Fig 1: Dysregulated genes in A549 cells after UBE2S knockdown. (A) A total of 458 genes were regulated (171 genes up-regulated and 287 genes down-regulated) by UBE2S knockdown in A549 cells (P < 0.05, fold change > 1.5). (B) Pathway enrichment was analyzed by IPA software. Orange, activated pathway. Blue, suppressed pathway. (C) Correlation map of up-regulated NUPR1 and its potential downstream targets. Red and orange box, up-regulated gene. Green box, down-regulated gene. Blue arrow, consistent repression. Orange arrow, consistent activation. Yellow arrow, inconsistent correlation. (D) Western blot analysis of P53, NUPR1 and ILK in shCtrl and shUBE2S A549 cells.
Fig 2: UBE2S induces the BrdU incorporation and suppresses apoptosis of lung cancer cells. (A) The BrdU incorporation of shCtrl, shUBE2S-1 and shUBE2S-2 A549 cells. **P < 0.01. (B) The BrdU incorporation of Control (Ctrl) and UBE2S overexpressed (UBE2S-OE) H1299 cells. **P < 0.01. (C, D) The apoptosis of shCtrl and shUBE2S-1 A549 cells was determined by Flow cytometry. (C) Representative images of the flow cytometry results, and (D) quantification of apoptosis. **P < 0.01. (E) Caspase 3/7 activity of shCtrl, shUBE2S-1 and shUBE2S-2 A549 cells. **P < 0.01. (F) Caspase 3/7 activity of Ctrl and UBE2S-OE H1299 cells. *P < 0.05. (G) Western blot analysis of cleaved-PURP1 in shCtrl, shUBE2S-1 and shUBE2S-2 A549 cells. (H) Western blot analysis of cleaved-PURP1 in Ctrl and UBE2S-OE H1299 cells.
Fig 3: UBE2S expression is increased in lung cancer tissues and cells and high expression of UBE2S predicts poor outcome of lung cancer patients. (A) Immuno-histochemical analysis of UBE2S expression in lung cancer and normal tissues. (B) UBE2S mRNA expression in lung cancer (n = 522) and normal tissues (n = 379) derived from the Cancer Genome Atlas (TCGA). P < 0.01. (C) UBE2S mRNA expression in lung cancer and adjacent normal tissues derived from the Cancer Genome Atlas (TCGA). FC, Fold Change, n = 57, P < 0.001. (D) UBE2S mRNA expression and protein level were determined respectively by qRT-PCR and Western blot assays in BEAS-2B, 95D, H1299, H1975 and A549 cells. **P < 0.01, ***P < 0.001. (E) Lung cancer patients were divided into UBES2 high (Green line) and low (Blue line) expression groups. Overall survival of patients was determined after operation. P < 0.001.
Fig 4: UBE2S knockdown suppresses the proliferation of A549 cells. (A, B) shCtrl, shUBE2S-1 and shUBE2S-2 A549 cells were subjected to qRT-PCR (A) and Western blot (B) analysis of UBE2S. GAPDH serve as internal control. **P < 0.01. (C, D) Control (Ctrl) and UBE2S over-expressed (UBE2S-OE) H1299 cells were subjected to qRT-PCR (C) and Western blot (D) analysis of UBE2S. GAPDH serve as internal control. **P < 0.01. (E, F) (E) Representative images of high-content screening (HCS) of shCtrl (top) and shUBE2S-1 (bottom) A549 cells from day 1 to day 5. HCS detected the numbers of viable cells. (F) Relative quantification of HCS results (Cell count). **P < 0.01, ***P < 0.001. (G) Viability of shCtrl, shUBE2S-1 and shUBE2S-2 A549 cells was determined by MTT assay. *P < 0.05, **P < 0.01. (H) Viability of Control (Ctrl) and UBE2S over-expressed (UBE2S-OE) H1299 cells was determined by MTT assay. *P < 0.05, **P < 0.01. (I) shCtrl, shUBE2S-1 and shUBE2S-2 A549 cells were subjected to colony formation assay. **P < 0.01. (J) Control (Ctrl) and UBE2S over-expressed (UBE2S-OE) H1299 cells were subjected to colony formation assay. *P < 0.05.
Fig 5: UBE2S promotes HCC growth and metastasis in vivo. (A) Tumors from the UBE2S‐shRNA and control‐shRNA groups in subcutaneous xenograft tumor models were removed 25 days after tumor inoculation, and photos of the tumors were taken. (B) The weights of tumors from the UBE2S‐shRNA and control‐shRNA groups in subcutaneous xenograft tumor models were measured. (C) The volumes of tumors in the UBE2S‐shRNA and control‐shRNA groups in subcutaneous xenograft tumor models were measured on different days after tumor inoculation. (D) The weights of mice from the UBE2S‐shRNA and control‐shRNA groups in subcutaneous xenograft tumor models were measured. (E) Tumors from the UBE2S‐overexpressing Bel‐7402 cells and control groups in subcutaneous xenograft tumor models were removed 16 days after tumor inoculation, and photos of the tumors were taken. (F) The weights of tumors from the UBE2S overexpression and control groups in subcutaneous xenograft tumor models were measured. (G) The volumes of tumors in the UBE2S overexpression and control groups in subcutaneous xenograft tumor models were measured on different days after tumor inoculation. (H) The weights of mice from the UBE2S overexpression and control groups in subcutaneous xenograft tumor models were measured. (I) Tumors from the UBE2S‐shRNA and control‐shRNA groups in orthotopic xenograft tumor models were removed 8 weeks after tumor inoculation, and photos of tumors were taken. (J) The tumors were excised from livers in orthotopic xenograft tumor models, and the weights and volumes of tumors in the UBE2S‐shRNA and control‐shRNA groups were measured. (K) Representative images of H&E staining of pulmonary metastatic nodules in the orthotopic HCC model (left panel). Quantitation of the pulmonary metastatic nodules is shown in the right panel. (L) Quantitation of the ratio of lung metastatic nodule number per tumor weight (g) for each mouse is shown. The data are presented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.
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