pc9 egfr  (ATCC)

Journal: Scientific Reports

Article Title: MicroRNA-130a-3p regulates osimertinib resistance by targeting runt-related transcription factor 3 in lung adenocarcinoma

doi: 10.1038/s41598-024-76196-1

Figure Lengend Snippet: Characteristics of EVs secreted by PC9 and PC9OR cells in the medium ( a ) TEM images of isolated EVs from PC9 and PC9OR cells. The black sphere image shows 40-nm gold colloids bound to the CD9 antibody. Scale bars:100 nm. ( b ) Protein concentrations of CD9 and CD81 (exosomal markers) in isolated EVs from PC9 and PC9OR cells. Original blots are presented in Supplementary Figure S4a. These images show that EVs were isolated ( a , b ). ( c ) Size distribution of isolated EVs from PC9 and PC9OR cells. ( d ) Protein concentrations of isolated total EVs from PC9 and PC9OR cells. ( e ) Number of CD63- and CD9-positive EVs from PC9 and PC9OR cells. The release of EVs differed in the PC9 and PC9OR cells ( c–e ). Data are presented as mean ± SEM ( n = 3–4) ( d , e ). * P < 0.05, compared with PC9 cells. P-values were determined using Student’s t-test. EVs, extracellular vesicles; TEM, transmission electron microscopy.

Article Snippet: PC9 cells ( EGFR exon 19 deletion/E746-A750) and H1975 cells ( EGFR L858R/T790M), which are human lung adenocarcinoma cell lines, were purchased from the Riken BioResource Center (Tsukuba, Japan) and American Type Culture Collection (ATCC, VA), respectively.

Techniques: Isolation, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: MicroRNA-130a-3p regulates osimertinib resistance by targeting runt-related transcription factor 3 in lung adenocarcinoma

doi: 10.1038/s41598-024-76196-1

Figure Lengend Snippet: Effects of PC9OR-EV- and PC9OR-EV-derived miRNAs on osimertinib resistance in PC9 cells ( a ) Fluorescence images of PC9 cells after exposure to red-labeled EVs from PC9 (PC9-EVs) and PC9OR cells (PC9OR-EVs). Scale bars: 50 μm. ( b ) Viability of PC9 cells treated with 5 µg of PC9-EVs and PC9OR-EVs in the absence or presence of 1 µM of osimertinib for 72 h. The controls are PC9 cells treated with PBS (vehicle). ( c ) The total concentration of miRNA derived from PC9-EVs and PC9OR-EVs. ( d ) Viability of PC9 cells transfected with 6–12 nM of miRNAs derived from PC9OR-EVs in the absence or presence of 1 µM osimertinib for 72 h. Data are presented as mean ± SEM ( n = 3–6) ( b–d ). * P < 0.05, compared with control or PC9 cells. P-values were determined using Student’s t-test ( c ) or ANOVA with Tukey–Kramer ( b , d ). ns , no significant difference. EVs, extracellular vesicles; ANOVA, analysis of variance.

Article Snippet: PC9 cells ( EGFR exon 19 deletion/E746-A750) and H1975 cells ( EGFR L858R/T790M), which are human lung adenocarcinoma cell lines, were purchased from the Riken BioResource Center (Tsukuba, Japan) and American Type Culture Collection (ATCC, VA), respectively.

Techniques: Derivative Assay, Fluorescence, Labeling, Concentration Assay, Transfection, Control

Journal: Scientific Reports

Article Title: MicroRNA-130a-3p regulates osimertinib resistance by targeting runt-related transcription factor 3 in lung adenocarcinoma

doi: 10.1038/s41598-024-76196-1

Figure Lengend Snippet: Identification of EV-miRNAs associated with osimertinib resistance in lung adenocarcinoma cell lines ( a ) Scatter plot for the miRNA array analysis of isolated EVs from PC9 and PC9OR cells. ( b ) A heat map illustrating the expression of 22 EV-miRNAs and demonstrating a 3.5-fold change between PC9 and PC9OR cells. ( c ) Relative EV-miRNA expression levels in PC9 and PC9OR cells. ( d ) Relative EV-miRNA expression levels in H1975 and H1975OR cells. ( e ) Relative intracellular miRNA levels in PC9 and PC9OR cells. miRNA levels were normalized using U6 levels determined by RT-qPCR ( c–e ). Data are presented as mean ± SEM ( n = 5–6). * P < 0.05, compared with PC9 or H1975 cells. P-values were determined using Student’s t-test. EVs, extracellular vesicles; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: PC9 cells ( EGFR exon 19 deletion/E746-A750) and H1975 cells ( EGFR L858R/T790M), which are human lung adenocarcinoma cell lines, were purchased from the Riken BioResource Center (Tsukuba, Japan) and American Type Culture Collection (ATCC, VA), respectively.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: MicroRNA-130a-3p regulates osimertinib resistance by targeting runt-related transcription factor 3 in lung adenocarcinoma

doi: 10.1038/s41598-024-76196-1

Figure Lengend Snippet: Effect of miR-130a-3p on osimertinib resistance in lung adenocarcinoma cell lines ( a , b ) Relative miR-130a-3p levels in PC9 and H1975 cells transfected with a negative control (NC) and an miR-130a-3p mimic (130a mimic). The miR-130a-3p concentrations were normalized using U6 levels relative to the levels in the cells transfected with the NC. ( c , d ) Viability of PC9 and H1975 cells transfected with the NC and miR-130a-3p mimic after exposure to various concentrations of osimertinib for 72 h. ( e , f ) Apoptotic rate in PC9 and H1975 cells after transfection with the NC and miR-130a-3p mimic at 24, 48, and 72 h in the absence or presence of 1 µM osimertinib. ( g ) Relative miR-130a-3p concentrations in PC9 and PC9OR cells transfected with the NC and miR-130a-3p inhibitor (130a inhibitor). The miR-130a-3p concentrations were normalized using U6 levels relative to the levels in PC9 cells transfected with the NC. ( h ) Cell viability in PC9 and PC9OR cells transfected with the NC and 130a inhibitor in the absence or presence of 1 µM osimertinib for 72 h. Cell viability is the percentage of viable cells relative to PC9 cells transfected with the NC in the absence of osimertinib. When the standard errors of the means are small, they are contained within the symbols. Data are presented as mean ± SEM ( n = 3–12). * P < 0.05, compared with NC. P-values were determined using Student’s t-test ( a , b ) or ANOVA with Tukey–Kramer ( c–h ). RLUs, relative luminescence units; ANOVA, analysis of variance.

Article Snippet: PC9 cells ( EGFR exon 19 deletion/E746-A750) and H1975 cells ( EGFR L858R/T790M), which are human lung adenocarcinoma cell lines, were purchased from the Riken BioResource Center (Tsukuba, Japan) and American Type Culture Collection (ATCC, VA), respectively.

Techniques: Transfection, Negative Control

Journal: Scientific Reports

Article Title: MicroRNA-130a-3p regulates osimertinib resistance by targeting runt-related transcription factor 3 in lung adenocarcinoma

doi: 10.1038/s41598-024-76196-1

Figure Lengend Snippet: Effect of RUNX3 on osimertinib resistance in PC9 cells ( a ) Identification of the target genes of miR-130a-3p using miRDB, TargetScan, and miRTarBase databases. ( b , c ) Protein concentrations of RUNX3 in PC9 and PC9OR cells normalized with β-actin. ( d ) Protein concentrations of RUNX3 and β-actin in PC9 cells transfected with the negative control (NC) and the miR-130a-3p mimic (130a mimic). ( e ) Relative mRNA expressions of RUNX3 in PC9 cells transfected with NC and miR-130a-3p inhibitor (130a inhibitor). ( f ) Protein concentrations of RUNX3 and β-actin in PC9 and PC9OR cells transfected with the NC and the 130a inhibitor. ( g ) Relative mRNA expression of RUNX3 in PC9 cells transfected with NC and si-RUNX3. The RUNX3 mRNA levels were normalized using the glyceraldehyde-3-phosphate dehydrogenase concentrations relative to the levels in PC9 cells transfected with the NC ( e , g ). ( h ) Protein concentrations of RUNX3 and β-actin in PC9 cells transfected with si-RUNX3. ( i ) Viability of PC9 cells transfected with si-RUNX3 in the absence or presence of 1 µM osimertinib for 72 h. Data are presented as mean ± SEM ( n = 3–6). * P < 0.05, compared with PC9 cells or NC. P-values were determined using Student’s t-test ( c , e , g ) or ANOVA with Tukey–Kramer ( i ). Original blots are presented in Supplementary Figures S4b-d. ANOVA, analysis of variance.

Article Snippet: PC9 cells ( EGFR exon 19 deletion/E746-A750) and H1975 cells ( EGFR L858R/T790M), which are human lung adenocarcinoma cell lines, were purchased from the Riken BioResource Center (Tsukuba, Japan) and American Type Culture Collection (ATCC, VA), respectively.

Techniques: Transfection, Negative Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: IC50 values for gefitinib in parental and gefitinib-resistant (GR) cell lines.

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: Aberrant activation of EGFR signaling in GR cells. ( a , b ) Western blot analysis of EGFR expression and activation levels in HCC827 ( a ) and PC9 ( b ) parental and GR cells, in the absence or presence of gefitinib at the indicated concentrations. An anti-α-tubulin antibody was used for normalization. ( c , d ) Western blot analysis of expression and activation levels of ERK and AKT in HCC827 ( c ) and PC9 ( d ) parental and GR cells, in the absence or presence of gefitinib at the indicated concentrations. An anti-GAPDH antibody was used for normalization. Densitometric value ratios were calculated for phospho-ERK/total ERK (pERK/ERK) and phospho-AKT/total AKT (pAKT/AKT).

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques: Activation Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: Evaluation of TGF-β1 expression in GR cells and its role in resistance and cell migration. ( a ) Immunofluorescence analysis of TGF-β1 expression (signaled in red) in HCC827 and PC9 parental and GR cell lines. Nuclei were stained with DAPI (signaled in blue). In the images, 20× magnification was used. ( b ) Immunoassay analysis of TGF-β1 levels in conditioned media from parental and GR cell lines. TGF-β1 levels were normalized for the cell number determined at the harvesting time and folds were calculated versus their respective parental cells. The values reported are the means from two independent experiments, each performed in duplicate. * p < 0.05 for comparison between parental versus GR cells (two-tailed Student’s t -test). ( c ) Cell proliferation assay on HCC827 parental and GR cells treated for 72 h with gefitinib (100 nM) and LY2109761 (LY, 5 µM), alone and in combination. *** p < 0.0005 for comparison between cells treated with gefitinib alone versus those treated with the drug combination (two-tailed Student’s t -test). ( d ) Analysis of wound-healing assays for HCC827 parental and GR-Low cells untreated and treated with LY (5 µM). *** p < 0.0005 for comparison between the treated versus untreated cells (two-tailed Student’s t -test).

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques: Expressing, Migration, Immunofluorescence, Staining, Comparison, Two Tailed Test, Proliferation Assay