Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 2. Changes in mRNA expression of BCL2, E2F1, E2F3, RB1 and P53 in K562 cells after cisplatin treatment. BCL2, E2F1, E2F3, RB1 and P53 were detected by (A) RT-PCR, (B) real-time PCR and (C) ELISA.

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 4. Correlative expression of miRNAs and oncogenes using antisense oligos (ASO). (A) RT-PCR, (B) real-time PCR. Correlative expression of (C) E2F1 and its targeted miR-17-5p, (D) E2F3 and its targeted miRNAs and (E) Bcl-2 and its targeted miRNAs (miR-16, 34a-c) using ELISA is shown. *Significant difference (p<0.05).

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 5. Expression of E2F1, E2F3, BCL2, RB1 and P53 genes regulated by miRNAs. Detection of the expression of (A) E2F1, (B) E2F3, (C) BCL2, (D) RB1 and (E) P53. *Significant difference (p<0.05).

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 2. Role of E2F1 in regulating the proliferation of PMCs. (A) The expression of E2F1-3 in palatal shelves on E12, E13 and E14 determined by Westernblot; Blots were cropped for figure construction. (B) Expression of E2F1-3 on E13.5 was measured by immunohistochemical staining (left panel, magnification ×100). The positive rate of E2F1 and E2F3 in palatal shelves was significantly higher than E2F2 (right panel, n = 6, *P < 0.01). (C) MTT assay showing the growth curve of PMCs with or without E2F1 knockdown. Values are mean ± SD of triplicate experiments in each group. *P < 0.01. (D) Left panel: immunofluorescence staining of Ki-67 and E2F1 in PMCs. Right panel: the quantification of Ki67 and E2F1 double positive cells. *P < 0.01.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemical staining, Staining, MTT Assay, Knockdown, Immunofluorescence

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 3. Positive regulation of miR-17-92 by E2F1. (A) The expression of miR-17-92 members in control and E2F1 overexpressing cells was analyzed by RT-qPCR. Values represent means ± SD. Error bars, SD. *P < 0.05. (B) To detect the direct regulation of E2F1 on miR-17-92, a ChIP assay was performed with 3 putative binding sites of E2F1 on the miR-17-92 promoter. Input DNA that was not enriched by immunoprecipitation was amplified as a positive control. The miR-106a ORF region was used as negative control. The gels were cropped for figure construction.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Expressing, Control, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Amplification, Positive Control, Negative Control

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 4. Negative regulation of E2F1 by miR-17-92. (A) The relative expression of E2F1-3 mRNA was evaluated by qPCR. Values represent means ± SD. Error bars, SD. *P < 0.05. (B) Protein levels of E2F1-3 were measured by Western blot analysis. GAPDH was used as the internal control. Blots were cropped for figure construction. (C) The location and sequences of predicted target sites in the 3′UTR of mouse E2F1 for miR- 17/20a. The seed sequences of miR-17/20a. The sequences of predicted target sites are conserved across species including human and mouse. (D) Luciferase assay was used to validate the predicted target sites of miR-17/20a on 3′UTR of E2F1. Data were presented as relative luciferase activities compared to normoxia after normalizing to Renilla luciferase activities. Experiments were performed in triplicate (*P < 0.05).

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Control, Luciferase

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 5. MiR-17-92 negatively regulates E2F1-induced cell cycle transition of PMCs. (A) MTT assay showing the role of miR-17-92 on the growth of PMCs. Values are mean ± SD of triplicate experiments in each group. *P < 0.01. (B) Cell cycle of PMCs transfected with miR-17-92 MI or miR SCR was analysed by flow cytometry. (C) Quantification analysis of cells in G0/G1 phase. Values are mean ± SD of triplicate experiments in each group. *P < 0.01. (D) Quantification analysis of cells in S phase. Values are mean ± SD of triplicate experiments in each group. *P < 0.01.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: MTT Assay, Transfection, Flow Cytometry

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 6. Schematic cartoon illustrating the negative feedback loop between E2F1 and miR-17-92 and its regulation on the cell cycle of PMCs.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques:

Journal: eBioMedicine

Article Title: YAP1 activation is associated with the progression and response to immunotherapy of non-muscle invasive bladder cancer

doi: 10.1016/j.ebiom.2022.104092

Figure Lengend Snippet: YAP1 knockdown inhibits the proliferation of bladder cancer cells in vitro. The expression of YAP1 subgroup target genes was partially restored by YAP1 expression. (a) UC3 cells were infected with lentivirus expressing shYAP1 or scrambled shRNA (scRNA). YAP1 mRNA and protein levels were detected using qRT–PCR and Western blotting, respectively. (b) Cell viability was analysed at 0, 24, and 48 h with the MTT assay to determine the proliferation of the UC3 cell line. The data are presented as the means ± standard deviations of three independent experiments. (c) A clonogenic assay was performed with control and YAP1 knockdown UC3 cells. Cells were stained with a crystal violet solution, and colonies were counted in three independent experiments. Invasion and migration assays of UC3 cells transfected with shNTS and shYAP1 for 24 h were performed using Boyden chamber assays. Data are presented as the means ± standard deviations of three experiments. (d) The YAP1 activation 1 (YA1) genes FOXM1, E2F1, CCNA2, AURKA, BIRC5, LMNB1, and TEAD2 were validated at the mRNA level after YAP1 knockdown in the UC3 cell line. The mRNA levels of CTGF, CYR61, and ZEB1 included in YAP1 activation 2 (YA2) group were verified using qRT–PCR. The YAP1 inactivation (YI) genes GATA2, SMAD3, and SMAD6 were validated in YAP1 knockdown cells using qRT–PCR. The data are presented as the means ± standard deviations of three independent experiments. (e) Levels of the YAP1, CTGF, CYR61, E2F1, and FOXM1 proteins were detected using Western blotting. (f) In qRT–PCR experiments, the levels of YA1 and YA2 subgroup genes were detected following YAP1 overexpression, and the levels of YA1 and YA2 subgroup genes were restored by YAP1 overexpression. This experiment was performed three times. ns , not significant; *, p < 0·05; **, p < 0·01; and ***, p < 0·001.

Article Snippet: Primary antibodies against GAPDH (Cat# 2118, RRID: AB_561053, Cell Signaling Technology, MA, USA), YAP1 (Cat# 4912, RRID: AB_2218911, Cell Signaling Technology, MA, USA), p-YAP1 (S127) (Cat# 4911, RRID: AB_2218913, Cell Signaling Technology, MA, USA), CYR61 (Cat# sc-374129, RRID: AB_10947399, Santa Cruz Biotechnology, CA, USA), CCNA2 (Cat# NBP1-31330, RRID: AB_10003781, Novus Biologicals, CO, USA), E2F1 (Cat# A300-766A, RRID: AB_2096774, Bethyl Laboratories, TX, USA), and FOXM1 (Cat# A301-533A, RRID: AB_999586, Bethyl Laboratories, TX, USA) were used.

Techniques: Knockdown, In Vitro, Expressing, Infection, shRNA, Quantitative RT-PCR, Western Blot, MTT Assay, Clonogenic Assay, Control, Staining, Migration, Transfection, Activation Assay, Over Expression