Journal: Cell reports

Article Title: Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer.

doi: 10.1016/j.celrep.2025.115267

Figure Lengend Snippet: Figure 4. Hypoxia-driven, CTCF-mediated inclusion of exon64A in COL5A1 mRNA pro- motes EMT in breast cancer cells (A) Schematic showing sgRNA sequence target- ing the CTCF motif present immediately down- stream of exon64A. (B and C) CTCF and RNA Pol II ChIP-qPCR on COL5A1 exon64A (B) and RT-qPCR analysis of COL5A1 exon64A and exon64B isoforms normalized to RPS16 and constitutive exon expression levels (C) in MCF7 cells transduced with CRISPR-Cas9-sgRNAs specific against CTCF motif immediately downstream of exon64A in comparison to the sgcontrol MCF7 cells under hypoxia. (D) Immunoblot of GFP (to confirm overexpression of COL5A1 isoforms), E-cad, and vimentin in MCF7 cells with empty vector (EV), COL5A1exon64A, or COL5A1exon64B ectopically expressed in nor- moxic MCF7 cells. (E and F) Invasion assay (scale bars, 200 mm) (E) with its quantification as a percentage of cells invaded (F) after overexpression of EV, COL5A1exon64A, or COL5A1exon64B in MCF7 cells under hypoxia. (G and H) Representative images (scale bars, 275 mm) (G) with their quantification (H) of 3D spheroid invasion assay in MCF7 cells over- expressing EV, COL5A1exon64A, or COL5A1ex- on64B spheroids under normoxic conditions. (I and J) Invasion assay (scale bars, 200 mm) (I) with its quantification (J) after overexpression of EV, COL5A1exon64A, or COL5A1exon64B iso- forms in CTCF-depleted MCF7 cells in compari- son to the shControl cells under hypoxia. Error bars, mean ± SEM; two-tailed t test; *p < 0.05, **p < 0.01, and ***p < 0.001; n = 3 biological replicates.

Article Snippet: Additionally, Addgene #71830 plasmid having non-targeting (NT) control guide RNAs (sgcontrol) was used as a control for dCas9-DNMT3A system.

Techniques: Sequencing, ChIP-qPCR, Quantitative RT-PCR, Expressing, Transduction, CRISPR, Comparison, Western Blot, Over Expression, Plasmid Preparation, Invasion Assay, Two Tailed Test

Journal: Cell reports

Article Title: Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer.

doi: 10.1016/j.celrep.2025.115267

Figure Lengend Snippet: Figure 5. Targeted methylation of CTCF binding site on exon64A by dCas9-DNMT3A blocks CTCF enrichment and leads to reduced inclusion of exon64A in COL5A1 mRNA and EMT under hypoxia (A) Schematic representation of targeting the pu- tative CTCF binding sites immediately down- stream of COL5A1 exon64A by dCas9-DNMT3A with specific sgRNAs to maintain methylation un- der hypoxia to abolish CTCF enrichment. (B) RT-qPCR of COL5A1 exon64A and exon64B in MCF7 cells transfected with dCas9-DNMT3A- sgRNAs or sgcontrol under hypoxia. (C and D) MeDIP-qPCR (C), CTCF-ChIP-qPCR and RNA Pol II ChIP-qPCR (D) in MCF7 cells transfected with either dCas9-DNMT3A-sgcontrol or dCas9-DNMT3A-sg3 under hypoxic condition. (E and F) Invasion assay (scale bars, 200 mm) (E) with its quantification (F) in MCF7 cells trans- fected with either dCas9-DNMT3A-sgcontrol or dCas9-DNMT3A-sg3 under hypoxic condition. (G and H) Representative images (scale bars, 275 mm) with their quantification (H) of 3D spheroid invasion assay in MCF7 cells transfected with either dCas9-DNMT3A-sgcontrol or dCas9- DNMT3A-sg3 under hypoxic condition. Error bars, mean ± SEM; two-tailed t test; *p < 0.05, **p < 0.01, and ***p < 0.001; n = 3 bio- logical replicates.

Article Snippet: Additionally, Addgene #71830 plasmid having non-targeting (NT) control guide RNAs (sgcontrol) was used as a control for dCas9-DNMT3A system.

Techniques: Methylation, Binding Assay, Quantitative RT-PCR, Transfection, Methylated DNA Immunoprecipitation, ChIP-qPCR, Invasion Assay, Two Tailed Test