Journal: Epigenetics & Chromatin

Article Title: The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

doi: 10.1186/s13072-022-00462-7

Figure Lengend Snippet: High-confidence interaction partners of ZFP57 in mouse ESCs. a Western blot analysis of ZFP57 and KAP1 in wild-type (WT), Zfp57 -AviTag-transfected and Zfp57 -/- ESCs. Note that AviTag-ZFP57 migrates as a 60 kDa band while the endogenous ZFP57 migrates as a 50 kDa-band. b Experimental workflow of the tagged protein-mass spectrometry approach used for identification of the ZFP57 interactors. The Zfp57 cDNA was cloned into the expression vector pEF6-Avitag-GGGx2-Avitag. Zfp57 -AviTag was transfected in stably BirA-expressing mouse ESCs and biotin-labelled pulled proteins were pulled down using streptavidin. Precipitated protein complexes were digested with trypsin for LC–MS/MS analysis. Proteins in common between Zfp57 -AviTag-transfected and Mock-transfected BirA-expressing E14 ESCs were excluded from further analyses. Images of petri dishes and eppendorf tubes are taken from https://doi.org/10.7875/togopic.2020.104 and https://doi.org/10.7875/togopic.2022.115 , respectively. BirA is depicted in light blue, biotin in red, ZFP57 in dark blue and ZFP57-interactors in grey, green, and olive. Zfp57 -AviTag-transfected and mock-transfected BirA-expressing E14 ESCs are depicted in red and green, respectively. c Chord diagram showing enriched GO clusters of ZFP57 interactors. The proteins identified by LC–MS/MS analysis ordered according to their relative enrichment are shown on the left, and the enriched GO clusters are indicated with different colours on the right. The number of peptides by which proteins were recognized by LC–MS/MS analysis is displayed in gradient red (2–20 peptides). d Western blot of proteins pulled down with streptavidin in WT and Zfp57 -AviTag-transfected ESCs and revealed with anti-MSH2 antibody. e Western blot of proteins pulled down with streptavidin in Msh2 -AviTag-transfected ESCs and revealed with anti-KAP1 antibody. f Western blot of proteins immunoprecipitated with anti-KAP1 antibody in WT and Zfp57-/- ESCs and revealed with anti-MSH2 antibody. Input corresponds to 1% of the cell lysate used for immunoprecipitation

Article Snippet: Streptavidin beads (Dynabeads MyOne streptavidin T1, ref 65,601, Invitrogen) and anti-MSH6 (Santa Cruz sc-137015), anti-H3K9me3 (Abcam 8898), anti-H3K4me3 (Abcam ab8580) antibodies or rabbit/mouse IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel.

Techniques: Western Blot, Transfection, Mass Spectrometry, Clone Assay, Expressing, Plasmid Preparation, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation

Journal: Epigenetics & Chromatin

Article Title: The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex

doi: 10.1186/s13072-022-00462-7

Figure Lengend Snippet: Parental origin-specific binding of MSH2 and MSH6 to the ICRs. a Box plot showing the methylation level of non-repetitive MSH2-bound regions (left) and MSH2-bound ICRs (right). b Electropherograms showing the DNA methylation level of the Inpp5f and Gnas ICRs in genomic DNA, ChIP DNA obtained with anti-H3K9me3, anti-H3K4me3 antibodies and Bio-ChIP DNA pulled down with streptavidin in Msh2 -AviTag-transfected ESCs, and revealed by bisulfite sequencing. Methylation levels were determined from the ratio between methylated (C) and unmethylated (T) cytosines. c Electropherograms showing the allele-specific binding of MSH6 in hybrid WT and Zfp57 -/- ESCs at the Plagl1 and Inpp5f ICRs. The SNVs associated with the maternal JF1 or paternal Black/6 (B6) alleles are shown at the top. The relative enrichment of the maternal over the paternal allele is reported below the electropherogram. Black arrows indicate the SNVs. d Bar plot showing relative enrichment of Biotin-tagged MSH2 protein to ICRs in WT and Zfp57 -/- ESCs. ChIP values were expressed as % input and normalized to the Smg7 region that is used as a positive control

Article Snippet: Streptavidin beads (Dynabeads MyOne streptavidin T1, ref 65,601, Invitrogen) and anti-MSH6 (Santa Cruz sc-137015), anti-H3K9me3 (Abcam 8898), anti-H3K4me3 (Abcam ab8580) antibodies or rabbit/mouse IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel.

Techniques: Binding Assay, Methylation, DNA Methylation Assay, Transfection, Methylation Sequencing, Positive Control

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: CircEZH2 interacts with m 6 A reader IGF2BP2 and blocks its ubiquitination-dependent degradation. A RNA pulldown was performed using biotin-labeled circEZH2 probe, followed by mass spectrometry analysis. B RNA pulldown assay was performed to verify the interaction between circEZH2 and IGF2BP2 in HCT116 and SW620 cells. C RBP immunoprecipitation (RIP) assay was performed using IGF2BP2 or IgG antibodies, followed by qRT-PCR assay for circEZH2 expression in HCT116 and SW620 cells. D Western blot was performed to detect the expression of IGF2BP2 in negative control (NC) and circEZH2-depleted (shRNA-1 and shRNA-2) HCT116 and SW620 cells. Hsp 70 served as a loading control. E qRT-PCR assay was performed to detect the mRNA levels of IGF2BP2 in negative control and circEZH2-depleted HCT116 and SW620 cells. F CircEZH2 knockdown accelerated the ubiquitination modification of IGF2BP2 in HCT116 cells transfected with ubiquitin (Ub) and treated with MG-132, which was detected by Western blot. G IHC staining of IGF2BP2 in tumors from control and circEZH2-OE AOM/DSS mice. Scale bar = 25 μm. H The mRNA level of IGF2BP2 in CRC ( n = 275) and adjacent normal ( n = 349) tissues was analyzed using TCGA database. I IHC staining of IGF2BP2 in CRC and adjacent normal tissues. Scale bar = 50 μm. J H score of IGF2BP2 in CRC and adjacent normal tissues ( n = 124). K Spearman’s correlation analysis revealed a positive association between IGF2BP2 protein expression and circEZH2 expression in CRC tissues (r = 0.4886; P < 0.01). L Kaplan-Meier analysis (log-rank test) of overall survival according to IGF2BP2 expression level ( P < 0.01). L Data were showed as mean ± SD. # P > 0.05, ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Ubiquitin Proteomics, Labeling, Mass Spectrometry, Immunoprecipitation, Quantitative RT-PCR, Expressing, Western Blot, Negative Control, shRNA, Control, Knockdown, Modification, Transfection, Immunohistochemistry

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: IGF2BP2 is a direct target of miR-133b in CRC. A Schematic illustration of IGF2BP2 3′-UTR wild-type (WT) and miR-133b binding site mutated (Mut) IGF2BP2 3′-UTR luciferases reporter vectors. B (Left panel) Relative luciferase activities were determined in HCT116 and SW620 cells transfected with IGF2BP2 3′-UTR WT or Mut luciferase reporter vectors and miR-133b or NC mimics. ( Right panel ) Relative luciferase activities were determined in HCT116 and SW620 cells transfected with IGF2BP2 3′-UTR WT or Mut luciferase reporter vectors and miR-133b inhibitor or inhibitor NC mimics. C IGF2BP2 mRNA expression was determined by qRT-PCR in HCT116 and SW620 cells transfected with miR-133b or NC mimics. D IGF2BP2 protein expression was determined by Western blot in HCT116 and SW620 cells transfected with miR-133b (50 nM and 100 nM) or NC mimics. Hsp 70 served as a loading control. E IGF2BP2 protein expression was determined by Western blot in control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE-treated HCT116 and SW620 cells. Hsp 70 served as a loading control. F-J CCK-8, plate colony-formation, EdU incorporation, wound healing and transwell assays were performed to determine the proliferation and migration abilities of control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE-treated HCT116 and SW620 cells. K Image of subcutaneous tumor xenografts in control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE groups. L The tumor growth curves of xenografts were plotted in control, miR-133b-OE, miR-133b-OE + IGF2BP2-OE-treated HCT116 groups. M The tumor weights of xenografts were evaluated. Data were showed as mean ± SD. ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Binding Assay, Luciferase, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, CCK-8 Assay, Migration

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: CircEZH2/IGF2BP2 enhances the stability of CREB1 mRNA in CRC. A MeRIP-seq (accession number: GSE179042) analysis was performed to discover the potential m 6 A modification profile in CRC tissues. m 6 A sites were displayed in 5′-UTR, CDS and 3′-UTR. B Schematic diagram based on MeRIP-seq showed the remarkable m 6 A modification site in the mRNA of CREB1. C qRT-PCR assay was performed to detect the expression of RGMA, FCGR1A, B4GALT3, NEK9, CLDN2, RTN4RL1, ZNF74, CENPL, RAB44 and CREB1 mRNAs in negative control (NC) and circEZH2-depleted HCT116 and SW620 cells. D Symbol showed the m 6 A motif in CRC tissues. E Stability of CREB1 mRNA was assessed by RNase R treatment followed by qRT-PCR in HCT116 and SW620 cells. F RBP immunoprecipitation (RIP) assay demonstrated the CREB1 mRNA enrichment precipitated by anti-IgG or anti-IGF2BP2 antibodies in negative control and circEZH2-depleted HCT116 and SW620 cells. G RIP assays demonstrated the CREB1 mRNA levels precipitated by anti-IgG or anti-m 6 A antibodies in NC and circEZH2 shRNA, control and miR-133-OE and NC and IGF2BP2 siRNA HCT116 cells. H Relative expression of CREB1 mRNA in CRC tissues and matched adjacent normal tissues was analyzed by qRT-PCR assays ( n = 124). I Spearman’s correlation analysis revealed a positive association between CREB1 mRNA expression and circEZH2 expression in CRC tissues (r = 0.5903; P < 0.01). J IHC staining of CREB1 in CRC and adjacent normal tissues. Scale bar = 50 μm. K H score of CREB1 in CRC and adjacent normal tissues ( n = 124). L Spearman’s correlation analysis revealed a positive association between CREB1 protein expression and circEZH2 expression in CRC tissues (r = 0.2831; P < 0.01). M The expression levels of miR-133b and CREB1 in tumors from control and circEZH2-OE AOM/DSS models were determined by qRT-PCR assay. N IHC staining of CREB1 in tumors from control and circEZH2-OE AOM/DSS mice. Scale bar = 25 μm. Data were showed as mean ± SD. # P > 0.05, ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Modification, Quantitative RT-PCR, Expressing, Negative Control, Immunoprecipitation, shRNA, Control, Immunohistochemistry

Journal: Molecular Cancer

Article Title: CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m 6 A-modified CREB1 mRNA

doi: 10.1186/s12943-022-01608-7

Figure Lengend Snippet: CircEZH2 aggravates CRC progression through modulating CREB1 expression. A CREB1 mRNA expression was determined by qRT-PCR in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 and SW620 cells. B CREB1 protein expression was determined by Western blot in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 and SW620 cells. C-G CCK-8, plate colony-formation, EdU incorporation, wound healing and transwell assays were performed to determine the proliferation and migration abilities of control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 and SW620 cells. H Image of subcutaneous tumor xenografts in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 groups. I The tumor growth curves of xenografts were plotted in control, circEZH2_shRNA and circEZH2_shRNA + CREB1-OE-treated HCT116 groups. J Schematic illustration indicates the mechanism by which circEZH2/miR-133b/IGF2BP2/CREB1 axis aggravates CRC progression. Data were showed as mean ± SD. ** P < 0.01

Article Snippet: The membranes were incubated with primary antibodies against human IGF2BP2 (Proteintech, 11,601–1-AP; 1: 3000), CREB1 (Proteintech, 12,208–1-AP; 1: 1000), Cyclin A2 (Proteintech, 18,202–1-AP; 1: 1000), MMP-9 (Proteintech, 10,375–2-AP; 1: 1000), GLUT3 (Proteintech, 20,403–1-AP; 1: 1000), BCL-2 (Proteintech, 12,789–1-AP; 1: 2000), GAPDH (Proteintech, 60,004–1-Ig; 1: 20000) or Hsp 70 (Proteintech, 10,995–1-AP; 1: 10000) at 4 °C overnight and followed by a 1 h incubation at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (Proteintech, SA00001–2; 1: 50000) or anti-mouse secondary antibody (Proteintech, SA00001–1; 1: 100000).

Techniques: Expressing, Quantitative RT-PCR, Control, shRNA, Western Blot, CCK-8 Assay, Migration