Fig. 1. USP2-AS1 suppresses cellular senescence and acts as an oncogenic lncRNA.

A, B A549 (A) and HCT116 (B) cells were infected with lentiviruses expressing control shRNA, USP2-AS1 shRNA#1, or USP2-AS1 shRNA#2. Ninety-six hours later, cells were subjected to β-galactosidase staining. The shown images are representative of three independent experiments. Data shown are mean ± SD (n = 3). ***P < 0.001. The knockdown efficiency of USP2-AS1 in these cells was shown in Supplementary Fig. S2C, D. C A549 and HCT116 cells were infected with lentiviruses expressing control shRNA, USP2-AS1 shRNA#1, or USP2-AS1 shRNA#2. Ninety-six hours later, cells were immunostained with anti-H3K9me3 antibody to examine senescence-associated heterochromatin foci (SAHF) formation. The nuclei were also visualized by DAPI staining. The number of SAHF per cell was counted and shown in Supplementary Fig. S2A, B. D–G A549 and HCT116 cells were infected with lentiviruses expressing control shRNA, USP2-AS1 shRNA#1, or USP2-AS1 shRNA#2. Ninety-six hours later, the extracellular levels of IL-6 and IL-8 were examined by using ELISA assay. Data shown are mean ± SD (n = 3). ***P < 0.001. H Shown are the growth curves of HCT116 cells expressing control shRNA, USP2-AS1 shRNA#1, or USP2-AS1 shRNA#2. Data shown are mean ± SD (n = 3). **P < 0.01 and ***p < 0.001. I Colonies of HCT116 cells expressing control shRNA, USP2-AS1 shRNA#1, or USP2-AS1 shRNA#2 were stained with crystal violet after 8 days of incubation. The shown images are representative of three independent experiments. Data shown are mean ± SD (n = 3). ***P < 0.001. J Shown are the growth curves of HCT116 cells expressing control or USP2-AS1. Data shown are mean ± SD (n = 3). ***P < 0.001. The successful overexpression of USP2-AS1 was shown in Supplementary Fig. S2K. K Colonies of HCT116 cells expressing control or USP2-AS1 were stained with crystal violet after 8 days of incubation. The shown images are representative of three independent experiments. Data shown are mean ± SD (n = 3). ***P < 0.001. L–O A total of 2 × 10⁶ HCT116 cells expressing either control shRNA or USP2-AS1 shRNA were individually injected to the left and right flanks of nude mice as indicated (n = 6 for each group). Representative photographs of mice and xenograft tumors were taken 24 days after injection (L). The excised tumors were weighed (M). Tumor sizes were measured at the indicated time points (N). RNA from the excised xenografts was also analyzed by both RT-PCR (O) and real-time RT-PCR (Supplementary Fig. S2O). ***P < 0.001. P–S A total of 2 × 10⁶ HCT116 cells expressing either control or USP2-AS1 were individually injected to the left and right flanks of nude mice as indicated (n = 6 for each group). Representative photographs of mice and xenograft tumors were taken 24 days after injection (P). The excised tumors were weighed (Q). Tumor sizes were measured at the indicated time points (R). RNA from the excised xenografts was also analyzed by both RT-PCR (S) and real-time RT-PCR (Supplementary Fig. S2P). ***P < 0.001.