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: Antibodies against SUV39H2 (11338-1-AP), p21 (10355-1-AP), glyceraldehyde-3-phosphate dehydrogenase (60004-1-Ig), SERPINE1 (13801-1-AP), PLK2 (15956-1-AP), ESRP1 (21045-1-AP), UPF1 (23379-1-AP), anti-mouse (SA00001-1), and anti-rabbit secondary antibodies (SA00001-2) were products from Proteintech.

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 ) Average distribution of ATAC-seq signal around promoters (±3 kb from TSS) and enhancers in MCF7-ABER cells treated with Ctr-ASO-M or SUV-ASO2-M (0.1 μM). ( B ) A volcano plot comparing Footprint TF activity between MCF7-ABER cells treated with Ctr-ASO-M or SUV-ASO2-M. ( C ) Footprint TF activity of NFYA between MCF7-ABER cells treated with Ctr-ASO-M or SUV-ASO2-M. ( D ) A volcano plot of DAPs between MCF7-ABER cells treated with Ctr-ASO-M and SUV-ASO2-M. ( E ) Category of DAPs according to their status (open or closed) and location (proximal or distal from TSS) in the genome. ( F ) Overlapping of up-regulated DEGs and open DAPs in MCF7-ABER cells treated with SUV-ASO2-M. The overlapped genes were analyzed using KEGG enrichment. ( G ) A heatmap of the overlapped genes enriched in the p53 pathway, senescence, or both processes. ( H ) IGV visualization of DAPs in p21 , GADD45A , and SERPINE1 of MCF7-ABER cells treated with Ctr-ASO-M or SUV-ASO2-M.

Article Snippet: Antibodies against SUV39H2 (11338-1-AP), p21 (10355-1-AP), glyceraldehyde-3-phosphate dehydrogenase (60004-1-Ig), SERPINE1 (13801-1-AP), PLK2 (15956-1-AP), ESRP1 (21045-1-AP), UPF1 (23379-1-AP), anti-mouse (SA00001-1), and anti-rabbit secondary antibodies (SA00001-2) were products from Proteintech.

Techniques: Activity Assay

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: Antibodies against SUV39H2 (11338-1-AP), p21 (10355-1-AP), glyceraldehyde-3-phosphate dehydrogenase (60004-1-Ig), SERPINE1 (13801-1-AP), PLK2 (15956-1-AP), ESRP1 (21045-1-AP), UPF1 (23379-1-AP), anti-mouse (SA00001-1), and anti-rabbit secondary antibodies (SA00001-2) were products from Proteintech.

Techniques: ChIP-sequencing, Over Expression, Control, Western Blot, Quantitative RT-PCR, Binding Assay, ChIP-qPCR, Transfection, Activity Assay, Two Tailed Test, 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 ) Volcano plots of the differentially expressed RBPs between EndoR and EndoR/PalR cells established from MCF7 and T47D cells ( GSE150997 ). ( B ) Overlapping of down-regulated RBPs between EndoR and EndoR/PalR cells established from MCF7 and T47D cells ( GSE150997 ). ( C ) Western blotting detection of ESRP1 and SUV39H2 protein levels in MCF7 and T47D cells transfected with Ctr siRNA or ESRP1 siRNA. ( D ) Western blotting detection of ESRP1 protein levels in MCF7-TAMR, MCF7-PALR, and MCF7-ABER cells. ( E and F ) RT-qPCR detection of SUV39H2 mRNA levels (E) and RT-PCR detection of SUV39H2 exon 2 (F) in MCF7 and T47D cells transfected with Ctr siRNA or ESRP1 siRNA. ( G ) Conservation analysis of the putative ESRP1-binding site in SUV39H2 exon 2. ( H ) Minigene assays of MCF7-ABER cells transfected with Ctr siRNA or ESRP1 siRNA in combination with pSPL3-SUV-W or pSPL3-SUV-M. RT-PCR was performed to detect SUV39H2 exon 2 in each group. ( I ) RIP-qPCR detection of ESRP1 binding to SUV39H2 mRNA in MCF7 and T47D cells. ( J ) Model illustrating the antitumor function of SUV-ASO2-M in CDK4/6i-resistant breast cancer cells. Free-uptake of SUV-ASO2-M promotes RNase H–dependent degradation of SUV39H2 RNA and skipping of SUV39H2 exon 2, resulting in decreased SUV39H2 protein expression in CDK4/6i-resistant breast cancer cells. Consequently, the H3K9me3 mark near the p21 distal enhancer is removed, followed by binding of the cohesin complex and interaction between the distal enhancer and the promoter of p21 . Next, the chromatin accessibility of the p21 promoter is increased to drive transcriptional activation of p21 . In (C) to (F) and (H) to (I), data were presented as means ± SD ( N = 3 biological replicates). The P values were calculated by 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: Antibodies against SUV39H2 (11338-1-AP), p21 (10355-1-AP), glyceraldehyde-3-phosphate dehydrogenase (60004-1-Ig), SERPINE1 (13801-1-AP), PLK2 (15956-1-AP), ESRP1 (21045-1-AP), UPF1 (23379-1-AP), anti-mouse (SA00001-1), and anti-rabbit secondary antibodies (SA00001-2) were products from Proteintech.

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Analysis, Binding Assay, Expressing, Activation Assay, Two Tailed Test, Comparison

Journal: Aging Cell

Article Title: Ghrelin Alleviates Aging‐Related Cognitive Impairment by Regulating Autophagy‐Related Signaling via the SHBG / JNK1 /Beclin1 Axis in Microglia

doi: 10.1111/acel.70732

Figure Lengend Snippet: The information on the antibody.

Article Snippet: p21 , 1:2000 , 10355‐1‐AP , Rabbit , Proteintech , USA.

Techniques: