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. 2021 Oct;10(10):3995–4011. doi: 10.21037/tlcr-21-767

Figure 1.

Figure 1

USP5 stabilizes CCND1 protein and upregulates it in non-small cell lung cancer (NSCLC) cells. (A,B) Flag-CCND1 plasmid (1 µg) was cotransfected with Myc-USP5 plasmid (1, 2, or 4 µg) or empty vector (EV) plasmid (4 µg) into HEK293T cells for 24 hours (A), and Flag-CCND1 plasmid (1 µg) was cotransfected with Myc-USP5 plasmid (1 µg) or EV plasmid (1 µg) into HEK293T cells for 12, 24, or 36 hours (B). Then, cell lysates were prepared and immunoblotting (IB) analysis of the expression levels of Flag (Flag-CCND1), Myc (Myc-USP5), and β-tubulin (loading control) with the indicated antibodies (Abs; primary Ab: anti-Flag, 1:5,000, anti-Myc, 1:5,000, or anti-β-tubulin, 1:5,000; secondary Ab: horseradish peroxidase-labeled goat anti-mouse or anti-rabbit IgG, 1:2,000). (C,D) Flag-CCND1 plasmid (1 µg) was cotransfected with Myc-USP5 plasmid (1 µg) or EV plasmid (1 µg) into HEK293T cells for 24 hours, followed by IB analysis of Flag (Flag-CCND1), Myc (Myc-USP5), and β-tubulin at 0, 0.5, 1, 2, or 4 hours post cycloheximide (CHX) treatment (final concentration: 100 µg/mL). Representative IB images (C) are shown. The decay curve of the CCND1 protein (D) was plotted to show the relative expression level of Flag-CCND1 over CHX treatment time. ***, P<0.001, Student’s t-test. (E) A549 and H1299 cells were transfected with 1 µg of Myc-USP5 or EV plasmid for 48 hours, and then IB analysis of the expression levels of Myc (Myc-USP5), USP5 (anti-USP5, 1:2,000), CCND1 (anti-CCND1, 1:1,000), and β-tubulin was performed with the indicated Abs. (F) SiUSP5 or siNC (150 nM) was transfected into A549 and H1299 cells for 48 hours, and then cellular lysates were collected for IB analysis of the expression levels of USP5, CCND1, and β-tubulin. (G,H) The expression of USP5 and CCND1 in six NSCLC cell lines and the human bronchial epithelial cell line HBE (normal control) was analyzed by IB. Representative IB images (G) are shown. The relationship between the protein expression levels of endogenous USP5 and CCND1 in NSCLC cells (H) is shown. **, P<0.01, Spearman rank correlation coefficient.