Journal: Journal of Cellular Physiology

Article Title: Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function

doi: 10.1002/jcp.29463

Figure Lengend Snippet: Effect of erlotinib on the intracellular and lysosomal accumulation of crizotinib and on the role of pH. Cells were treated with 0.1% dimethyl sulfoxide as control, 10 µM erlotinib, 5 µM crizotinib or their combination for 24 hr. Bafilomycin (50 nM) was used to perturb the lysosomal function. (a) Effect of erlotinib on the intracellular crizotinib concentration in pmol/µg protein. (b) Intracellular erlotinib concentration in fmol/µg protein. Bars represent mean ± standard error of mean of three separate tests. (c) Quantification of pHrodogreen intensity in relative fluorescent units with FIJI from d, e. All the p ‐values are summarized in Table S1. (d, e) Intracellular effect of drugs on HCC827 and HCC827GR5, respectively, cells were stained for 1 hr with 5 µM sunitinib and 0.5 µM Lysotracker Red, and for 30 min with pHrodoGreen. Each sample was divided in multiple focus planes (z‐stack). Z‐stacks were imaged using a Leica TCS SP8 STED 3× microscope. Image panels of d from left to right: sunitinib staining of HCC827 control cells; sunitinib staining of HCC827 treated with 10 µM erlotinib and 5 µM crizotinib; Phrodogreen and Lysotracker red staining of HCC827 control cells; Phrodogreen and Lysotracker red staining of HCC827 treated with 10 µM erlotinib and 5 µM crizotinib. Image panels of e from left to right: sunitinib staining of HCC827GR5 control cells; sunitinib staining of HCC827GR5 treated with 10 µM erlotinib and 5 µM crizotinib; Phrodogreen and Lysotracker red staining of HCC827GR5 control cells; Phrodogreen and Lysotracker red staining of HCC827GR5 treated with 10 µM erlotinib and 5 µM crizotinib. Control: 0.1% DMSO, E: 10 µM erlotinib, C: 5 µM crizotinib, Combo: 10 µM erlotinib + 5 µM crizotinib, B: 50 nM Bafilomycin A1. * p < .05 as compared to control, ** p < .01; *** p < .001 as compared to control

Article Snippet: The intracellular drug concentration was measured as previously described (Honeywell et al., ).

Techniques: Control, Concentration Assay, Staining, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phase variation in Mycobacterium tuberculosis glpK produces transiently heritable drug tolerance

doi: 10.1073/pnas.1907631116

Figure Lengend Snippet: Differential tolerance of HN878 SC and LC variants to supra-MIC concentrations of antibiotics and to hydrogen peroxide. M. tuberculosis HN878 SC and LC strains grown in 7H9 supplemented with OADC and Tween 80 to midlog phase (OD600 = 0.6 to 0.7) were diluted to ∼5 × 106 or 5 × 107 CFU/mL and treated in the presence of glycerol with (A) 0.4 µg/mL MXF (4× MIC) or (B) 1 µg/mL INH (20× MIC). Viability was determined by CFU counts. (C) Fold-change with P value of survival differences between SC and LC identified in A and B. (D) Differential susceptibility of HN878 SC and LC strains to H2O2 compared to control was determined by OD600 measurements. Both strains were grown in 7H9 medium containing OADC and Tween 80 with (+) or without (−) glycerol to midlog phase (OD600 = 0.6 to 0.7). The cultures were diluted 1/20 and treated with different amounts of H2O2. The treated and control cultures were incubated for 6 d at 37 °C. OD600 were recorded and normalized to the corresponding control without oxidant treatment. The values of 3 independent experiments are shown for each graph. Significant differences of survival frequencies were calculated using 1-tailed Student’s t test, *P < 0.05.

Article Snippet: Briefly, the first column of wells of a 96-well plate (Costar) was filled with 200 µL of 7H9 containing the drug at maximum concentration to be tested.

Techniques: Incubation