Optical adhesive film

The NTUH-K2044, NTUH-K2044ΔterC, NTUH-K2044 pVector, NTUH-K2044ΔterC pVector, and NTUH-K2044ΔterC pTerZ-F strains were cultured overnight from single colonies in M9-Glu. Overnight cultures were diluted to 10⁷ CFU/mL into M9-Glu with a 2× concentration of strain-appropriate antibiotics. Then, metals, ROS generators, antibiotics, biocides, or K₂TeO₃ was diluted in M9-Glu to a 2× final concentration. 100 μL of this solution were plated into a single well of a U bottom 96-well plate in triplicate and then 2-fold serially diluted into M9-Glu 10 times, discarding 50 μL of the last dilution to achieve a final volume of 50 μL in each well, leaving the last well as only medium. Finally, 50 μL of the 2× culture dilution were plated across each serial dilution to achieve a final cell density of 5× 10⁶ CFU/mL. This plate was sealed using optical adhesive film (Applied Biosystems, Waltham, MA) and incubated at 37°C for 24 h. After 24 h, the MIC of each compound was defined as the lowest concentration that fully inhibited bacterial growth. Due to the opacity of the high concentration metal solutions, the MICs of the metals were confirmed via replicate plating onto LB-agar and overnight culture at 37°C.

MBP-tagged TDP-43 (10 μM) was incubated in the presence or absence of 5 µM DNAJB6 WT or LGMDD1 disease mutants for 10 min at 37 °C. The aggregation was induced by cleavage of the MBP solubility tag with 0.1 μM TEV protease and monitored at 37 °C by measuring light scattering at 360 nm as a function of time. All reactions were performed in 50 mm of HEPES pH 7.4 buffer supplemented with 100 mm of KCl and 1 mM DTT. Assays were acquired in an area scan mode with a 3 × 3 matrix for each well in clear, flat-bottom, 96-well plates (Nunc) sealed with optical adhesive film (Applied Biosystems). Assays were conducted in triplicate and the mean ± standard deviation is reported.