Fig 1.
TRIM59 is highly expressed in NSCLC cell lines.
(A) The mRNA expression levels of 60 TRIM family genes in four NSCLC cell lines (H1299, H292, SPC-A1 and A549) and normal human bronchial epithelial (HBE) cell line were determined by Q-PCR. The data were organized in a heat map by using MEV software. The relative expression levels of the genes were shown in the color scale of 0–4.0435486 in green-red-black color scheme. (B) The mRNA expression levels of TRIM genes including TRIM3, TRIM7, TRIM14, TRIM16, TRIM21, TRIM22, TRIM29, TRIM59, TRIM66 and TRIM70 were significantly upregulated in NSCLC cell lines compared with that in HBE cells. (C) Schematic depiction of TRIM59. TRIM59 contains a RING finger domain (RING), a B-box2 domain (B2), two coiled-coil domains (CC) and a transmembrane domain (TM). (D) The expression of TRIM59 in 14 kinds of normal tissues was checked by western blot using TRIM59 antibody. (E) The expression of TRIM59 protein in four NSCLC cell lines and HBE cell line. Lysates from the cell lines were subjected to immunoblot analysis with TRIM59 antibody.
Fig 2.
TRIM59 promotes the proliferation of NSCLC cells.
(A) Low serum assay. The indicated NSCLC cell lines transiently transfected with TRIM59 siRNA-1, TRIM59 siRNA-2, control siRNA or untransfected were cultured in RPMI 1640 medium supplemented with 1% FBS. At the indicated times, cells were trypsinized and counted. The data represent the average of three independent experiments (mean±SD) (Top figures). Immunoblot of TRIM59 to check the knockdown efficiency of TRIM59 siRNAs in the indicated cell lines (Bottom figures). (B) Saturation density assay. The indicated NSCLC cells transiently transfected with TRIM59 siRNA-1, TRIM59 siRNA-2, control siRNA or untransfected were cultured in RPMI 1640 supplemented with 10% FBS for 6 days, trypsinized and counted. The data represent the average of three independent experiments (mean±SD). (C) Colony formation assay. Five hundred H1299 cells transiently transfected with TRIM59 siRNA-1, TRIM59 siRNA-2, control siRNA or untransfected were seeded in 6-well plates in RPMI 1640 with 5% FBS. After two weeks, cells were fixed and stained with crystal violet. Representative wells were photographed and shown.
Fig 3.
TRIM59 promotes the migration of NSCLC cells.
(A) H1299 cells transiently transfected with TRIM59 siRNA-1, control siRNA or untransfected were cultured to create a confluent monolayer of 90–100% confluence, then the monolayer was scraped in a straight line to create a “scratch”. The extent of cell migration was photographed at the indicated times (Left figures). The transverse scratch wounds were re-examined and analyzed using image J software at 3 different sites from each wound area of gaps at each time point. Results are presented as mean ± standard error (Right figure). (B) H1299 cells transiently transfected with control siRNA or TRIM59 siRNA-1 or TRIM59 siRNA-2 and cultured with RPMI 1640 containing 10% FBS for 48hrs. Then cells were trypsinized and seeded in transwell chambers. After incubation for 10hrs, cells were fixed, stained, photographed and counted in five random views.
Fig 4.
Knocking down of TRIM59 arrests NSCLC cell cycle in G2 phase.
HBE and H1299 cells were transiently transfected with TRIM59 siRNA-1, TRIM59 siRNA-2, control siRNA or untransfected were cultured in RPMI 1640 with 10% FBS for 48 hrs. (A) Adherent cells were collected and cell cycle analysis was done by flow cytometry. The inserts showed the proportion of cells for each phase and are marked with different colors (pink: G0/G1 phase, green: S phase, and gray: G2/M phase). (B) The ratio of the cells in each phase was counted. (C) H1299 cells were fixed and stained with anti-PCNA antibody (red) and DAPI (blue). (D)The protein expression levels of PCNA in H1299 cells were checked by western blot.
Fig 5.
Knocking down of TRIM59 decreased the expression of cell cycle proteins.
H1299 cells transiently transfected with TRIM59 siRNA-1, TRIM59 siRNA-2 or control siRNA were cultured in RPMI 1640 with 10% FBS for 48 hrs. (A) The mRNA expression levels of G2/M phase related genes CDC2, CCNA1, CCNB1, CCNB2 and CDC25C were tested by quantitative Real-Time PCR. (B) The protein expression levels of CDK1 (also known as CDC2), CDC25C and cyclin B1 were checked by western blot with indicated antibodies.
Fig 6.
TRIM59 promotes NSCLC cell growth not through p53 signaling pathway.
Four types of indicated NSCLC cells and HBE cells transiently transfected with TRIM59 siRNA-1, TRIM59 siRNA-2, control siRNA or untransfected were cultured in RPMI 1640 with 10% FBS for 48 hrs. The protein expression levels of p53 were checked by western blot using anti-p53 and anti-TRIM59 antibodies.