positive and negative controls, respectively. Data points are mean for one experiment performed in triplicate and represent the results of the three similar experiments.

Fig 4: Erythrocytes sequester infective ISAV particles and inhibit serial infection of cultured cells. (A) Density gradient-purified erythrocytes (RBC) and plasma samples were harvested from ISAV-infected fish (13 d.p.i. in the 2020 trial) and inoculated on ASK cells. Data points represent samples from individual fish. ** p = 0.079, Mann–Whitney U. (B) Serially diluted RBC from healthy fish were added to infected ASK cells 24 h p.i. and incubated another 24 h before supernatants were transferred to new ASK cells. Infection of ASK cells 72 h.p.i. was measured by immunostaining for ISAV NP and automated quantification of the number of infected cells (Spectramax i3x plate reader, minimax 300 Imaging Cytometer module, Molecular devices, CA, USA). The infectivity graphs show medians (dots) and 95% confidence intervals (bars), representative of two independent experiments. *** p < 0.001, ** p < 0.01, one sample t test. (C) Serially diluted RBC from healthy fish were incubated with ISAV. The total levels of ISAV RNA associated with the diminishing RBC pellets were measured by qPCR (red, left y-axis). ISAV RNA relative to the RBC content in the pellet was calculated by the ??CT method, using ELF1a as reference gene and the highest dilution of RBC with ELF1a CT < 35 (considered the reliable limit of detection) as reference sample (black, right y-axis, open circle indicates ELF1a CT > 35). Data show the means of technical duplicates representative of two independent experiments.

Fig 5: Effects of GPA and inhibition of AKT on phosphorylation of AKT and expression level of NRF2 and its downstream genes. (A) Cells were co-pretreated with GPA (50 µM) and MK2206 (10 µM) for 24 h and then exposed to 750 µM H2O2 for 4 h. Western blotting was performed to identify the corresponding genes’ expression levels. (B) HaCaT cells were pretreated with GPA (50 µM), and SC66 (5 µM) for 24 h, followed by treatment with H2O2 (750 µM) for another 4 h. Cell lysates were collected and then used for Western blotting analysis. (C) Under the same experimental conditions, intracellular ROS generation was measured by a fluorescence microscope using a CM-H2DCFDA probe. (D) In a parallel experiment, the DCF fluorescence intensity was measured with a SpectraMax i3X microplate reader (Molecular Devices, CA, USA) and compared to the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.