Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Epidermal growth factor receptor-tyrosine kinase inhibitor-insensitive A549 cells harbor high cilia incidence. (A and B) A549 cells were treated with increasing concentrations of (A) Gef or (B) Dac, and the cell viability related to untreated control (Ctrl) was measured at 48 h post-treatment. (C and D) HCC827 cells were treated with increasing concentrations of (C) Gef or (D) Dac, and the cell viability related to untreated control was measured at 48 h post-treatment. (E and F) A549 and HCC827 cells were exposed to increasing concentrations of Gef for 48 h, and the immunoblotting analysis was performed to measure the protein expression levels of ERK and p-ERK at Thr202/Tyr204. α-tubulin (α-tub) was used as a loading control. (G) Representative images of primary cilia labeled with ARL13B (red) and basal bodies labeled with γ-tub (green) in A549 and HCC827 cells, and the cilia incidence was calculated. The nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. All quantitative data were obtained from three replicates and shown as the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with 0 µM (0.1% DMSO). Gef, gefitinib; Dac, dacomitinib; p-, phosphorylated; SD, standard deviation.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Control, Western Blot, Expressing, Labeling, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Epidermal growth factor receptor-tyrosine kinase inhibitors promote ciliogenesis in dose-dependent manner in A549 but not HCC827 cells. (A) Representative images of primary cilia labeled with ARL13B (red) and basal bodies labeled with γ-tub (green) in A549 cells treated with increasing doses of Gef or Dac for 48 h. (B and C) Quantification of (B) ciliated cells and the (C) cilium length of A549 cells treated with 0 (n=117), 1 (n=176), 5 (n=133), 10 (n=216), 25 (n=210), 50 (n=95) µM Gef for 48 h. **P<0.01 and ****P<0.0001 compared with 0 µM; ns, not significant. (D and E) Quantification of (D) ciliated cells and the (E) cilium length of A549 cells treated with 0 (n=133), 0.5 (n=153), 1 (n=138), 5 (n=163), 7.5 (n=130), 10 (n=80) µM Dac for 48 h. *P<0.05 and ****P<0.0001 compared with 0 µM; ns, not significant. (F) Representative images of primary cilia labeled with ARL13B (red) and basal bodies labeled with γ-tub (green) in HCC827 cells treated with 0.01 µM Gef or 0.001 µM Dac for 48 h. (G) Quantification of ciliated cells in HCC827 cells in (F). ns, not significant compared with DMSO. The nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. All quantitative data were obtained from three replicates and shown as the mean ± SD. Error bars, ± SD. Gef, gefitinib; Dac, dacomitinib; p-, phosphorylated; SD, standard deviation; ns, not significant.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Labeling, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Gef treatment leads to G1 phase arrest and senescence. (A and B) Cell cycle distribution assay of A549 cells treated with DMSO (0.1%) or 25 µM Gef for 1, 2, and 3 days (d) by flow cytometry with (A) PI staining; (B) quantification data. (C and D) Cell cycle distribution assay of HCC827 cells treated with DMSO (0.1%) or 0.05 µM Gef for 1, 2, and 3 days by flow cytometry with (C) PI staining; (D) quantification data. (E) Immunoblotting analysis of the expression levels of CCNA, CCNB, CCND, CCNE, CDK1, CDK2, p16 and p21 in A549 cells treated with DMSO, Gef (25 µM), or Dac (5 µM) for 1 and 3 days. α-tubulin (α-tub) and GAPDH were used as loading controls. (F) Representative PI staining images of A549 cells exposed to DMSO (0.1%) or Gef (25 µM) for 1, 2, 3 days, the dead cells (red) were stained with PI and indicated with red arrows. Scale bar, 100 µm. (G) Immunoblotting analysis of the expression levels of IFT88, p-ERK (Thr202/Tyr204) and BCL2 in A549 cells treated with DMSO (0.1%) or Gef (25 µM) for 1, 2, 3 days. α-tubulin (α-tub) was used as a loading control. The values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. (H and I) The (H) cellular senescence assay of A549 cells treated with DMSO (0.1%) or Gef (25 µM) for 1–3 d labeling by senescence-tracker (Sen-Tra) fluorescence probe, and the (I) quantification data. Scale bar, 100 µm. Data are expressed as the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01 and ***P<0.001 compared with DMSO. Gef, gefitinib; PI, propidium iodide; CCNA, Cyclin A2; CCNB, Cyclin B1; CCND, Cyclin D1; CCNE, Cyclin E1; Dac, dacomitinib; BF, bright field; p-, phosphorylated; SD, standard deviation.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Flow Cytometry, Staining, Western Blot, Expressing, Control, Software, Labeling, Fluorescence, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Inhibition of ciliogenesis sensitizes A549 cells to epidermal growth factor receptor-tyrosine kinase inhibitors. (A) Immunoblotting analysis of the protein expression levels of IFT88 and ARL13B in A549 cells transfected with siRNAs against IFT88 (siIFT88-1/2), ARL13B (siARL13B-1/2), or NC. GAPDH was employed as a loading control. The values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. (B-D) Immunofluorescence labeling of primary cilia (ARL13B, red) and basal bodies (γ-tub, green) in A549 control cells (Ctrl) and cells transfected with siIFT88-1/2, siARL13B-1/2, or NC following treated with 10 µM Gef for 48 h, (C) the proportion of ciliated cells and (D) cilium length in each group. Ctrl, n=52; Gef-NC, n=59; Gef-siIFT88-1, n=57; Gef-siIFT88-2, n=64; Gef-siARL13B-1, n=56; Gef-siARL13B-2, n=69. The nuclei were stained with DAPI (blue). (E and F) A549 cells transfected with NC, siIFT88-1/2, or ARL13B-1/2 siRNAs were exposed to (E) Gef (0, 5, 10 µM) or (F) Dac (0, 1, 5 µM) for 48 h, the cell viability related to untreated control was measured. (G and H) A549 cells transfected with indicated siRNAs were exposed 10 µM Gef for 48 h and stained with PI to detect the dead cells and quantification data for each group. Scale bar, 100 µm. Data are expressed as the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01 and ***P<0.001 compared with NC. siRNA, small interfering RNA; NC, negative control; Gef, gefitinib; Dac, dacomitinib; BF, bright field; SD, standard deviation.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Control, Software, Immunofluorescence, Labeling, Staining, Small Interfering RNA, Negative Control, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Epidermal growth factor receptor-tyrosine kinase inhibitors induce AC3 expression and ciliary localization. (A) Immunoblotting analysis of the expression levels of AC3 and ARL13B in A549 cells exposed to DMSO (0.1%), Gef (10 µM) and Dac (5 µM) for 1 and 3 days. (B and C) Immunofluorescence labeling of primary cilia (Arl13b, green) and AC3 (red, indicated by white arrows) in A549 cells administrated with DMSO (0.1%), Gef (10 µM), and Dac (5 µM) for 3 days, and quantification of AC3 positive (AC3 + ) cilia in each group. The nuclei were stained with DAPI (blue). Scale bar, 10 µm. Data are expressed as the mean ± SD. Error bars, ± SD. ***P<0.001 compared with DMSO. (D) Immunoblotting analysis of the expression levels of AC3 and ARL13B in HCC827 cells exposed to DMSO (0.1%), Gef (0.05 µM) and Dac (0.01 µM) for 3 days. (E) Immunoblotting analysis of the protein expression levels of AC3 and IFT88 in Gef-treated A549 cells transfected with siIFT88-1 (siIFT88) or NC siRNA for 3 days. (F) Immunofluorescence labeling of primary cilia (Arl13b, red) and AC3 (green) in cells in (E). Scale bar, 5 µm. GAPDH was used as a loading control; the values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. Gef, gefitinib; Dac, dacomitinib; SD, standard deviation; si-, small interfering; NC, negative control; AC3, adenylate cyclase 3.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Expressing, Western Blot, Immunofluorescence, Labeling, Staining, Transfection, Control, Software, Standard Deviation, Negative Control

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Silencing AC3 expression enhances cellular sensitivity to Gef. (A) Immunoblotting analysis of the expression levels of AC3 and ARL13B in A549 cells transfected with siRNAs targeting AC3 (siAC3-1/2/3) or NC for 48 h. GAPDH was used as a loading control. The values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. (B) A549 cells transfected NC or siAC3-1/2/3 were exposed to Gef (0, 5, 10 µM) for 48 h, the cell viability related to untreated control was measured. (C and D) Immunofluorescence labeling of primary cilia (Arl13b, red, indicated by white arrows) in A549 cells transfected with NC and siAC3-1 following administrated with Gef (10 µM) for 3 days, and quantification of ciliated cells in each group. The nuclei were stained with DAPI (blue). Scale bar, 10 µm. Data are expressed as the mean ± SD. Error bars, ± SD. *P<0.05 and **P<0.01 compared with NC. AC3, adenylate cyclase 3; Gef, gefitinib; si-, small interfering; NC, negative control; SD, standard deviation; ns, not significant.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Expressing, Western Blot, Transfection, Control, Software, Immunofluorescence, Labeling, Staining, Negative Control, Standard Deviation