Journal: Reproductive biology and endocrinology : RB&E

Article Title: KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation.

doi: 10.1186/s12958-023-01129-y

Figure Lengend Snippet: Fig. 1 The expression and subcellular localization of KRT18 in preimplantation embryos. A-F Embryos at the 2-cell, 4-cell, 8-cell, morula and blastocyst stages were harvested for immunofluorescence staining. Anti-KRT18 antibody (green), phalloidin (red) and Hoechst (blue) were used to determine the expression of the corresponding proteins. A-E The expression level of KRT18 gradually increased during embryo development. KRT18 signalling was clearly observed in blastocysts trophoblasts. F A negative control experiment was performed by replacing the anti-KRT18 antibody with IgG. Scale bar, 20 µm

Article Snippet: KRT18 fusion protein (Ag1260) and E-cadherin fusion protein (Ag15085) were purchased from Proteintech (Wuhan, Hubei, P.R.C.). siRNA transfection and lentivirus infection Control siRNA and KRT18 siRNA were obtained from Shanghai GenePharma (Shanghai, China).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control

Journal: Reproductive biology and endocrinology : RB&E

Article Title: KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation.

doi: 10.1186/s12958-023-01129-y

Figure Lengend Snippet: Fig. 2 Knockdown of KRT18 impairs mouse embryo adhesion and implantation. A-C Knockdown of KRT18 with lentivirus reduced KRT18 expression at the mRNA (A) and protein levels (B) and (C). The histogram shows the quantitative analysis of western blotting (C). D Schematics of the experimental mouse embryo adhesion and in vivo implantation process. E Representative picture of embryos treated with lentivirus for 48 h. Scale bar, 20 µm. F Representative picture of embryos that adhered to a monolayer of Ishikawa cells. Scale bar, 50 µm. G The attachment rate of the mouse embryos was plotted as a histogram. *, p < 0.05. Blastocysts treated with control or siKRT18-2 lentivirus were transferred into pseudopregnant mice. Seventy-two hours after transfer, the number of implantation sites in the uteri was counted. (H) Representative picture of implantation sites in the uteri. I The number of implantation sites in the uteri with transferred embryos treated with control or siKRT18-2 lentivirus (n = 6). The data are presented as the mean ± SE. **P < 0.01

Article Snippet: KRT18 fusion protein (Ag1260) and E-cadherin fusion protein (Ag15085) were purchased from Proteintech (Wuhan, Hubei, P.R.C.). siRNA transfection and lentivirus infection Control siRNA and KRT18 siRNA were obtained from Shanghai GenePharma (Shanghai, China).

Techniques: Knockdown, Expressing, Western Blot, In Vivo, Control

Journal: Reproductive biology and endocrinology : RB&E

Article Title: KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation.

doi: 10.1186/s12958-023-01129-y

Figure Lengend Snippet: Fig. 3 Knockdown of KRT18 inhibits trophoblast cell migration and invasion. A and B Knockdown of KRT18 with siRNAs reduced KRT18 mRNA (A) and protein (B) (C) expression. D and E Twenty-four hours after siKRT18-3 transfection, three scratches were made onto a monolayer of HTR8/ SVneo cells, and images of the initial scratch and the scratch after 24 h were taken (D). Scale bar, 200 µm. The histogram shows the statistical results of the cell scratch assay (E). F and G HTR8/SVneo cell invasiveness was determined using Matrigel transwell assays after 24 h of siKRT18-3 transfection (F), and the histogram shows the statistical results (G). Scale bar, 100 µm. *, p < 0.05

Article Snippet: KRT18 fusion protein (Ag1260) and E-cadherin fusion protein (Ag15085) were purchased from Proteintech (Wuhan, Hubei, P.R.C.). siRNA transfection and lentivirus infection Control siRNA and KRT18 siRNA were obtained from Shanghai GenePharma (Shanghai, China).

Techniques: Knockdown, Migration, Expressing, Transfection, Wound Healing Assay

Journal: Reproductive biology and endocrinology : RB&E

Article Title: KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation.

doi: 10.1186/s12958-023-01129-y

Figure Lengend Snippet: Fig. 4 Knockdown of KRT18 impairs JEG-3 spheroid adhesion and outgrowth. A-C Knockdown of KRT18 with siRNAs reduced KRT18 expression at the mRNA (A) and protein levels (B). The histogram shows the quantitative analysis of western blotting (C). D After treatment with siKRT18-3 for 24 h, JEG-3 spheroids were generated and transferred onto a monolayer of Ishikawa cells. After centrifugation of the plate at 145 rpm for 10 min and removal of the spheroids that did not bind, the attachment rate was determined (the number of attached spheroids divided by the total number of input spheroids). The JEG-3 spheroid adhesion rates were plotted as a histogram. E and F The outgrowth ability of JEG-3 spheroids. The spheroids that attached to the Ishikawa cell monolayer were photographed at 1 h and 48 h after being transferred onto the Ishikawa cell monolayer (E). Scale bar, 100 µm. The histogram shows the outgrowth rate of the two groups (F). *, p < 0.05

Article Snippet: KRT18 fusion protein (Ag1260) and E-cadherin fusion protein (Ag15085) were purchased from Proteintech (Wuhan, Hubei, P.R.C.). siRNA transfection and lentivirus infection Control siRNA and KRT18 siRNA were obtained from Shanghai GenePharma (Shanghai, China).

Techniques: Knockdown, Expressing, Western Blot, Generated, Centrifugation

Journal: Reproductive biology and endocrinology : RB&E

Article Title: KRT18 regulates trophoblast cell migration and invasion which are essential for embryo implantation.

doi: 10.1186/s12958-023-01129-y

Figure Lengend Snippet: Fig. 5 Knockdown of KRT18 impairs the expression of E-cadherin. A and B MST results of His-E-cadherin fusion protein vs. GST-KRT18 fusion protein. The fluorescence change upon switching the laser on and off at 20% intensity is shown. C HTR8/SVneo cells were treated with the control or siKRT18 siRNA. After incubation for 48 h, the cells were harvested for immunofluorescence staining and then photographed with confocal laser scanning microscopy. Anti-KRT18 antibody (green), anti-E-cadherin antibody (red), and Hoechst (blue) were used to determine the expression of the corresponding proteins. The results shown are representative of 3 independent experiments. Scale bar, 20 µm. D Quantification of mean fluorescence intensity. KRT18 and E-cadherin fluorescence was normalized with the mean fluorescence intensity of the control group. *P < 0.05

Article Snippet: KRT18 fusion protein (Ag1260) and E-cadherin fusion protein (Ag15085) were purchased from Proteintech (Wuhan, Hubei, P.R.C.). siRNA transfection and lentivirus infection Control siRNA and KRT18 siRNA were obtained from Shanghai GenePharma (Shanghai, China).

Techniques: Knockdown, Expressing, Fluorescence, Control, Incubation, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: CBFA2T2 recognizes non-mutated or un-modified histone H3K27. A Schematic diagram of the domain structure in mouse CBFA2T2, with amino acid positions indicated. B Validation of bPPI-seq results by reconstituting active GFPs through co-expression of the GFPC-fused reading protein and GFPN-fused histones. C In vitro peptide pull-down assays using indicated histone peptides and FLAG-tagged full-length (FL) CBFA2T2 purified from insect cells ( Hi5 ) or E.coli . D Nucleosome-binding assays using indicated designer nucleosomes assembled in vitro and HA-tagged CBFA2T2 FL purified from insect cells. E - J Peptide pull-down assays using various histone peptides and recombinant proteins purified from insect cells ( Hi5 ): FLAG-CBFA2T2 FL ( E - F ), FLAG-tagged truncated fragments of CBFA2T2 ( G ), FLAG-tagged NHR2 and MYND domains of CBFA2T2 ( H ), CBFA2T2 internal deletion mutants ( I ), FLAG-CBFA2T2(aa 300-429) ( J ). K Surface Plasma Resonance (SPR) experiments showing H3.3 K27M exhibited a drastic decrease in its binding affinity to NHR2 as compared to H3.3 WT. L In vitro peptide pull-down assay performed in the presence of competing non-biotinylated histone peptides

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: Modification, Biomarker Discovery, Expressing, In Vitro, Purification, Binding Assay, Recombinant, Clinical Proteomics, Pull Down Assay

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: The NHR2 domain of CBFA2T2 interacts with histone H3.3K27. A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) of CBFA2T2 NHR2 and H3.3 WT interaction. B Predicted mode of binding between NHR2 and H3.3 WT based on Alphafold2. C 1H-15N HSQC result of apparent signal intensity decline after the addition of unmodified H3.3 peptide based on transverse relaxation optimized spectroscopy (TROSY) method. D Crystallographic structure of NHR2 (PDB: 9K22) reveals it is composed of a tetrameric α-helical bundle and crystal packing indicates its tendency to oligomerize in a head-to-tail conformation. E Peptide pull-down assays using various histone peptides and recombinant proteins purified from insect cells ( Hi5 )

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: Mass Spectrometry, Binding Assay, Spectroscopy, Recombinant, Purification

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: CBFA2T2 does not bind H3K27me3 in cells. A Heatmap analysis of ChIP-seq peaks in H3.3WT and Cbfa2t2 KO cells, ranked by CBFA2T2 peaks in H3.3WT cells. B Genome browser snapshot of selected CBFA2T2 and H3K27me3 ChIP-seq peaks in H3.3WT and Cbfa2t2 KO cells. C Aggregation plot showing the average enrichment of CBFA2T2 in H3.3WT and Cbfa2t2 KO cells. D Venn diagram analysis of CBFA2T2 bound genes and 2,370 transcriptionally up regulated genes (FC ≥ 1.5, P < 0.05) upon the knock-out of CBFA2T2. E KEGG analysis of 698 overlapped genes in Fig. 3D. F RT-qPCR analysis of mRNA levels of the indicated genes in H3.3WT and H3.3WT Cbfa2t2 KO cells. Mean ± SD ( n = 3), two-tailed t-test: * P < 0.05, ** P < 0.01, *** P < 0.001 and**** P < 0.0001. G Genome browser snapshot of CBFA2T2 target genes in H3.3WT and Cbfa2t2 KO cells

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: ChIP-sequencing, Knock-Out, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: CBFA2T2 directly regulates metabolic genes to impact H3K27me3 level. A Heatmap analysis of Spike-in normalized H3K27me3 ChIP-seq signals in H3.3WT and Cbfa2t2 KO cells. B Western-blot of the indicated proteins in H3.3WT and Cbfa2t2 KO cells. C Venn diagram analysis of upregulated genes upon CBFA2T2 KO and H3K27me3-bound genes in H3.3 WT cells. D KEGG comparisons of 698 CBFA2T2-bound up- and 522 H3K27me3-bound up-regulated genes using CompareCluster. E Abbreviated schematic of the α-KG generation and its role in regulating H3K27me3 demethylation. F Western-blot of the indicated proteins in H3.3WT, Cbfa2t2 KO and Cbfa2t2- Idh1 DKO , Cbfa2t2-Idh2 DKO , Cbfa2t2-Slc2a1 DKO , Cbfa2t2-Pfkp DKO , Cbfa2t2-Gpi1 DKO cells. G Western-blot of various H3 modifications in H3.3WT and Cbfa2t2 KO cells. H , I Western-blot of the indicated proteins in Cbfa2t2 KO and Rescue( Cbfa2t2 KO +CBFA2T2) cells

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: ChIP-sequencing, Western Blot

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: CBFA2T2 transcriptionally regulates metabolites through its binding to H3K27. A , B Western-blot of H3K27me3 in Cbfa2t2 KO and H3.3WT cells cultured under the specified concentrations of α-KG, succinate and glutamine. C , D Western-blot analysis to detect the time-course of H3K27me3 in Cbfa2t2 KO and H3.3WT cells cultured with 4.5g/L glucose. E LC-MS quantification of the α-KG/succinate ratio in the indicated cell lines. Data are mean ± SD ( n = 3), two-tailed t-test: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. F Metabolic flux analysis of cells cultured for 8 hours in medium containing 1 mg/mL U-¹³C₆-glucose. G Labeled a-KG(m+5)/succinate(m+4) and a-KG(m+4)/succinate(m+4) ratios in H3.3WT, Cbfa2t2 KO and rescued cells. Mean ± SD ( n = 3), two-tailed t test: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. H Western-blot of the indicated proteins in Cbfa2t2 KO cells rescued with CBFA2T2 WT or the binding-defective mutant(E334A+E338A). I Labeled α-KG(m+5)/succinate(m+4) and α-KG(m+4)/succinate(m+4) ratios in Cbfa2t2 KO , RescueWT( Cbfa2t2 KO +CBFA2T2) and RescueMut( Cbfa2t2 KO +CBFA2T2 E334A+E338A ) cells. Mean ± SD ( n = 4), two-tailed t-test: * P <0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. J Real-time glycolytic proton efflux rate (GlycoPER) recorded with a Seahorse XFe96 analyzer in H3.3WT, Cbfa2t2 KO , RescueWT and RescueMut cells. Mean ± SD ( n = 4-6). K , L Basal and Compensatory glycolysis derived from ( J ). Mean ± SD ( n = 4-6), two-tailed t test: * P < 0.05, ** P <0.01, *** P < 0.001 and **** P < 0.0001. M Labeled α-KG(m+5)/succinate(m+4) ratio in Cbfa2t2 KO , Cbfa2t2-Idh1 DKO , Cbfa2t2-Idh2 DKO , Cbfa2t2-Slc2a1 DKO and Cbfa2t2-Pfkp DKO cells. Mean ± SD ( n = 4), two-tailed t-test: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. N Western-blot of the indicated proteins in Cbfa2t2 KO cells treated with the specified concentrations of the KDM6A/B inhibitor GSK-J4. O Western-blot of indicated proteins in H3.3WT, Cbfa2t2 KO , Cbfa2t2-Kdm6a DKO and Cbfa2t2-Kdm6b DKO cells. P Western-blot of H3K27me3 in various cell lines with or without CBFA2T2 overexpression. Q Labeled α-KG(m+5)/succinate(m+4) and α-KG(m+4)/succinate(m+4) ratios in 293T and 293T OE-CBFA2T2 cells. Mean ± SD ( n = 4), two-tailed t-test as above

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: Binding Assay, Western Blot, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Labeling, Mutagenesis, Derivative Assay, Over Expression

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: CBFA2T2 suppresses tumorigenesis. A KEGG comparisons of up-regulated genes in Cbfa2t2 KO vs H3.3WT and down-regulated genes in Rescue( Cbfa2t2 KO +CBFA2T2) vs Cbfa2t2 KO , analyzed using CompareCluster. B Heatmap analysis of the genes (FC ≥ 1.5, P < 0.05) involved in the KEGG terms for carbon metabolism, glycolysis/gluconeogenesis, TCA cycle, and central carbon metabolism in cancer by Metware Cloud. Two biological replicates for each cell line were used. C The expression levels of tumorigenesis-related genes upon CBFA2T2 KO in tumor (GBM/LGG) and control data from TCGA and GTEx database. The box plot was generated using GEPIA2 (http://gepia2.cancer-pku.cn/). Tumor color red, Normal color grey. GBM:num(T)=163;num(N)=207, LGG:num(T)=518;num(N)=207. |Log2FC| Cut-off=1, P -value Cutoff = 0.01. D Subcutaneous injection of the indicated cell lines. E-F Volume and Weight measurements of the implanted subcutaneous tissue. Mean ± SD ( n = 6), two-tailed t-test: * P < 0.05, ** P < 0.01. G Immunohistochemistry (IHC) demonstrating levels of Ki67, IDH1 and H3K27me3 in indicated tumors. Scale bars = 50 μm

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: Expressing, Control, Generated, Injection, Two Tailed Test, Immunohistochemistry

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: CBFA2T2 regulates metabolism in H3.3K27M cells. A Heatmap analysis of HA-tagged H3.3 and H3.3K27M peaks in H3.3WT and H3.3K27M cells, ranked by the 5,486 overlapping CBFA2T2 peaks identified in both cell types. B Sequential ChIP-qPCR using the CBFA2T2 antibody followed by anti-HA beads in H3.3WT and H3.3K27M cells. Mean ± SD ( n =3), two-tailed t-test: * P < 0.05, ** P < 0.01. C Venn diagram analysis of the commonly up-regulated genes in H3.3K27M cells and Cbfa2t2 KO cells compared to H3.3WT cells (FC ≥ 1.5, P <0.05). D KEGG analysis of the 1,096 co-up regulated genes. E Heatmap analysis of the genes (FC ≥ 1.5, P < 0.05) involved in carbon metabolism, TCA cycle and glycolysis/gluconeogenesis by Metware Cloud. Two biological replicates were used for each cell lines. F Venn diagram analysis of CBFA2T2 and H3.3K27M ChIP-seq peaks in human H3.3K27M DMG SHSMU-DIPG-01 cells. G Heatmap analysis of CBFA2T2 and H3.3K27M ChIP-seq peaks in human H3.3K27M DMG SHSMU-DIPG-01 cells. H Western-blot of H3K27me3 in H3.3K27M and H3.3K27M CBFA2T2 overexpressing cells. I Heatmap analysis of Spike-in normalized H3K27me3 ChIP-seq signals in H3.3WT and Cbfa2t2 KO cells. J KEGG comparisons of up-regulated genes in H3.3K27M vs H3.3WT and down-regulated genes in CBFA2T2 OE-H3.3K27M vs H3.3K27M cells, analyzed using CompareCluster. K Heatmap analysis of the genes (FC ≥ 1.5, P -value < 0.05) involved in carbon metabolism, glycolysis/gluconeogenesis, TCA cycle, and central carbon metabolism in cancer by Metware Cloud. Two biological replicates for each cell line were used. L Western-blot of H3K27me3 in H3.3K27M, H3.3K27M OE-CBFA2T2-WT and H3.3K27M OE-CBFA2T2 Mutant E334A+E338A cells. M Labeled α-KG(m+5)/succinate(m+4) and α-KG(m+4)/succinate(m+4) ratios in H3.3K27M, H3.3K27M OE CBFA2T2-WT and H3.3K27M OE CBFA2T2-Mutant E334A+E338A cells. Mean ± SD ( n = 4), two-tailed t-test: * P < 0.05. N Western-blot of indicated proteins in SHSMU-DIPG-01 (H3.3K27M) and SHSMU-DIPG-01 (H3.3K27M) overexpressing CBFA2T2-WT and CBFA2T2-Mutant E334A+E338A cells. O Western-blot of indicated proteins in SU-DIPG-XVII(H3.3K27M) and SU-DIPG-XVII(H3.3K27M) overexpressing CBFA2T2-WT and CBFA2T2-Mutant E334A+E338A cells

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: ChIP-qPCR, Two Tailed Test, ChIP-sequencing, Western Blot, Mutagenesis, Labeling

Journal: Molecular Cancer

Article Title: CBFA2T2: a novel H3K27 reader regulating metabolism and tumor growth

doi: 10.1186/s12943-026-02593-x

Figure Lengend Snippet: Overexpression of CBFA2T2 suppresses H3.3K27M tumor growth. A Subcutaneous injection of the indicated cell lines. B , C Volume and Weight measurements of the implanted subcutaneous tissue. Mean ± SD ( n = 6), two-tailed t-test: * P < 0.05, ** P < 0.01. D Immunohistochemistry (IHC) demonstrating levels of Ki67, IDH1 and H3K27me3 in indicated subcutaneous tissue. Scale bars = 50μm. E Transwell invasion assay of SU-DIPG-XVII control and SU-DIPG-XVII overexpressing CBFA2T2. F Kaplan-Meier survival curves of NOD-SCID mice transplanted with 1 × 10⁶ SHSMU-DIPG-01 control and SHSMU-DIPG-01 overexpressing CBFA2T2 in the pons. G NOD-SCID mouse pups implanted with 2 × 10⁵ SHSMU-DIPG-01-luc(control) or SHSMU-DIPG-01-luc overexpressing CBFA2T2(CBFA2T2 OE) cells respectively. Total flux of bioluminescence values of control ( n = 10) and CBFA2T2 OE ( n = 10) mice at 57 days post-treatment. Mean ± SD, two-tailed t-test: P = 0.0143. H Western-blot of indicated proteins in SHSMU-DIPG-01 and CBFA2T2-overexpressing SHSMU-DIPG-01 cells

Article Snippet: The antibodies used are listed below: CBFA2T2(1:2000; #A303-593A, Bethyl Laboratories); H3K4me1(1:1000; #39297,Active motif); H3K4me3 (1:1000; # 9751S, Cell Signaling Technology); H3K9me1(1:1000;# ab9045,Abcam); H3K9me2(1:1000; # ab1220,Abcam); H3K9me3(1:1000;# ab8898,Abcam); H3K27ac(1:1000;# ab4729,Abcam); H3K27me1(1:1000;# 61015,Active motif); H3K27me2 (1:1000;# 9728,Cell Signaling Technology); H3K27me3 (1:1000;# 9733,Cell Signaling Technology); H3K27M (1:1000;# ab190631,Abcam); H3K36me2(1:1000;# 39255,Active motif); H3K36me3 (1:1000;# 61101,Active motif); Acetyl-H3(1:1000; # A17917, Abclonal); H3K14ac(1:1000, # A7254, Abclonal); H3 (1:6000;# ab1791,Abcam); EZH2(1:1000;# 5246,Cell Signaling Technology); SUZ12(1:1000 ;# 3737,Cell Signaling Technology); EED (1:1000;# 85322,Cell Signaling Technology); UTX (1:1000;# 33510,Cell Signaling Technology); KDM6B (1:1000;# A12763,ABclonal); IDH1 (1:1000; # 8137,Cell Signaling Technology); IDH2 (1:1000;#56439,Cell Signaling Technology); OGDH (1:1000;# 15212-1-AP, Proteintech); GLUT1/Slc2a1(1:1000;# 66290-1-IG, Proteintech); Gpi1(1:1000;# A17044,ABclonal); Pfkp(1:1000;# A20983,ABclonal); HA-Tag(1:1000;# 3724,Cell Signaling Technology); Lamin B1 (1:3000;# 66095-1-Ig, Proteintech);α-Tubulin(1:3000; # 2144,Cell Signaling Technology); Vinculin (1:2000;# 13901,Cell Signaling Technology); DYKDDDDK (FLAG ® tag)(1:3000, # SLAB0101,Smart-Lifesciences), Goat anti-Rabbit IgG Secondary Antibody HRP conjugated(1:3000; # L3012, Signalway), Goat anti-Mouse IgG Secondary Antibody HRP conjugated (1:3000;# L3032, Signalway).

Techniques: Over Expression, Injection, Two Tailed Test, Immunohistochemistry, Transwell Invasion Assay, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Reprogramming Chromosome Ends by Functional Histone Acetylation

doi: 10.3390/ijms25073898

Figure Lengend Snippet: ZSCAN4 binds to telomeres, leading to increased histone H3 acetylation. The “% Input” values represent the enrichment of ZSCAN4, Alu, or histone acetylation on a specific region from each sample: without (Dox−) or with ZSCAN4 induction (Dox+): ( A ) ZSCAN4 interaction with the telomeres was assessed by ZSCAN4-ChIP, followed by telomere qPCR and ( B ) Alu qPCR as control. ( C ) The effect of ZSCAN4 induction for 48 h on telomere-histone H3 acetylation was assessed by ChIP using H3K14ac or H3K18ac antibodies, followed by telomere qPCR. ( D ) Alu repeats were used as controls. Data are shown as mean ± S.E.M. * p < 0.05. **** p < 0.0001. ns—not significant.

Article Snippet: Antibodies used for ChIP: Anti-ZSCAN4 (10 μg) (Origene); anti-H3K14ac, H3K18ac, or H3K27ac (4 μg) (Cell signaling, Danvers, MA) and the provided anti-RNA Polymerase II (1 μg) and a Rabbit IgG (10 μg) were used as controls.

Techniques: Control