Journal: Advanced Science

Article Title: Novel Evolutionarily Conserved Oncogene COA4 is Driven by KRAS Mutant and Promotes Cancer Metastasis Through Dual Mitochondrial Metabolism‐Dependent and ‐Independent Mechanisms

doi: 10.1002/advs.202507533

Figure Lengend Snippet: E2F1 directly binds to COA4 promoter and promotes its expression under KRAS mutation. A,B) Western blot (A) and RT‐qPCR (B) analyses of COA4 expression in A549 cells following E2F1 overexpression or knockdown. ( n = 3; ** p < 0.01; **** p < 0.0001) C) Luciferase reporter assay confirming that E2F1 directly regulates COA4 transcription in 293T and A549 cells. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) D) Luciferase reporter assay demonstrating that KRAS enhances E2F1‐mediated transcriptional activation of the COA4 promoter in 293T cells. ( n = 3; **** p < 0.0001) E) ChIP‐qPCR analysis identifying the specific E2F1 binding region within the COA4 promoter in A549 cells. ( n = 3; *** p < 0.001) F) AlphaFold 3 molecular docking of the COA4 promoter with E2F1, supporting their direct interaction. G) Chromatin immunoprecipitation followed by qPCR (ChIP‐qPCR) was performed to evaluate E2F1 binding to the COA4 promoter in normal tissues and KRAS‐mutant tumor tissues. Data are presented as mean ± SD ( n = 6, *** p < 0.001) H) Spearman's rank correlation analysis of COA4 and E2F1 expression in LUAD using the GEPIA2 database. I. IHC analysis of E2F1 expression in LUAD tumor versus normal tissues ( n = 12 per group; **** p < 0.0001) J) ATP quantification in E2F1 ‐overexpressing A549 cells following COA4 knockdown.( n = 3; ** p < 0.01) K) Complex IV enzyme activity in E2F1 ‐overexpressing A549 cells after COA4 knockdown. ( n = 3; ** p < 0.01) L,M) Western blot (L) and RT‐qPCR (M) analyses of COA4 expression in KRAS G12C ‐overexpressing A549 cells following E2F1 knockdown. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) N) Western blot analysis of COA4 expression in A549 cells treated with the PI3K agonist 740Y‐P(15 µM) for 48 h (left), and in 740Y‐P‐treated A549 cells following E2F1 knockdown (right).( n = 3) O) Western blot analysis of COA4 expression in A549 and H23 cells treated with the PI3K inhibitor LY294002 following E2F1 overexpression. ( n = 3) P) RT‐qPCR analysis of COA4 mRNA levels in A549 cells treated with LY294002 following E2F1 overexpression (left), and in A549 cells treated with 740Y‐P following E2F1 knockdown (right). ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) Q) Transwell assays assessing migration of COA4 ‐overexpressing A549 and H23 cells following E2F1 knockdown (top), and of COA4‐knockdown cells following E2F1 overexpression (bottom). Scale bar: 275 µm (n = 3; ** p < 0.01; **** p < 0.0001) R) Transwell assays analyzing migration of A549 and H23 cells treated with 740Y‐P following E2F1 knockdown (left), and with LY294002 following E2F1 overexpression (right). Scale bar: 275 µm ( n = 3) S) Transwell assays evaluating migration and invasion in A549 and H23 cells following KRAS G12D overexpression, E2F1 knockdown, and COA4 overexpression. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) T) Analysis of E2F1 expression in LUAD tissues: left, comparing KRAS wild‐type versus KRAS mutant samples using TCGA data; right, comparing LUAD tissues with high versus low KRAS expression using combined datasets ( GSE30219 , GSE37745 , GSE118370 , and GSE140797 ). U) Survival analysis based on COA4 and E2F1 expression levels using the GSE30219 database. V) Combined analysis of COA4 and E2F1 expression with survival status and overall survival time, performed using the linkET package on the GSE30219 dataset. The data are given as mean ± SD and compared by Student's t test (A–E, G, I–T), Spearman's rank correlation analysis (H). The Log‐rank (Mantel–Cox) test was used for survival analyses (U,V). Significance levels are indicated as follows: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Primary antibodies: ACTIN (1:5000, Proteintech, 81115‐1‐RR); Coa4 (1:2500, Invitrogen, PA5‐59076); CDC42 (1:1000, Proteintech, 10155‐1‐AP); E2F1 (1:1000, Proteintech, 12171‐1‐AP); MTCO1 (1:500, ABclonal, A17889); MTCO2 (1:3000, Proteintech, 55070‐1‐AP); MTCO3 (1:2000, Proteintech, 55082‐1‐AP); KRAS (1:10000, Proteintech, 12063‐1‐AP); FAK (1:5000, ABclonal, A11131); p‐FAK‐Y397 (1:300, ABclonal, AP0302).

Techniques: Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR, Over Expression, Knockdown, Luciferase, Reporter Assay, Activation Assay, ChIP-qPCR, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Migration

Journal: bioRxiv

Article Title: The BRD4S-LOXL2-MED1 interaction at the forefront of cell cycle transcriptional control in triple-negative breast cancer

doi: 10.1101/2022.05.27.493725

Figure Lengend Snippet: ( A ) Representative cell cycle profile of MDA-MB-231 cells infected either with shControl (C) or shLOXL2 (KD). DNA content was analyzed by FACS following Hoechst staining. Three biological replicates were analyzed for each condition. ( B ) High-throughput immunofluorescence of H3S10p mitotic marker in C or KD transduced MDA-MB-231 cells. Mitotic cells (black dots) showed on average an H3S10p signal higher than the population median + 3S.D. Interphase cells are represented with gray dots. H3S10p intensity is represented as the normalized median. ( C ) Representative cell cycle profile of MDA-MB-231 cells treated either with DMSO or PXS5382 for 96h. DNA content was analyzed by FACS following Hoechst staining. Three biological replicates were analyzed for each condition. ( D ) MDA-MB-231 cells expressing SLBP-mTurquoise2 and H1-Maroon1 were treated with DMSO or PXS5382 for 96h. The percentage of mTurquoise2 nuclei in each well is shown, representing cells in G1-S. The difference between the PXS5382 and DMSO treated cells was significant (p-value <0.005) under a linear model comparing the percentage of mTurquoise2-positive cells, across time, in the two conditions. The estimated increase due to PXS5382 on the Area Under Curve (AUC), is 13.3 au. Quantification was performed every 12h. Six biological replicates were performed using at least 200 cells/replicate for the analysis. ( E ) PD of endogenous Lin9, B-Myb, and FOXM1 in MDA-MB-231 cells. Precipitates were analyzed by Western blot with the indicated antibodies. Irrelevant IgGs were used as a negative control (ns: non-specific). Three biological replicates were performed. ( F ) Differential gene essentiality between high and low LOXL2-expressing cell lines (CCLE) as calculated by analyzing the Achilles’ dataset. Cell lines with low LOXL2 expression are significantly more sensitive to depletion of genes represented in the right part of the X-axis as compared to cell lines with high LOXL2 expression, which are more sensitive to depletion of genes represented in the left part of the X-axis. Significance was determined using the Student’s t-test with BH multiple hypothesis correction. Significant threshold is based on adjusted p-value <0,05; black dots represent significant essentialities while gray dots are not significant. The dot size is proportional to the adjusted p-value of each gene.

Article Snippet: Nuclear extracts were quantified using Pierce BCA Protein Assay Kit (Thermo Scientific; PIER23225) and 1mg of nuclear extract was incubated with the indicated antibodies: 2.5ug of BRD4 (Abcam; ab128874), 5ug of MED1 (Bethyl Laboratories; A300-793A), 4ug of Lin9 (Proteintech; 17882-1-AP), 4ug of B-Myb (Proteintech; 18896-1-AP) 4ug of FOXM1 (Proteintech;13147-1-AP) O/N, followed by incubation with Protein A Dynabeads (Thermo Scientific; 10002D) for 1h at 4oC.

Techniques: Infection, Staining, High Throughput Screening Assay, Immunofluorescence, Marker, Expressing, Western Blot, Negative Control

Journal: Folia Histochemica et Cytobiologica

Article Title: EID1 plays a protective role in early-onset pre-eclampsia via promoting proliferation and invasion in trophoblast cells

doi: 10.5603/fhc.a2022.0001

Figure Lengend Snippet: Figure 1. EP300 interacting inhibitor of differentiation 1 (EID1) is downregulated in placental tissues of patients with pre-ec- lampsia, especially early-onset pre-eclampsia. A. Real-time PCR was used to detect the mRNA levels of EID1 in placental tissues of women with early-onset pre-eclampsia, late-onset pre-eclampsia, and normal pregnancy control. B. The repre- sentative images of immunohistochemical staining for detection of EID1 in placental tissues (arrows indicate EID1-positive cells). C. TUNEL assay was performed to examine cell apoptosis in placental tissues, and representative images were shown (arrows indicate TUNEL-positive cells). (Abbreviations: PE — pre-eclampsia. The scale bar represents 50 μm. *p < 0.05.

Article Snippet: The sections were incubated with rabbit antibodies against EID1 (1:50; Proteintech, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Control, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Folia Histochemica et Cytobiologica

Article Title: EID1 plays a protective role in early-onset pre-eclampsia via promoting proliferation and invasion in trophoblast cells

doi: 10.5603/fhc.a2022.0001

Figure Lengend Snippet: Figure 2. EID1 promoted proliferation and cell cycle transition of trophoblast cells. A. Western blot was used to confirm the effectiveness of EID1 overexpression or knockdown in HTR-8/SVneo cells. B. Viability of HTR-8/SVeno cells was detected by CCK-8 after culture for 48 h. C. The expression of Ki-67 in HTR-8/SVneo cells after EID1 ectopic expression or knockdown was determined by immunofluorescent staining. D. Flow cytometry was carried out to detect cell cycle phase distribution of HTR-8/SVneo cells. E. The expression of cyclin D1 and cyclin B1 was detected by Western blot. The scale bar represents 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001 compared with pcDNA3.1 or si-NC group.

Article Snippet: The sections were incubated with rabbit antibodies against EID1 (1:50; Proteintech, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Western Blot, Over Expression, Knockdown, CCK-8 Assay, Expressing, Staining, Flow Cytometry

Journal: Folia Histochemica et Cytobiologica

Article Title: EID1 plays a protective role in early-onset pre-eclampsia via promoting proliferation and invasion in trophoblast cells

doi: 10.5603/fhc.a2022.0001

Figure Lengend Snippet: Figure 3. EID1 enhanced migration and invasion of trophoblast cells. A. Transwell assay was performed to detect migration and invasion of HTR-8/SVneo cells after overexpression or silencing of EID1. B. The expression of MMP2 and MMP9 in HTR-8/SVneo cells was detected by Western blot. The scale bar represents 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 compared with pcDNA3.1 or si-NC group.

Article Snippet: The sections were incubated with rabbit antibodies against EID1 (1:50; Proteintech, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Migration, Transwell Assay, Over Expression, Expressing, Western Blot

Journal: Folia Histochemica et Cytobiologica

Article Title: EID1 plays a protective role in early-onset pre-eclampsia via promoting proliferation and invasion in trophoblast cells

doi: 10.5603/fhc.a2022.0001

Figure Lengend Snippet: Figure 4. EID1 activated Akt/b-catenin signaling. A, B. Western blot was used to detect the levels of Akt, p-Akt (Ser 473), GSK3b, and p-GSK3b (Ser9) in HTR-8/SVneo cells with overexpression or knockdown of EID1. C. The nuclear b-catenin levels in HTR-8/SVneo cells were detected. D. Immunofluorescent staining was performed to detect the distribution of b-catenin in HTR-8/SVneo cells. The scale bar represents 50 μm. **p < 0.01, ***p < 0.001 compared with pcDNA3.1 or si-NC group.

Article Snippet: The sections were incubated with rabbit antibodies against EID1 (1:50; Proteintech, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Western Blot, Over Expression, Knockdown, Staining

Journal: Folia Histochemica et Cytobiologica

Article Title: EID1 plays a protective role in early-onset pre-eclampsia via promoting proliferation and invasion in trophoblast cells

doi: 10.5603/fhc.a2022.0001

Figure Lengend Snippet: Figure 5. The suggested mechanism of EID1 function. Reduction of EID1 expression deactivated Akt/b-catenin signaling, causing the decrease of proliferation and invasion of trophoblast cells, resulting in pre-eclampsia. On the contrary, the high expression of EID1 promoted proliferation and invasion of trophoblast cells by inactivating Akt/b-catenin signaling, which may be helpful for successful gestation.

Article Snippet: The sections were incubated with rabbit antibodies against EID1 (1:50; Proteintech, Wuhan, Hubei, China) at 4°C overnight.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: KRAS G12D -driven pentose phosphate pathway remodeling imparts a targetable vulnerability synergizing with MRTX1133 for durable remissions in PDAC

doi: 10.1016/j.xcrm.2025.101966

Figure Lengend Snippet:

Article Snippet: Monolith NT.115 system (NanoTemper Technologies GmbH, Germany) was used to quantify the interaction of E2F1 (Abcam, #ab82207) and Rb (Proteintech, #Ag11211), p53 (MCE, #HY-P72257) and G6PD (Proteintech, #Ag21862), p53 and E2F1.

Techniques: Mutagenesis, Recombinant, Virus, CCK-8 Assay, Membrane, Concentration Assay, Modification, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation, Extraction, Bicinchoninic Acid Protein Assay, Staining, RNA Extraction, H&E Stain, Viability Assay, Reporter Assay, Labeling, Western Blot, Sequencing, Control, Software

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