. (B) H1975 or H1975/OR cells were stained with a FITC‐conjugated CSPG4 antibody targeting an extracellular epitope. Left panel : Histograms of CSPG4‐FITC staining from one replicate are shown, where unstained correspond to cells without addition of the CSPG4‐FITC antibody. Right panel : Quantification of the FITC median fluorescence intensity. Data are based on three experiments. No statistical difference was found by multiple comparison testing (one‐way Anova) when comparing the median fluorescent intensities of the untreated and treated cells, or when comparing the two cell lines. (C) H1975/OR cells were transiently transfected with siRNA targeting CSPG4 (siCSPG4) or non‐targeting siRNA (siNT) alone or combined with osimertinib (0.1 µM). Left panel : Real time quantitative PCR assessment of CSPG4 mRNA expression at 24 h post siRNA transfection. Data shown are from four biological experiments, with significance analysed by t test, ***; p ≤ 0.001. Right panel : Cell viability was assessed at 48 h post transfection by trypan blue counting. The number of viable cells from four biological replicates are shown in percentage relative to the respective siNT controls. One‐way Anova was performed, *; p value ≤ 0.05, **; p value ≤ 0.01. " width="100%" height="100%">
Journal: Journal of Extracellular Vesicles
Article Title: Profiling of Extracellular Vesicles of Non‐Small Cell Lung Cancer Reveals Proteins Associated With Osimertinib Resistance
doi: 10.1002/jev2.70219
Figure Lengend Snippet: Chondroitin sulfate proteoglycan 4 is expressed on mutant EGFR expressing non‐small cell lung cancer cells and influences their cell viability . (A) Bar chart showing expression levels of the osimertinib refractoriness EV associated proteins in TMT pro‐labelled H1975 and H1975/OR cell extracts prior‐ and post osimertinib (0.1 µM; 48 h) as revealed by mass spectrometry (MS) quantification. The fold change in mean protein abundance is shown in H1975/OR versus H1975 cell extracts. Data from three biological replicates is shown. Two‐way Anova, ◇ p value ≤ 0.05 versus H1975, ◯ p value ≤ 0.05 versus H1975 + Osi 0.1 µM, ◻ p value ≤ 0.05 versus H1975/OR. For data normalization, see Supporting Information (S1) . (B) H1975 or H1975/OR cells were stained with a FITC‐conjugated CSPG4 antibody targeting an extracellular epitope. Left panel : Histograms of CSPG4‐FITC staining from one replicate are shown, where unstained correspond to cells without addition of the CSPG4‐FITC antibody. Right panel : Quantification of the FITC median fluorescence intensity. Data are based on three experiments. No statistical difference was found by multiple comparison testing (one‐way Anova) when comparing the median fluorescent intensities of the untreated and treated cells, or when comparing the two cell lines. (C) H1975/OR cells were transiently transfected with siRNA targeting CSPG4 (siCSPG4) or non‐targeting siRNA (siNT) alone or combined with osimertinib (0.1 µM). Left panel : Real time quantitative PCR assessment of CSPG4 mRNA expression at 24 h post siRNA transfection. Data shown are from four biological experiments, with significance analysed by t test, ***; p ≤ 0.001. Right panel : Cell viability was assessed at 48 h post transfection by trypan blue counting. The number of viable cells from four biological replicates are shown in percentage relative to the respective siNT controls. One‐way Anova was performed, *; p value ≤ 0.05, **; p value ≤ 0.01.
Article Snippet: The program applied was 25°C, 10 min, 37°C, 1 h, 95°C for 5 min. CSPG4 mRNA expression was analysed by real time quantitative PCR in which 1 μL of cDNA was added to Taqman fast advanced mastermix (cat. 4444557, Applied Biosystems / Thermo Fisher Scientific) and Taqman FAM‐labelled primer/probe mix for CSPG4 (Hs05636647_s1; Thermo Fisher Scientific, cat. #4331182) or GAPDH (Hs02758991_g1; Thermo Fisher Scientific).
Techniques: Mutagenesis, Expressing, Mass Spectrometry, Quantitative Proteomics, Staining, Fluorescence, Comparison, Transfection, Real-time Polymerase Chain Reaction
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