Fig 2: miR-590-5p functions as a tumor suppressor in GC. a Ectopic transfection of miR-590-5p significantly suppressed proliferation in GC cell lines (**, P < 0.001). The mean and SDs obtained from six wells were plotted. b miR-590-5p transfection significantly inhibited anchorage-dependent colony formation ability (**, P < 0.001). Experiments were performed in triplicate. Error bars represent SDs. c Cell invasion ability was suppressed by miR-590-5p significantly (*, P < 0.05; **, P < 0.001). SDs were achieved from visions randomly selected. d Cell cycle distribution was examined by flow cytometry, which suggested G1 phase arrest in miR-590-5p-transfected cells. Experiments were conducted in triplicate. Statistical analysis of cell cycle percentages were presented by histograms (*, P < 0.05; **, P < 0.001). e Western blot analysis demonstrated the increased level of p21, p27, and reduction of pRb. Key factors in ATM signaling were activated in the miR-590-5p transfectants. f Immunofluorescence showed that ?H2AX was significantly increased in GC cells with ectopic miR-590-5p expression. g Drug sensitivity was enhanced by miR-590-5p (*, P < 0.05). The cell viability was detected with different concentrations of Cisplatin. The mean and SDs were obtained from six wells. The largest mean was defined as 100% and the smallest mean defined as 0%. IC50 values were calculated and listed in tables. h The expression correlation of miR-590-5p and related EMT markers in TCGA dataset. i The xenograft formation ability with stable miR-590-5p abundance was significantly inhibited compared with the negative control (*, P < 0.05). Black circles indicate the negative controls and red circles show the xenografts derived from miR-590-5p-transfected cells

Fig 3: NBT arrests synchronized cells in G1/S phase by decreasing E2F1 activity (A) HeLa cells were treated with NBT (5 µM) for 12 or 24 h. Relative E2F1 (left panel) /Rb1 (middle panel) mRNA levels (normalized to 18S mRNA) were analyzed by quantitative RT-PCR. Right panel: HeLa cells were treated with NBT (5 µM) for the indicated time periods (2, 4, 6, 8, and 10 h). Samples were analyzed by western blotting for p-Rb, E2F1, and GAPDH (loading control). (B,C) HeLa cells arrested in G1/S phase were released (double thymidine release, DT. Rel.) into fresh medium containing NBT (5 µM) or DMSO. (B) Cells were immunostained for E2F1 protein (green) and DAPI (blue) at the indicated times. Scale bars: 20 µm, Magnification: 30X. (C) Western blot analysis was performed after the preparation of nuclear (N) and cytosolic (C) fractions. (D) HeLa cells were treated with NBT (5 µM) for 12 or 24 h. Relative cyclin A and cyclin E mRNA levels (normalized to 18S) were analyzed by quantitative RT-PCR. The data shown are the means ± SD from 2 independent experiments and were analyzed using ANOVA (**P < 0.01, ***P < 0.001).

Fig 4: Pin1–Rb interaction is induced by G1-S Cyclin-mediated phosphorylation. (a) U2-OS cells were treated with vehicle (dimethyl sulfoxide, DMSO) or 30 µM roscovitine for 18 h. Cell lysates (500 µg total protein) were pulled down by GST-Pin1, followed by western blotting for Rb. Total protein (30 µg) was loaded as an input control. Hyper-pRb, hyperphosphorylated Rb; Hypo-pRb, hypophosphorylated Rb. (b) Diagram of RbLP highlighting seven pS/T-P putative Pin1-binding sites within the Rb C-pocket (RbC), as well as two double-point mutations to Ala (S807A;S811A, denominated 2S, and T821A;T826A, denominated 2T) and mutation of all Ser or Thr residues to Ala residues (7A) used for RbC-Pin1-binding studies. (c) U2-OS cells were transfected with WT or mutant RbLP. Cell lysates were subjected to GST-Pin1 pull-down assay and western blotted for Rb to assess binding. (d) Saos-2 cells were co-transfected with Rb along with Cyclin A, Cyclin B1, Cyclin D1, Cyclin E or a vector control, as shown. Cell lysates were subjected to GST-Pin1 pull down followed by western blotting for Rb

Fig 5: Knocking down AMOTL1 exerts anti-tumor effects.a The KD efficiency of siAMOTL1 was confirmed by qRT-PCR. b AMOTL1 KD, p21, and p27 were upregulated, whereas pRb was reduced. c MTT assay suggested that siAMOTL1 suppressed GC cellular growth (**P < 0.001). d Monolayer colony formation assay indicated a slower proliferation of GC cells after AMOTL1 KD (**P < 0.001). e Cellular invasive ability was retarded by AMOTL1 deactivation (**P < 0.001). f Re-expression of AMOTL1 partly restored the KD effect of siRNAs on AGS. g siAMOTL1 resulted in G1-phase arrest in GC cell lines (*P < 0.05). h AMOTL1 KD increased cellular apoptosis (**P < 0.001). i During the starvation/FBS stimulation, GC cells with siAMOTL1 exhibited lower levels of ERK, pERK, CCND1, and pRb, while the expressions of p21 and p27 were elevated.