• Wed. Feb 12th, 2025

Dang provided pCMV-FLAG-N3DA, and pHES1-luciferase constructs

Byacusticavisual

Oct 17, 2024

Dang provided pCMV-FLAG-N3DA, and pHES1-luciferase constructs. find a kinase-dependent physical association between the Notch3 and EGFR receptors and tyrosine phosphorylation of Notch3. This could explain the worsened survival observed in some studies of erlotinib treatment at early-stage disease, and suggests that specific dual targeting might overcome this adverse effect. luciferase constructs were provided by Graham Carpenter (Vanderbilt University). Dr. Thao P. Dang provided pCMV-FLAG-N3DA, and pHES1-luciferase constructs. The TP1-luc reporter construct contains 12 tandem repeats of CSL binding sites upstream of luciferase. Co-immunoprecipitation, immunoprecipitation and western blotting Cells were washed twice in ice-cold phosphate buffered saline, harvested and lysed with NP40 buffer (10 mM phosphate buffer, 120 mM NaCl, 2.7 mM KCl, 1% Nonidet P40, 10% glycerol) for co-immunoprecipitation experiments or lysed with RIPA buffer (10 mM phosphate buffer, 120 Bozitinib mM NaCl, 2.7 mM KCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS) supplemented with complete mini-EDTA free protease inhibitor mixture (Roche) and phosphatase inhibitor mixture cocktails 2 and 3 (sigma), 2 mM NaF and pervanadate for immunoprecipitation for detection of phosphorylation. Equal amount of lysates were precipitated using appropriate antibodies and protein G magnetic beads, or equal amounts of protein were mixed with SDS sample buffer and separated on SDS-PAGE prior to Western analysis. Aldefluor assay and Flow cytometry The aldefluor assay kit (Stem cell Technologies) was used to determine the ALDH+ cells. The assay was performed according to manufacturers instructions with modifications. Cells were suspended in aldefluor assay buffer and divided into two groups. One group was pretreated for 10 min with ALDH-specific inhibitor Diethylaminobenzaldehyde (DEAB) before incubation with ALDH enzyme substrate Bodipy-Aminoacetaldehyde (BAA) for 45 minutes at 37 C. Cells were centrifuged and re-suspended in a fresh aldefluor assay buffer to remove the unutilized substrate. Cells were analyzed on a FACSCalibur (BD Biosciences) Flow Cytometer. For the analysis of ALDH+ cells, DEAB treated sample was used as a negative control and ALDH activity in presence of DEAB was considered as a baseline. Pulmosphere formation assay To study the stem-like cell phenotype, sphere formation assays were performed as described previously (25) with modifications. HCC827 cells treated with vehicle control or erlotinib were trypsinized and counted using Luna automated cell counter. Cells were seeded in 96-well plates at 1000 cells per well in RPMI supplemented with 10% fetal bovine serum, 35 g/ml bovine pituitary extract (Life Technologies), N2 supplement (Invitrogen), 20 ng/ml EGF, 20 ng/m (Life Technologies), basic fibroblast growth Bozitinib factor (Roche) and 50% Geltrex LDEV-free, hESC-qualified, reduced growth factor basement membrane matrix (Geltrex) (Life Technologies). Cells containing the semi solid medium were seeded in triplicate. The culture was allowed to solidify at 37C for 30 min followed by layering of 200 l of similar growth medium without 50% geltrex and incubated for one to 3 weeks. Pulmosphere number was determined using the GelCount mammalian cell colony counter (Oxford Optronix). Soft agar assay To measure tumorigenicity due to erlotinib treatment treated and untreated H358 cells at a density of 10,000 cells per well in 6-well plate were plated in soft agar, in triplicate. The assay was performed using 0.5% and 0.35% agar in RPMI1640 supplemented with 10% FBS as the base and top layers, respectively. Cells were incubated for 21 days and medium was refreshed twice per week. Colonies were counted using GelCount (Oxford Optronix). The colony efficiency Comp was calculated as proportion of colonies per total number of seeded cells. The data was analyzed using GelCount software. Results Pharmacological inhibition of EGFR Bozitinib increases the fraction of ALDH+ cells in lung cancer cell.