Journal: Advanced Science

Article Title: Topoisomerase I Inhibition in ETV4‐overexpressed Non‐Small Cell Lung Cancer Promotes Replication and Transcription Mediated R‐Loop Accumulation and DNA Damage

doi: 10.1002/advs.202409307

Figure Lengend Snippet: ETV4 is located at the origins of LAMB2 and MCM4 DNA replication and plays a role in ORC loading at the origins of NSCLC cells. A) Gene body enrichment heat map of anti‐Flag ChIP‐seq signals in A549‐shETV4 cells transfected with Flag‐ETV4 plasmids. B) Pie chart demonstrating the percentage of ETV4 peak distribution. C) Functional analysis showing the top KEGG pathways significantly associated with genes of ETV4 binding by ChIP‐seq assay. D,E) IGV tracks showing the co‐occupancy of ETV4 and c‐MYC with the chromatin accessibility at the LAMB2 origin and MCM4 origin (‐1Kb) regions from our ETV4 ChIP‐seq, MYC ChIP‐seq (ENCSR537FDJ), and ATAC‐seq (ENCSR220ASC) from A549 cells. F,G) ChIP‐qPCR assay showing the binding of ETV4 at LAMB2 origin and MCM4 origin regions in A549‐shETV4 cells transfected with Flag‐ETV4 plasmids (mean ± SD, n = 3; two‐tailed unpaired t ‐test). * p < 0.05, ** p < 0.01. H,I) IP of ETV4 and Immunoblots against ORC1‐6 subunits from whole‐cell proteins or chromatin‐bound (Chrom.) proteins of A549 cells. J) Predicting model of the structural arrangement of ETV4 and ORC by superimposing the structure of ETV4 (PDB code 4co8) with the ORC complex (PDB code 7mca). dsDNA, double‐strand DNA. K,L) Schematic of the protocol for cells synchronized and released using a double thymidine block. The cell cycle distribution was detected by Flow cytometry. M) IP of ETV4 and Immunoblots against ORC1 and ORC6 from thymidine block and released synchronized A549 cells. N,O) Quantification of ETV4‐ORC1 or ORC6 binding in M. P) ChIP‐qPCR assay showing the binding of ORC1 at LAMB2 and MCM4 origins in A549 cells transfected with ORC6 siRNA or NC siRNA. (mean ± SD, n = 3; two‐tailed unpaired t ‐test) * p < 0.05, ** p < 0.01. Q) ChIP‐qPCR assay showing the binding of ORC1 at LAMB2 and MCM4 origins in A549‐shETV4 cells transfected with ETV4 plasmids or combined with ORC6 siRNA, respectively. (mean ± SD, n = 3; one‐way ANOVA followed by Tukey's multiple comparisons test). * p < 0.05, ** p < 0.01.

Article Snippet: Blots were blocked with 5% nonfat milk in Tris‐Buffered Saline and Tween 20 (TBST), and incubated with antibodies specific for ETV4 (10684‐1‐AP, Proteintech), MCM2 (3619, CST), MCM3 (15597‐1‐AP, Proteintech), MCM4 (13043‐1‐AP, Proteintech), MCM5 (11703‐1‐AP, Proteintech), MCM6 (13347‐2‐AP, Proteintech), MCM7 (11225‐1‐AP, Proteintech), MCM10 (12251‐1‐AP, Proteintech), ORC1 (NBP100‐121, Novus), ORC2 (12739‐1‐AP, Proteintech), ORC3 (sc‐374231, Santa Cruz), ORC4 (13026‐1‐AP, Proteintech), ORC5 (11542‐1‐AP, Proteintech), ORC6 (17784‐1‐AP, Proteintech), Histone‐H3 (17168‐1‐AP, Proteintech), PCNA (13110, CST), phospho‐MCM2 S40 (ab133243, Abcam), CDC45 (15678‐1‐AP, Proteintech), SUPT16H (28598‐1‐AP, Proteintech), SSRP1 (15696‐1‐AP, Proteintech), γ‐H2AX (ET‐1602, HUABio), Flag (14793, CST), His (66005‐1‐Ig, Proteintech), HA (sc‐7392, Santa Cruz), GST (66001‐2‐lg, Proteintech), GAPDH (60004‐1‐Ig, Proteintech) and β‐actin (66009‐1‐Ig, Proteintech) was used as a loading control for Western blot.

Techniques: ChIP-sequencing, Transfection, Functional Assay, Binding Assay, ChIP-qPCR, Two Tailed Test, Western Blot, Blocking Assay, Flow Cytometry

Journal: Biomedicines

Article Title: Tephrosin Suppresses the Chemoresistance of Paclitaxel-Resistant Ovarian Cancer via Inhibition of FGFR1 Signaling Pathway

doi: 10.3390/biomedicines11123155

Figure Lengend Snippet: The effect of tephrosin on downregulation of XIAP expression and independent intracellular reactive oxygen species levels in SKOV3-TR cells. ( A ) SKOV3-TR cells (5.0 × 10 3 ) were seeded in 96-well plates for 24 h. Cells were then treated with serially diluted paclitaxel (0–500 nM) with N-acetylcysteine (NAC) (5 mM), tephrosin (10 µM), and combination treatment with tephrosin and NAC for 48 h. Cell viability was measured using the WST-1 assay. ( B ) Cells (5.0 × 10 4 ) were seeded in 24-well plates. After 24 h of incubation, cells were treated with paclitaxel (0, 25, 50, 100, 200, and 400 nM), with NAC (5 mM), tephrosin (10 µM), and combination treatment with tephrosin and NAC for 48 h. The visualization of cell viability was determined using the crystal violet assay. ( C ) Cells were treated with paclitaxel (200 nM), tephrosin (10 µM), and combination treatment with paclitaxel and tephrosin for 48 h. Immunoblotting was performed to analyze the expressions of BCL-2, BCL-XL, MCL-1, BAX, Survivin, XIAP, caspase 9, caspase 3, cleaved caspase 3, and cleaved PARP. Actin was used as a loading control. Protein expression levels were quantified relative to the control, following normalization to the corresponding expression of actin using ImageJ software. ( D ) Cells were treated with paclitaxel and tephrosin as shown in ( C ), and the mRNA expressions of BCL-2, BCL-XL, MCL-1, BAX, XIAP, and SURVIVIN were analyzed using RT-PCR tests. GAPDH was used as a loading control. ( E ) Relative quantifications of BCL-2, BCL-XL, MCL-1, BAX, XIAP, and SURVIVIN were analyzed using RT-qPCR tests, and the mRNA relative expression was normalized using GAPDH . All experiments were repeated three times. Significant differences were calculated using one-way ANOVA. * p ≤ 0.05, as analyzed using the concentration, was considered significant, and p > 0.05 indicated no significant (ns) difference. PTX, paclitaxel; Teph, tephrosin; NAC, N-acetylcysteine.

Article Snippet: Antibodies against Actin (sc-47778), FRS2 (sc-17841), XIAP (sc-55551), and caspase 9 (sc-17784) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

Techniques: Expressing, WST-1 Assay, Incubation, Crystal Violet Assay, Western Blot, Control, Software, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay