control non targeting sirna  (Santa Cruz Biotechnology)

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: 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 shRNA, 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 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: Control, Western Blot, Concentration Assay, Alamar Blue Assay, Enzyme-linked Immunosorbent Assay, Apoptosis Assay, Derivative Assay, shRNA, Transfection, Plasmid Preparation, Sequencing

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

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: 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.

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: Transfection, Expressing, Plasmid Preparation, Construct, Cell Culture, Cell Fractionation, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Apoptosis Assay, Small Interfering RNA

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: 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 shRNA, 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 RNA.

Article Snippet: For acute knockdown studies, control non-targeting siRNA or pool of siRNAs specifically targeting TLE1 (Santa Cruz Biotechnology, Inc.) or AES (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into A549 cells (2x10 5 ) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37˚C in a humidified incubator with 5% CO 2 for 24 h followed by subsequent experimentation ( , ).

Techniques: Control, Western Blot, Concentration Assay, Alamar Blue Assay, Enzyme-linked Immunosorbent Assay, Apoptosis Assay, Derivative Assay, shRNA, Transfection, Plasmid Preparation, Sequencing

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: For acute knockdown studies, control non-targeting siRNA or pool of siRNAs specifically targeting TLE1 (Santa Cruz Biotechnology, Inc.) or AES (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into A549 cells (2x10 5 ) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37˚C in a humidified incubator with 5% CO 2 for 24 h followed by subsequent experimentation ( , ).

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

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: 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.

Article Snippet: For acute knockdown studies, control non-targeting siRNA or pool of siRNAs specifically targeting TLE1 (Santa Cruz Biotechnology, Inc.) or AES (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected into A549 cells (2x10 5 ) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37˚C in a humidified incubator with 5% CO 2 for 24 h followed by subsequent experimentation ( , ).

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Cell Culture, Cell Fractionation, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Apoptosis Assay, Small Interfering RNA

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: 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 shRNA, 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 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: Control, Western Blot, Concentration Assay, Alamar Blue Assay, Enzyme-linked Immunosorbent Assay, Apoptosis Assay, Derivative Assay, shRNA, Transfection, Plasmid Preparation, Sequencing

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

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: 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.

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: Transfection, Expressing, Plasmid Preparation, Construct, Cell Culture, Cell Fractionation, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Apoptosis Assay, Small Interfering RNA

Journal: Oncology Letters

Article Title: Hsa_circ_0009910 knockdown in HeLa cells increases miR‑198 expression levels and decreases c‑Met expression levels and cell viability

doi: 10.3892/ol.2024.14820

Figure Lengend Snippet: Primer sequences.

Article Snippet: HeLa cells were seeded in 6-well plates at 80% confluence and subsequently transfected with si-circ9910 (50 nM) or siRNA negative control using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and harvested 48 h later for further analysis.

Techniques: Sequencing

Journal: Oncology Letters

Article Title: Hsa_circ_0009910 knockdown in HeLa cells increases miR‑198 expression levels and decreases c‑Met expression levels and cell viability

doi: 10.3892/ol.2024.14820

Figure Lengend Snippet: Hsa_circ_0009910 knockdown increases miR-198 and decreases c-Met mRNA expression levels. (A) Relative expression of hsa_circ_0009910 in HeLa cells in the HeLa si-circ9910 and HeLa si-NC groups. (B) Binding site of hsa_circ_0009910/miR-198, depicting the sequence of hsa_circ_0009910 (red letters) and the sequence of miR-198 (blue letters). The binding type is 7mer-m8. (C) Expression levels of miR-198 in HeLa si-circ9910 and HeLa si-NC. (D) Basal expression levels of c-Met in HaCaT and HeLa cells. (E) Expression levels of c-Met in HeLa si-circ9910 and HeLa si-NC. Data presented are from at least three independent experiments and expressed as the mean ± standard deviation. *P<0.05. circ, circular; miR, microRNA; si-circ9910, small interfering RNA against hsa_circ_0009910; NC, negative control.

Article Snippet: HeLa cells were seeded in 6-well plates at 80% confluence and subsequently transfected with si-circ9910 (50 nM) or siRNA negative control using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and harvested 48 h later for further analysis.

Techniques: Knockdown, Expressing, Binding Assay, Sequencing, Standard Deviation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Hsa_circ_0009910 knockdown in HeLa cells increases miR‑198 expression levels and decreases c‑Met expression levels and cell viability

doi: 10.3892/ol.2024.14820

Figure Lengend Snippet: Primer sequences.

Article Snippet: To perform the hsa_circ_0009910 knockdown, a small interfering RNA (siRNA) targeting hsa_circ_0009910 (si-circ9910) and siRNA negative control (si-NC) were synthesized by Integrated DNA Technologies, Inc. ( ).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: Hsa_circ_0009910 knockdown in HeLa cells increases miR‑198 expression levels and decreases c‑Met expression levels and cell viability

doi: 10.3892/ol.2024.14820

Figure Lengend Snippet: Hsa_circ_0009910 knockdown increases miR-198 and decreases c-Met mRNA expression levels. (A) Relative expression of hsa_circ_0009910 in HeLa cells in the HeLa si-circ9910 and HeLa si-NC groups. (B) Binding site of hsa_circ_0009910/miR-198, depicting the sequence of hsa_circ_0009910 (red letters) and the sequence of miR-198 (blue letters). The binding type is 7mer-m8. (C) Expression levels of miR-198 in HeLa si-circ9910 and HeLa si-NC. (D) Basal expression levels of c-Met in HaCaT and HeLa cells. (E) Expression levels of c-Met in HeLa si-circ9910 and HeLa si-NC. Data presented are from at least three independent experiments and expressed as the mean ± standard deviation. *P<0.05. circ, circular; miR, microRNA; si-circ9910, small interfering RNA against hsa_circ_0009910; NC, negative control.

Article Snippet: To perform the hsa_circ_0009910 knockdown, a small interfering RNA (siRNA) targeting hsa_circ_0009910 (si-circ9910) and siRNA negative control (si-NC) were synthesized by Integrated DNA Technologies, Inc. ( ).

Techniques: Knockdown, Expressing, Binding Assay, Sequencing, Standard Deviation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Overexpression of HYOU1 is associated with cisplatin resistance and may depend on m 6 A modification in patients with cervical cancer

doi: 10.3892/ol.2024.14823

Figure Lengend Snippet: Validation of HYOU1 in promoting CC survival and DDP resistance. Kaplan-Meier OS analysis of patients with high and low expression levels of HYOU1 in the (A) TCGA-CC 2 and (B) TCGA-CC 3 datasets. (C) Point plot of the correlation analysis between the mRNA expression level values of HYOU1 and IC 50 values of DDP in the Genomics of Drug Sensitivity in Cancer database. (D) Survival curves of parental HeLa and HeLa/DDP cells that were subjected to different concentrations of DDP, as measured using the CCK-8 assay (n=5). (E) Representative western blot showing the HYOU1 protein expression levels in HeLa and HeLa/DDP cells. (F) Semi-quantified expression levels of HYOU1 in HeLa and HeLa/DDP cells (n=3). (G) Proliferation of HeLa/DDP cells treated with DDP and siRNA (HYOU1 siRNA or siRNA NC) or DDP alone using the CCK-8 assay (n=5), using one-way analysis of variance. (H) Bar plot of GSEA of HYOU1 -associated genes; orange represents the activation pathway and blue represents the inhibition pathway. (I) GSEA results for the activation pathways. ***P<0.001. The statistical difference between two group was analyzed using the unpaired student's t-test, whereas the statistical difference among multiple groups was analyzed using one-way analysis of variance and Tukey's test. CC, cervical cancer; DDP, cisplatin; GSEA, gene set enrichment analysis; IC 50 , half-maximal inhibitory concentration; OS, overall survival; TCGA, The Cancer Genome Atlas; NC, negative control; siRNA, small interfering RNA; HYOU1, hypoxia-upregulated 1 gene; HR, hazard ratio; CI, confidence interval; CCK-8, Cell Counting Kit-8; HeLa/DDP, DDP-resistant HeLa cells.

Article Snippet: The siRNA sequences (Wanleibio Co., Ltd.) used were as follows: HYOU1 sense: 5′-AAGCUGCUGCGUGAGGCUAAUC-3′; anti-sense: 5′-GAUUAAGCCUCACGAGCAGCUU-3′; HYOU1 siRNA-2 sense: 5′-AGCUGGGGAAGAACAUCAAU-3′; anti-sense: 5′-AUUGUUCUUCCCAUCAUCG-3′; and siRNA negative control (NC) sense: 5′-AUAAACAUCGACUCAAU-3′; anti-sense: 5′-AUUGAGCUCGAUUGUUAU-3′.

Techniques: Expressing, CCK-8 Assay, Western Blot, Activation Assay, Inhibition, Concentration Assay, Negative Control, Small Interfering RNA, Cell Counting