Journal: Science Advances

Article Title: Targeting SUV39H2 exon skipping reverses resistance to CDK4/6 inhibitors via transcriptional activation of p21

doi: 10.1126/sciadv.aea2001

Figure Lengend Snippet: ( A ) MCF7-PALR and MCF7-ABER cells were treated with Ctr-ASO-M or SUV-ASO-Ms (0.1 μM). CCK8 assays reveal the cell growth under PAL (0.5 μM) or ABE (0.1 μM) treatment ( N = 3 biological replicates). ( B ) CCK8 assays calculating IC 50 values of SUV-ASO-Ms in MCF7-TAMR, MCF7-PALR, and MCF7-ABER cells ( N = 3 biological replicates). ( C ) Synergy Finder is calculating ZIP scores in MCF7-TAMR to study the synergistic effect between SUV-ASO-Ms and CDK4/6i (PAL and ABE). ( D and E ) MCF7-PALR and MCF7-ABER cells were treated with Ctr-ASO-M or SUV-ASO-Ms (0.1 μM) alone or in combination with CDK4/6i (PAL, 0.5 μM and ABE, 0.1 μM). Flow cytometry analysis (D) and β-Gal activity assays (20× magnification) (E) showing the cell cycle distribution and senescence of the cells. ( F ) Schematic depicting the experimental design of the MCF7-ABER xenograft tumors conducted to assess the efficacy of SUV-ASO2-M or a combination of SUV-ASO2-M with ABE in vivo. ( G to I ) After being treated with Ctr-ASO-M or SUV-ASO2-M in combination with Veh or ABE, the tumor growth curves (G), the harvested tumors (H), and the tumor weight (I) were measured. ( J and K ) RT-qPCR and Western blotting detection of SUV39H2 mRNA (J) and protein (K) in tumors from xenografts. ( L ) IHC staining of p21 in tumors from xenografts. H scores were calculated for p21 in all samples. Data were presented as means ± SD [(A to E), N = 3 biological replicates; (G), (I), (J) to (L), N = 5 biological replicates]. The P values were calculated by one-way ANOVA followed by Tukey’s test for multiple-group comparison.

Article Snippet: The cell proliferation was detected by the CCK8 kit (Dojindo, CK04).

Techniques: Flow Cytometry, Analysis, Activity Assay, Xenograft Assay, In Vivo, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Comparison

Journal: Science Advances

Article Title: Targeting SUV39H2 exon skipping reverses resistance to CDK4/6 inhibitors via transcriptional activation of p21

doi: 10.1126/sciadv.aea2001

Figure Lengend Snippet: ( A ) H3K9me3 signals (CUT&Tag) around promoters and enhancers in MCF7-ABER cells treated with Ctr-ASO-M or SUV-ASO2-M (0.1 μM). ( B ) RAD21 signals (ChIP-seq) around enhancers in MCF7 cells ( GSE25021 and GSE72082 datasets). ( C ) RAD21 signals (ChIP-seq) around SUV39H2-dependent and independent H3K9me3 peaks. ( D ) A volcano plot of DEGs between the RAD21 overexpression and the control group (MCF7-ABER). ( E ) GSEA of transcriptome between RAD21 overexpression and the control. ( F and G ) Western blotting (F) and RT-qPCR (G) detection of RAD21 and p21 protein levels, and p21 mRNA levels in MCF7-PALR and MCF7-ABER cells with RAD21 overexpression. ( H ) IGV visualization of H3K9me3, RAD21, STAG1, and SMC1A signals in the p21 promoter ( p21 -Pro) and distal enhancer ( p21 -SE). Blue box: cohesin binding site; gray box: differential H3K9me3 peak. ( I and J ) ChIP-qPCR of H3K9me3 (I) and RAD21 (J) in p21 -Pro and p21 -SE of MCF7-PALR and MCF7-ABER cells treated with Ctr-ASO-M or SUV-ASO2-M (0.1 μM). ( K ) ChIP-qPCR of SUV39H2 in p21 -Pro and p21 -SE of MCF7-PALR and MCF7-ABER cells. ( L ) Western blotting of RAD21, SUV39H2, and p21 protein levels in MCF7-ABER cells transfected with Ctr siRNA or RAD21 siRNA and then treated with Ctr-ASO-M or SUV-ASO2-M (0.1 μM). ( M ) MCF7-ABER cells were transfected with Ctr siRNA or RAD21 siRNA1. CCK8 assays showing cell growth in cells treated with Ctr-ASO-M or SUV-ASO2-M (0.1 μM) in combination with ABE (0.1 μM). ( N ) MCF7-ABER cells were transfected with Ctr siRNA or RAD21 siRNA1. β-Gal activity assays (20× magnification) revealing the senescent cells treated with Ctr-ASO-M or SUV-ASO2-M (0.1 μM). In (F), (G), (I) to (N), data were presented as means ± SD ( N = 3 biological replicates). The P values were calculated using a two-tailed unpaired Student’s t test for two-group comparison and one-way ANOVA followed by Tukey’s test for multiple-group comparisons.

Article Snippet: The cell proliferation was detected by the CCK8 kit (Dojindo, CK04).

Techniques: ChIP-sequencing, Over Expression, Control, Western Blot, Quantitative RT-PCR, Binding Assay, ChIP-qPCR, Transfection, Activity Assay, Two Tailed Test, Comparison