Cyanide 3 chlorophenylhydrazone

The MICs of Imipenem was determined in a range of 1 to 512 μg/ml for all Imipenem resistant Acinetobacter isolates. Determination of Imipenem MIC was repeated in the presence of efflux pumps inhibitor, cyanide 3-chlorophenylhydrazone (CCCP), in order to investigate the role of efflux pumps in Imipenem resistant isolates. To determine the appropriate concentration of CCCP, an MIC determination of CCCP was also carried out and a concentration equals to 1/2 and 1/4 of the determined MIC was used as non-inhibitory concentration. Determination of MIC values was carried out using the broth microdilution method in 96 well micro plates according to CLSI standards (12 ). Imipenem and cyanide 3-chlorophenylhydrazone powder were purchased from Sigma Aldrich, United States of America.

N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), β-actin antibody, oligomycin, rotenone, RU486, ascorbate, succinic acid, digitonin, cyanide 3-chlorophenylhydrazone (CCCP), antimycin A and 4-hydroxytamoxifen (4-HOT) were acquired from Sigma (St Louis, MO, USA). Rapamycin was from AG Scientific, Inc (San Diego, CA, USA); (³H) 2-deoxyglucose was acquired from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Phospho-Akt, Phospho-AMPK, total AMPK, Glut-4, hexokinase I, GPX-1, p70S6K (total and phosphorylated form) and α/β tubulin antibodies were from Cell Signaling Technology, Inc (Danvers, MA, USA). Cytochrome c, Akt total, SCO2, Glut-1, hexokinase II antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). MitoProfile® Total OXPHOS Human WB Antibody Cocktail was obtained from Mitosciences (Eugene, OR, USA); 7-AAD, MitoSOX™ Red, MitoTracker™ Green and rhodamine-123 were purchased from Invitrogen/Molecular Probes (Carlsbad, CA, USA). p70S6K and MigR1 plasmids were from Addgene (Cambridge, MA, USA) and shRNA P70S6K were from Thermo Scientific/Dharmacon (Lafayette, CO, USA) and were transfected to the cells according to the method provided by the companies.