Journal: Molecular and Cellular Biology

Article Title: Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

doi: 10.1128/mcb.00872-09

Figure Lengend Snippet: FIG. 6. C/EBP regulates the expression of E2F family genes in E-treated uterine epithelial cells. (A) LE cells were isolated from uteri of ovariectomized WT or C/EBP/ mice following treatment with E for 0, 12, and 15 h. (a to c) Expression levels of E2F1 (a), E2F2 (b), and E2F3 (c) were determined by real-time PCR. The fold changes indicate mRNA expression levels of the E2F genes relative to those of E-treated WT cells at 0 h. (d) Relative steady-state levels of mRNAs for E2Fs 1, 2, and 3 in primary LE cells obtained from uteri treated with E for 15 h are shown following normalization with the 36B4 mRNA level. Statistically significant differences (P 0.05) are indicated by asterisks. (B) Uteri from WT and C/EBP-null mice treated with E for 15 h were collected, and sections were subjected to IHC analysis using anti-E2F3 antibody. Positive staining for E2F3 is indicated in red, and DAPI-stained nuclei are shown in blue. (C) ChIP analysis. LE cells were isolated from uteri of ovariectomized WT mice following treatment with E for 12 h. ChIP was performed as described in Materials and Methods using antibodies against C/EBP, RNA polymerase II (RNAP II), and rabbit IgG. Relative levels of recruitment at various sites on the E2F3 promoter were determined by real-time PCR and normalized to input DNA and RNA polymerase II values.

Article Snippet: Sections were incubated with the following primary antibodies diluted in blocking solution (0.25% bovine serum albumin [BSA], 0.3% Triton X-100, sterile PBS) overnight at 4°C: BrdU and Ki67 (BD Pharmingen), phospho-Ser10 histone H3 (Upstate Biotechnology); cyclin E, cyclin A, and Rad18 (Abcam); cyclin D1 (LabVision NeoMarkers); E2F3 (Santa Cruz Biotechnology); p27 (BD Transduction Laboratories); and phospho(Ser1981)-ATM, phospho-(Ser139)-H2AX, caspase 3, and cleaved caspase 3 (Cell Signaling Technology).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular and Cellular Biology

Article Title: Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

doi: 10.1128/mcb.00872-09

Figure Lengend Snippet: FIG. 10. Molecular pathways regulated by C/EBP during E-in- duced proliferation of uterine epithelial cells. In mouse uterine epi- thelial cells, the expression of C/EBP is stimulated by E and opposed by simultaneous treatment with P. C/EBP controls the entry of E- stimulated epithelial cells into the S phase of the cell cycle by upregu- lating the expression of E2F3 and cyclin E. It also promotes the nuclear localization of cyclin E, which enables the formation of an active cyclin E-cdk2 complex critical for DNA replication. The lack of C/EBP allows the nuclear accumulation of p27, which contributes to the cell cycle arrest. In E-stimulated C/EBP-null uterine epithelial cells, stalled DNA replication activates a DNA damage response path- way involving ATM/ATR, Chk1/Chk2, Rad18, and p53. Activated p53 maintains the block in DNA replication by enhancing the synthesis of the cell cycle inhibitor p21 and also promotes the removal of damaged cells via caspase-dependent apoptosis.

Article Snippet: Sections were incubated with the following primary antibodies diluted in blocking solution (0.25% bovine serum albumin [BSA], 0.3% Triton X-100, sterile PBS) overnight at 4°C: BrdU and Ki67 (BD Pharmingen), phospho-Ser10 histone H3 (Upstate Biotechnology); cyclin E, cyclin A, and Rad18 (Abcam); cyclin D1 (LabVision NeoMarkers); E2F3 (Santa Cruz Biotechnology); p27 (BD Transduction Laboratories); and phospho(Ser1981)-ATM, phospho-(Ser139)-H2AX, caspase 3, and cleaved caspase 3 (Cell Signaling Technology).

Techniques: Expressing, Blocking Assay

Journal: Molecular medicine reports

Article Title: MicroRNA‑141 inhibits the differentiation of bone marrow‑derived mesenchymal stem cells in steroid‑induced osteonecrosis via E2F3.

doi: 10.3892/mmr.2022.12750

Figure Lengend Snippet: Figure 3. Luciferase assays. Relative luciferase activity was assessed in bone marrow‑derived mesenchymal stem cells following co‑transfection with miR‑141 and E2F3 WT or mut plasmids. **P<0.01 vs. control. miR, microRNA; E2F3, E2F transcription factor 3, NC negative control; WT, wild‑type; mut, mutant.

Article Snippet: Western blot analysis of E2F3 protein expression. at day 3, 7 and 14, after discarding the RASMX‐90021 medium (Cyagen; cat. no. RASMX‐90021) and α‐MEM (HyClone; Cytiva; SH30265) the cells were lysed in RIPA lysis buffer at 4 ̊C overnight.

Techniques: Luciferase, Activity Assay, Control, Negative Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑141 inhibits the differentiation of bone marrow‑derived mesenchymal stem cells in steroid‑induced osteonecrosis via E2F3.

doi: 10.3892/mmr.2022.12750

Figure Lengend Snippet: Figure 4. MRNA expression of miR‑141 and E2F3 (A) Expression levels of miR‑141 in group of control and ONFH at 3d, 7d and 14d. (B) MRNA expression levels of E2F3 in group of NC, miR‑141, ONFH+ inhibitor NC and ONFH+ miR‑141 inhibitor (left part). mRNA expression levels of E2F3 in group of control and ONFH at 3d, 7d and 14d (right part). *P<0.05, **P<0.01 vs. control. miR, microRNA; E2F3, E2F transcription factor 3, NC negative control; ONFH, osteonecrosis of the femoral head.

Article Snippet: Western blot analysis of E2F3 protein expression. at day 3, 7 and 14, after discarding the RASMX‐90021 medium (Cyagen; cat. no. RASMX‐90021) and α‐MEM (HyClone; Cytiva; SH30265) the cells were lysed in RIPA lysis buffer at 4 ̊C overnight.

Techniques: Expressing, Control, Negative Control

Journal: Molecular medicine reports

Article Title: MicroRNA‑141 inhibits the differentiation of bone marrow‑derived mesenchymal stem cells in steroid‑induced osteonecrosis via E2F3.

doi: 10.3892/mmr.2022.12750

Figure Lengend Snippet: Figure 5. Protein expression levels of E2F3. (A) Protein expression of E2F3 in BMSCs from the control or ONFH rat at day 3, 7 and 14. (B) Protein expression of E2F3 in BMSCs from the normal rat transduced with NC or miR‑141 mimic lentivirus and BMSCs from the ONFH rat transduced with the inhibitor NC or miR‑141 inhibitor lentivirus. *P<0.05, **P<0.01 vs. control. miR, microRNA; E2F3, E2F transcription factor 3, NC negative control; ONFH, osteonecrosis of the femoral head; BMSCs, bone marrow‑derived mesenchymal stem cells.

Article Snippet: Western blot analysis of E2F3 protein expression. at day 3, 7 and 14, after discarding the RASMX‐90021 medium (Cyagen; cat. no. RASMX‐90021) and α‐MEM (HyClone; Cytiva; SH30265) the cells were lysed in RIPA lysis buffer at 4 ̊C overnight.

Techniques: Expressing, Control, Transduction, Negative Control

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