Fig 1: TRIM59 induced EMT program and ERK phosphorylation. (A) H1299 and A549 cells tranfected with shCtrl or shTRIM59 plasmid were subjected to western blotting for detection of E‐cadherin, N‐cadherin, Snail, Slug, and Vimentin expression. (B) Western blotting measurement of activation of ERK signaling by TRIM59 knockdown in H1299 and A549 cells. (C) DNA synthesis of H1299 and A549 cells treated with negative control and U0126 (10 μmol/L) was measured using an EdU‐incorporation assay. Scale bars, 100 μm. Results are expressed as means ± SEM. Data were assessed by Student's t test. *P < 0.05; **P < 0.01. (D) In vitro invasion assays using transwell chambers in H1299 and A549 cells treated with negative control and U0126 (10 μmol/L). Scale bars, 100 μm. Values are means ± SEM from three independent experiments. Significance was determined by Student's t test
Fig 2: TRIM59 is overexpressed in lung cancer and is correlated with poor survival. (A) Up‐regulation of TRIM59 was found in 8 of 20 cancer types. (B) The TRIM59 expression is up‐regulated in lung cancer tissue compared with normal lung tissue as revealed by Oncomine data‐mining analysis in TCGA Lung 2's dataset. (C) Scores indicate TRIM59 levels in representative tumour tissues. The scores were calculated by intensity and percentage of stained cells as described in the materials and methods. Scale bars, 100 μm. (D) Kaplan‐Meier survival curves for overall survival (OS) in lung cancer patients according to TRIM59 expression. Patients with high TRIM59 expression (score 2‐3) have poorer overall survival compared with patients with low TRIM59 expression (score 0‐1)
Fig 3: Roles of TRIM59 in promoting lung cancer growth and metastasis. (A) Expression levels of TRIM59 in the indicated lung cancer cell lines were analysed by western blotting (upper). Confirmation of TRIM59 knockdown in A549 and H1299 cells (lower). (B) CCK8 assays were used to analyse proliferation of cells stably transfected with Scrambled shRNA (shCtrl) or TRIM59 shRNA (shTRIM59). (C) EdU incorporation assay was used to analyse DNA synthesis rates of each transfected cell population. Scale bars, 100 μm. (D, E) Invasion of lung cells stably infected with shCtrl or shTRIM59 was assessed by the transwell invasion assays (scale bars, 100 μm) and wound‐healing assays (scale bars, 200 μm). Error bars indicate means ± SEM. All these experiments have been repeated three times. The significance was determined by Student t test. *P < 0.05; **P < 0.01
Fig 4: Coexpression of TRIM59 and CDK6 in lung cancer. (A, B) Co‐expression data from Oncomine (www.oncomine.org). We used the following filters: gene “TRIM59” Analysis Type: “Co‐expression analysis” Cancer Type: “Lung cancer.” The colour changed according to a weaker (blue) or higher (red) expression in wang cell line 2 and Lee lung
Fig 5: TRIM59 drives tumour growth and metastasis in vivo. (A) Representative images of two xenograft groups. A549 cells tranfected with shCtrl or shTRIM59 plasmid were subcutaneously injected into the back of athymic mice to estimate carcinogenesis. (B) Tumour volumes were measured on the indicated days to assess the effects of TRIM59 on subcutaneous tumour growth. (C) Representative images of tumour samples stained with haematoxylin & eosin (H&E) (200× magnification). (D) The weight of the xenotransplanted tumours in two groups were compared at 30 days postimplantation (n = 8). (E) Representative photos of Ki‐67 immunostaining and statistical analyses of the Ki‐67‐positive index in subcutaneous tumour sections. (F) Proteins of subcutaneous tumours were used for western blotting of CDK6 expression. (G) Western blot evaluations were used to evaluate E‐cadherin, N‐cadherin, Snail, Slug, and Vimentin expression in xenograft tumours. Results are expressed as means ± SEM. The significance was determined by Student's t test. *P < 0.05; **P < 0.01
Supplier Page from Sino Biological, Inc. for Human TRIM59 Gene ORF cDNA clone expression plasmid, N-GFPSpark tag