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: For immunoblotting experiment, the primary antibodies of ERK (1:1,000; cat. no. ab184699) and phosphorylated-ERK (p-ERK; 1:1,000; cat. no. ab201015) were purchased from Abcam; primary antibodies of BCL2 (1:2,000; cat. no. 12789-1-AP), α-tubulin (α-tub; 1:40,000; cat. no. 11224-1-AP), IFT88 (1:1,000; cat. no. 60227-1-Ig), AC3 (1:1,000; cat. no. 19492-1-AP), Cyclin A2 (CCNA; 1:1,000; cat. no. 18202-1-AP), Cyclin B1 (CCNB; 1:1,000; cat. no. 55004-1-AP), Cyclin D1 (CCND; 1:3,000; cat. no. 60186-1-Ig), Cyclin E1 (CCNE; 1:1,000; cat. no. 11554-1-AP), CDK1 (1:1,000; cat. no. 19532-1-AP), CDK2 (1:1,000; cat. no. 10122-1-AP), p21 (1:1,000; cat. no. 10355-1-AP), p16 (1:1,000; cat. no. 10883-1-AP), GAPDH (1:50,000; cat. no. 60004-1-Ig), and the secondary antibodies including HRP-conjugated goat anti-mouse IgG (1:5,000; cat. no. SA00001-1), HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. SA00001-2) were supplied by Proteintech Group, Inc. For immunofluorescence staining, the primary antibodies of ADP ribosylation factor such as GTPase 13B (ARL13B; 1:500; cat. no. 17711-1-AP/66739-1-Ig), AC3 (1:500; cat. no. 19492-1-AP) and γ-tubulin (γ-tub; 1:500; cat. no. T6557) were purchased from Proteintech Group, Inc. and Sigma-Aldrich; Merck KGaA respectively.

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