Fig 2: ZAR1 functions in the resistance to the fungal pathogen Botrytis cinerea. (A) ZAR1 functions in the resistance to the fungal pathogen Botrytis cinerea. Lesions on the leaves of zar1-3, zar1-4 mutant, and Col-0 plant were observed at 48 h after inoculation with B. cinerea. Lesion sizes were analyzed. Error bars represent SEM from means of 16 leaves from each line. Asterisks indicate significant differences compared with Col-0 plant (****P < 0.0001, Student’s t-test). Scale bar = 1 cm. (B) Chlorophyll fluorescence imaging and quantification of FPSII was measured in zar1-3 mutant, cerk1 mutant, and Col-0 plant, at 36 h after B. cinerea spray. Error bars represent SEM of 16 leaves from each line. Asterisks indicate significant differences between each mutant and Col-0 plant (*P < 0.05, **P < 0.01, Student’s t-test). (C) Visualization of cell death in infected leaves at 36 h post-infection. Representative images of trypan blue (TPB) stained leaves are shown. Scale bar = 500 µm. (D) Chitin-triggered MAPK activation in zar1-3 mutant, cerk1 mutant, and wild type Col-0. 10-day-old seedlings were treated with 200 µg/mL chitin at the indicated time points. MAPK activation was detected with an anti-pERK antibody. cerk1 mutant was included as a control. Equal loading is demonstrated by CBB (Coomassie brilliant blue) staining of Rubisco (below). The experiments were repeated three times, with similar results. (E) Leaves of the 4-week-old zar1-3 mutant, cerk1 mutant, and Col-0 plants were treated with 200 µg/ml chitin and incubated with luminol and horseradish peroxidase to detect ROS. Luminescence was recorded at different time points as indicated. Error bars represent SEM of data derived from replicate samples (n = 12). The chemiluminescence was measured in a time-course manner using a microplate reader (Varioskan LUX, Thermo Fisher Scientific Inc., MA, United States).

Fig 3: Impact of exogenous supplementation of fatty acids and phospholipids on the activity of Rom. S. aureus HG001 was grown in BM with Rom (8 µg/mL) and various supplementations. For panel A, supplementations included the following FAs: 4 µg/mL of C15:0 (˜16 µM), 4 µg/mL of C18:0 (˜14 µM), 4 µg/mL of C16:0 (˜16 µM), 10 µg/mL of C16:1 (˜39 µM), and 21 µg/mL of C18:2 (˜75 µM). For panel B, supplementations included 8 µg/mL of the lipid glyceryl tripalmitate (˜9.9 µM) and phospholipids as follows: 4 µg/mL of PG 18:0 (˜4.6 µM) and 4 µg/mL of Lys-PG 18:1 (˜4 µM). Wells containing tryptic soy broth (TSB) only and bacteria with Rom (8 µg/mL) alone were included as negative and positive controls, respectively. Growth of the bacteria was measured at OD578 every 4 h for 48 h using a Varioskan Lux microplate reader (Thermo Scientific) in a 48-well plate. Each point in the graph is the mean ± SD from three independent biological replicates.

Fig 4: VitD and TXNIP silencing prevent high glucose-induced accumulation of triglycerides and lipid droplets. HUVEC were cultured in medium containing 5 mM (CTR), 11.1, or 30 mM of d-glucose (-) and either after TXNIP silencing or after the administration of 20 nM VitD. l-glucose (30 mM) was used as a control of osmolarity. (A) Triglyceride accumulation was measured by the Triglyceride Quantification Kit, as described in the methods. (B) The cells were stained with Oil Red O, and after the image acquisition using the FLoid Cell Imaging Station (Thermo Fisher Scientific) (left panel), the cells were solubilized, and the triglycerides were quantified by measuring the absorbance at 500 nm using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific) (right panel). The results are the mean of three experiments that were repeated in triplicate ± SD. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Fig 5: Hydrolysis of remdesivir by human liver S9 fractions and by recombinant CES1 or CES2 and hydrolysis-based cytotoxicity. (A) Hydrolysis of remdesivir by human-liver S9 fractions. Remdesivir (1 µM) was incubated at 37°C for 0 or 60 minutes with individual human S9 fraction (0.1 µg/µL) (n = 26) The reactions were terminated by two volumes of termination buffer (acetonitrile and methanol: 50:50) containing the internal standard tenofovir (adenine-13C[U]) (438 ng/mL). The reaction mixtures were subjected to centrifugation at 4°C for 15 minutes to remove the proteins, and the supernatants were analyzed for the decrease of remdesivir at mass transition of m/z 603.2 to 200 and internal standard at 293.2 to 181.1 by liquid chromatography with tandem mass spectrometry (TSQ Fortis). The quantification was determined with the standard curve generated with remdesivir. To specify the level of CES1 or CES2, S9 fractions (0.5 µg) were analyzed by western blotting and normalized with the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The correlation of hydrolysis by S9 fractions was made by SPSS software (IBM, Armonk, NY). Potential outliers are indicated by arrows. (B) Hydrolysis of remdesivir by recombinant CES1 or CES2. Reactions were set up as previously with cell lysates (0.005 µg/µL) from cells transfected with CES1, CES2, or the corresponding vector. The expression of CES1 and CES2 in the lysates was confirmed by western blotting. (C) Implicated cytotoxicity of excessive hydrolysis of remdesivir cells (parent, nontransfected HEK293T; vector, vector-transfected HEK293T; and CES1, CES1 transfected HEK293T-CES1) were seeded as 8,000 per well in a 96-well plate. After overnight, cells were treated with remdesivir (1 µM) for 72 hours and analyzed for proliferation using a Cell Proliferation kit (Sigma-Aldrich, St. Louis, MO). The spectrophotometric absorbance of sample was measured using a microplate reader (Varioskan Lux; Thermo Fisher Scientific, Waltham, MA). To verify the expression of CES1, cell lysates (6.25 µg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the expression of CES1 and GAPDH was analyzed by western blotting (×250 magnification). All experiments were performed in triplicate. *P < 0.05. **P < 0.01.