Resazurin

The measurement of the minimum inhibitory concentration (MIC) of compounds 1 and 2 was determined using the resazurin reduction microplate assay (REMA) as described previously (Palomino et al., 2002 (link)). M. smegmatis, E. coli and P. putida were grown to mid-log phase, and the inoculum was standardized to 1 × 10⁶ colony forming units (CFU)·mL⁻¹ before addition to the prepared 96-well flat-bottom microtiter plate with twofold serial dilutions of each drug in media. An antibiotic control was also added to each plate (rifampicin for M. smegmatis, ampicillin for E. coli and tetracycline for P. putida), and the last column of the plate was used as a control without the addition of compound. In the case of M. smegmatis, the plates were incubated for 24 h at 37°C without shaking before an addition of 25 μL resazurin [one tablet of resazurin (VWR) dissolved in 30 mL of sterile PBS]. Following a further incubation at 37°C, the plates were assessed for colour development The MIC values were determined as the lowest concentration of drug that prevented the colour change of resazurin (blue – no bacterial growth) to resorufin (pink – bacterial growth). The MIC values were determined with five independent experimental repeats, and data are presented as mean ± SEM.

Epoxomicin, MG132, and lactacystin were purchased from Cayman Chemical. SP600125 was purchased from Cell Signaling. Trehalose was purchased from Calbiochem. Bafilomycin A1 was purchased from LC Laboratories. Resazurin and Calcein-AM were purchased from VWR. The polyclonal tau antibody was purchased from Dako. 12E8 (pSer262) was a gift from P. Seubert (Prothena, Inc) and PHF-1 (pSer396/404) was a gift from P. Davies (The Feinstein Institute for Medical Research). The other antibodies used in the study were AT-180 (pThr231, Pierce), anti-LC3B (Cell Signaling), anti-HSPB8 (Cell Signaling), anti-BAG3 (Proteintech), anti-β-actin (Millipore), and anti-α-tubulin (Pierce Biotechnology).