Journal: Biomedical Reports
Article Title: TLE1 corepressor promotes gefitinib resistance in lung cancer A549 cells via E‑cadherin silencing
doi: 10.3892/br.2024.1914
Figure Lengend Snippet: Gefitinib-resistant A549 (A549GR) cells display EMT and increased TLE1 expression, and knockdown of TLE1 attenuates the EMT phenotype and gefitinib resistance of A549GR cells. (A) The parental A549 and gefitinib-resistant A549GR cells were subjected to (Aa) phase contrast light microscopy to assess their morphology, (Ab and Ac) Borden chamber assay to evaluate migration potential, and (Ad) western blotting to measure protein expression of different EMT markers including TLE1 with specific antibodies. (B) A549 and A549GR cells were cultured in the presence or absence of 10 mmol/l gefitinib for 48 h followed by cell death ELISA apoptosis assay. (C) A549 and A549GR cells were subjected to (Ca) RT-qPCR analysis to measure TLE1 mRNA expression level; (Cb) A549GR cells transfected with a pool of TLE1 specific, or control siRNAs were cultured in the presence of absence of 10 mmol/l gefitinib for 48 h followed by cell death ELISA apoptosis assay. The control siRNA or TLE1 siRNA transfected A549GR cells were also subjected to (Cc) RT-qPCR analysis to assess E-cadherin mRNA level and (Cd and Ce) Boyden chamber migration assay. (D) A549GR cells transfected with a E-cadherin expressing or vector construct were subjected to (Da and Db) a Boyden chamber migration or cultured in the presence of absence of 10 mmol/l gefitinib for 48 h followed by (Dc) cell death ELISA apoptosis assay. In , , , , and , cells were added to the upper compartment of the Boyden chamber, and after 12 h, cells attached on the underside of the membrane were stained with 0.1% crystal violet, counted (4Ab, Cd and Da), and images were captured (4Ac, Ce, and Db). In the aforementioned experiments, the results are representative of three independent experiments. * P<0.05 and ** P<0.01 [Student's t test (Ca) and one-way ANOVA with post hoc Tukey's test (Cb, Cc, Cd, Da and Dc)]. Error bars indicate SD. EMT, epithelial-mesenchymal transition; TLE1, transducin-like enhancer of split 1; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.
Article Snippet: Meanwhile, to generate the stable A549 TLE1 shRNA and control shRNA cells, the parental A549 cell line was transfected with 0.5 µg of the control short hairpin RNA (shRNA) or TLE1-specific shRNA construct (OriGene Technologies, Inc.) cells in OPTI-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cultured at 37˚C humidified incubator with 5% CO 2 .
Techniques: Expressing, Knockdown, Light Microscopy, Boyden Chamber Assay, Migration, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Apoptosis Assay, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Construct, Membrane, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA
![TLE1 regulates the sensitivity of A549 cells to gefitinib. (A and B) Control GFP and GFP-TLE1 pool of A549 cells were subjected to (A) western blotting against specific antibodies to GFP and B-actin and treated with the indicated concentration of gefitinib (mmol/l) for 48 h followed by (B) Alamar blue assay. Data are expressed as a percentage of the value for untreated cells. (C) The control GFP and GFP-TLE1 cells were treated with 10 mmol/l gefitinib at the indicated times followed cell death ELISA apoptosis assay. (D-F) The A549 derived control <t>shRNA,</t> TLE1 shRNA, and TLE1 shRNA cells transfected with a TLE1 plasmid containing silent mutations in the shRNA target sequence were subjected to (D) western blotting with the indicated antibodies, (E) Alamar blue assay 48 h post-treatment with indicated concentration of gefitinib, and (F) cell death ELISA apoptosis assay at the indicated times following treatment with 10 mmol/l gefitinib. The results are representative of three independent experiments. * P<0.05 and ** P<0.01 [(B and C) Student's t-test and (E and F) one-way ANOVA with post hoc Tukey's test]. Error bars indicate SD. TLE1, transducin-like enhancer of split 1; shRNA, short hairpin <t>RNA.</t>](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4834/pmc11704834/pmc11704834__br-22-03-01914-g01.jpg)
![Gefitinib-resistant A549 (A549GR) cells display EMT and increased TLE1 expression, and knockdown of TLE1 attenuates the EMT phenotype and gefitinib resistance of A549GR cells. (A) The parental A549 and gefitinib-resistant A549GR cells were subjected to (Aa) phase contrast light microscopy to assess their morphology, (Ab and Ac) Borden chamber assay to evaluate migration potential, and (Ad) western blotting to measure protein expression of different EMT markers including TLE1 with specific antibodies. (B) A549 and A549GR cells were cultured in the presence or absence of 10 mmol/l gefitinib for 48 h followed by cell death ELISA apoptosis assay. (C) A549 and A549GR cells were subjected to (Ca) RT-qPCR analysis to measure TLE1 mRNA expression level; (Cb) A549GR cells transfected with a pool of TLE1 specific, or control siRNAs were cultured in the presence of absence of 10 mmol/l gefitinib for 48 h followed by cell death ELISA apoptosis assay. The control siRNA or TLE1 siRNA transfected A549GR cells were also subjected to (Cc) RT-qPCR analysis to assess E-cadherin mRNA level and (Cd and Ce) Boyden chamber migration assay. (D) A549GR cells transfected with a E-cadherin expressing or vector construct were subjected to (Da and Db) a Boyden chamber migration or cultured in the presence of absence of 10 mmol/l gefitinib for 48 h followed by (Dc) cell death ELISA apoptosis assay. In , , , , and , cells were added to the upper compartment of the Boyden chamber, and after 12 h, cells attached on the underside of the membrane were stained with 0.1% crystal violet, counted (4Ab, Cd and Da), and images were captured (4Ac, Ce, and Db). In the aforementioned experiments, the results are representative of three independent experiments. * P<0.05 and ** P<0.01 [Student's t test (Ca) and one-way ANOVA with post hoc Tukey's test (Cb, Cc, Cd, Da and Dc)]. Error bars indicate SD. EMT, epithelial-mesenchymal transition; TLE1, transducin-like enhancer of split 1; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4834/pmc11704834/pmc11704834__br-22-03-01914-g03.jpg)
![Cell death effector Bit1 enhances gefitinib-induced apoptosis in A549 cells by targeting nuclear TLE1 protein to the cytoplasm. (A) A549 cells transfected with a C-terminally myc-tagged mitochondrial Bit1 expressing or vector construct were cultured in the presence or absence of 10 µmol/l gefitinib for 16 h followed by Cell Fractionation assay. The resulting mitochondrial, nuclear, and cytoplasmic fractions were subjected to western blotting with the indicated antibodies. (B) A549 cells transfected with a mitochondrial Bit1 expressing or vector construct were cultured in the presence of absence of 10 mmol/l gefitinib for 48 h followed by (Ba) Alamar blue or (Bb) cell death ELISA apoptosis assays. In (Bb), mitochondrial Bit1 expressing cells were pretreated with or without 20 mmol/l Z-VAD-fmk or transfected with a pool of AES specific or control siRNAs followed by gefitinib treatment and cell death Elisa apoptosis assay. (C) The mitochondrial Bit1 expressing A549 cells were transfected with TLE1 expressing or vector construct, and 24 h post-transfection cells were cultured in the presence or absence of 10 mmol/l gefitinib for 48 h followed by cell death Elisa apoptosis assay. The results are representative of three independent experiments. * P<0.05 and ** P<0.01 [one-way ANOVA with post hoc Tukey's test (Ba, Bb, C)]. Error bars indicate SD. Bit1, Bcl-2-inhibitor of transcription 1; TLE1, transducin-like enhancer of split 1; AES, Amino Enhancer Split; siRNA, small interfering RNA; ns, not significant.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4834/pmc11704834/pmc11704834__br-22-03-01914-g04.jpg)