Journal: Respiratory research

Article Title: Involvement of miRNA-34a regulated Krüppel-like factor 4 expression in hyperoxia-induced senescence in lung epithelial cells.

doi: 10.1186/s12931-022-02263-8

Figure Lengend Snippet: Fig. 5 miR-34a-5p inhibitor reduces hyperoxia-induced KLF4 protein expression in cultured lung epithelial cells. MLE-12 cells were transfected with miR-34a-5p inhibitor (Inh) or negative control (NC) at 20 nM. 24 h after transfection, MLE-12 cells were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 24 h. A–D E2F3, E2F1, CCND1, and KLF4 protein levels were measured by Western blot. Densitometry of bands was normalized using calnexin (CNX) levels. E Transcription levels of Klf4 were measured by RT-qPCR. Data are expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 versus air NC, group, †p < 0.05 versus NC in O2 group, RT-qPCR, quantitative real-time PCR

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% milk and then probed with primary antibodies against p53 (ab131442, Abcam), p21 (ab109199, Abcam), p16 (ab108349, Abcam), MDM4 (ab222905, Abcam or sc-74468, Santa Cruz), E2F1 (ab137415, Abcam), E2F3 (sc-56665, Santa Cruz), CCND1 (ab134175, Abcam), KLF4 (ab129473, Abcam), β-actin (ab8227, Abcam) and calnexin (ADI-SPA-860-F, Enzo, New York, NY, USA) to determine the corresponding proteins.

Techniques: Expressing, Cell Culture, Transfection, Negative Control, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

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: Cell cycle (Georgetown, Tex.)

Article Title: E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis.

doi: 10.4161/cc.7.3.5286

Figure Lengend Snippet: Figure 3. Both E2F1 and E2F3 contribute to E1A-induced apoptosis and p53 induction. (A) Serum-starved wildtype (Wt) and E2f1-/- MAFs were infected with AdCMV empty vector or AdE1A at MOI of 100. 48 h post-infection caspase 3 activity was measured with the fluorescence substrate Ac-DEVD-AFC and the average of three experiments is presented. Bars indicate standard error and *indicates statistical significance compared to E1A-infected wildtype MAFs (p < 0.05). (B) The same cells were treated as above or exposed to 10 Gy of IR one h prior to harvest. Western blot analysis was performed on protein lysates (50 μg) using antibodies to phospho-serine 15 p53, total p53, E1A and β-actin as indicated. (C) Primary NHFs were transfected with control siRNA or siRNA directed to E2F3 under starvation conditions and 24 h later infected with AdCMV or AdE1A at MOI of 100. 48 h post-infection caspase 3 activity was measured with the fluorescence substrate Ac-DEVD-AFC and the average of three experiments is presented. Bars indicate standard error and *indicates statistical significance compared to E1A-infected NHFs (p < 0.05). (D) NHFs were transfected with control (lanes 1 and 2) or E2F3 (lanes 3 and 4) siRNA and then infected with AdCMV (lanes 1 and 3) or AdE1A (lanes 2 and 4) as described above. Western blot analysis was performed on protein lysates (50 μg) using antibodies to E2F3, E2F1, phospho-serine 15 p53, γH2AX and H2B as indicated.

Article Snippet: NHFs were plated at 4,000 cells/cm2 in 6 cm plates and allowed to recover overnight in complete media, 10% FBS DMEM with antibiotics and transfected with 200–250 pmol of E2F3 siRNA or control siRNA (Santa Cruz biotechnology, Inc.) using Oligofecatamin reagent (invitrogen) according to the manufacturer’s protocol.

Techniques: Infection, Plasmid Preparation, Activity Assay, Fluorescence, Western Blot, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis.

doi: 10.4161/cc.7.3.5286

Figure Lengend Snippet: Figure 6. DNA breaks induced by E1A are unaffected by loss of E2F1 but reduced by knockdown of E2F3. (A) Wildtype (Wt) and E2f1-/- MAFs were infected with AdCMV empty vector or AdE1A at MOI of 100. 48 h post-infection cells were subjected to the comet assay and analyzed as previously described. Bars indicate standard error and *indicates statistical significance compared to AdCMV infected cells of the same genotype (p < 0.05). (B) NHFs were transfected with control siRNA or E2F3 siRNA 24 h prior to infections with AdCMV or AdE1A at MOI of 100. 48 h post-infection cells were subjected to the comet assay and analyzed as previously described. Bars indicate standard error and *indicates statistical significance compared to control siRNA treated cells infected with AdE1A.

Article Snippet: NHFs were plated at 4,000 cells/cm2 in 6 cm plates and allowed to recover overnight in complete media, 10% FBS DMEM with antibiotics and transfected with 200–250 pmol of E2F3 siRNA or control siRNA (Santa Cruz biotechnology, Inc.) using Oligofecatamin reagent (invitrogen) according to the manufacturer’s protocol.

Techniques: Knockdown, Infection, Plasmid Preparation, Single Cell Gel Electrophoresis, Transfection, Control

Journal: Genes, chromosomes & cancer

Article Title: E2F3b Over-Expression in Ovarian Carcinomas and in BRCA1 Haplosufficient Fallopian Tube Epithelium

doi: 10.1002/gcc.21990

Figure Lengend Snippet: E2F3 and KI67 over-expression in TP53 foci of histologically normal FTE. Adjacent sections of TP53 foci were stained for TP53, E2F3 and KI67. A. Brown immunostain demonstrates a clear demarcation of a TP53 focus in normal FTE from a BRCA1 mutation carrier. B. E2F3 is evident as brown nuclear staining and is more frequent in the TP53 focus than in adjacent normal FTE. C. KI67 over-expression is evident in the TP53 focus compared to normal adjacent FTE. E2F3 and KI67 overexpression occurred more frequently in TP53 foci from BRCA1 mutated cases (4/18) than BRCA2 mutated cases (1/21, P = 0.04), and compared to wildtype cases (0/28, P = 0.002). B1: specimen with BRCA1 mutations. B2: specimen with BRCA2 mutations. RRSO: risk reducing salpingo-oophorectomy. CA: ovarian carcinoma. WT: wildtype for BRCA1 and BRCA2.

Article Snippet: TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA) were used for BRCA1 (Hs00173233_m1), E2F3 (Hs01076040_g1), E2F3a (Hs01076037_m1), and E2F3b (primers and probe sequences same as described previously ( Reimer et al. 2010 )), and GAPDH (402869) was used as the reference gene.

Techniques: Over Expression, Staining, Mutagenesis

Journal: Genes, chromosomes & cancer

Article Title: E2F3b Over-Expression in Ovarian Carcinomas and in BRCA1 Haplosufficient Fallopian Tube Epithelium

doi: 10.1002/gcc.21990

Figure Lengend Snippet: Correlation between expression of E2F3 and BRCA1 message. A. There was a significant correlation between expression of total E2F3 and BRCA1 message in all three genetic subgroups. B. There was no significant correlation between expression of E2F3a and BRCA1 message in any genetic subgroup. C. There was a significant correlation between expression of E2F3b and BRCA1 message in all three genetic subgroups.

Article Snippet: TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA) were used for BRCA1 (Hs00173233_m1), E2F3 (Hs01076040_g1), E2F3a (Hs01076037_m1), and E2F3b (primers and probe sequences same as described previously ( Reimer et al. 2010 )), and GAPDH (402869) was used as the reference gene.

Techniques: Expressing

Journal: Genes, chromosomes & cancer

Article Title: E2F3b Over-Expression in Ovarian Carcinomas and in BRCA1 Haplosufficient Fallopian Tube Epithelium

doi: 10.1002/gcc.21990

Figure Lengend Snippet: E2F3 protein over-expression in ovarian carcinoma and E2F3 copy number. Cases with 3 or 4 copies of E2F3 have significantly higher E2F3 expression than cases with 2 copies (P = 0.01, t = 2.61, unpaired t-test).

Article Snippet: TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA) were used for BRCA1 (Hs00173233_m1), E2F3 (Hs01076040_g1), E2F3a (Hs01076037_m1), and E2F3b (primers and probe sequences same as described previously ( Reimer et al. 2010 )), and GAPDH (402869) was used as the reference gene.

Techniques: Over Expression, Expressing

Journal: Genes, chromosomes & cancer

Article Title: E2F3b Over-Expression in Ovarian Carcinomas and in BRCA1 Haplosufficient Fallopian Tube Epithelium

doi: 10.1002/gcc.21990

Figure Lengend Snippet: E2F3 protein over-expression in wildtype, BRCA1 mutated and BRCA2 mutated ovarian carcinomas. Paraffin embedded ovarian carcinoma sections were stained with pan-E2F3 antibody. There was no significant difference in the distribution of cases with negative (0–10% neoplastic staining), medium (11–30% neoplastic staining) and higher (31–100% neoplastic staining) E2F3 expression between the genetic subgroups.

Article Snippet: TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA) were used for BRCA1 (Hs00173233_m1), E2F3 (Hs01076040_g1), E2F3a (Hs01076037_m1), and E2F3b (primers and probe sequences same as described previously ( Reimer et al. 2010 )), and GAPDH (402869) was used as the reference gene.

Techniques: Over Expression, Staining, Expressing