Journal: Cancers

Article Title: Keratin-14 (KRT14) Positive Leader Cells Mediate Mesothelial Clearance and Invasion by Ovarian Cancer Cells

doi: 10.3390/cancers11091228

Figure Lengend Snippet: ( A ) Parallel endpoint Boyden chamber assays. Boyden chamber assays using labelled mesothelial cells overlaid with individual patient-derived ovarian cancer spheroids; we observe no invasion of the mesothelial cells at MALDI imaging collection points; n = 3 wells/sample of one representative experiment. ( B ) Haemotoxylin and Eosin (H&E) staining of the invasive interface. H&E staining identifying the invading interface of ovarian cancer spheroid mesothelial co-cultures and the interface used for MALDI imaging mass spectrometry (IMS). ( C ) MALDI IMS of the invading interface. MALDI IMS identifies: CDCA8, HNRN, keratin-14 (KRT14) and FNDC3B expressed at the invading interface of ovarian/mesothelial co-cultures. ( D ) Representative qRT-PCR of MALDI identified candidates using fresh-frozen confirmed primary high-grade serous ovarian tumours or normal whole ovary ( n = 3/group) where individual data points represent individual patient samples. ( E ) Representative IHC of individual MALDI identified candidates in HGSC primary ovarian samples.

Article Snippet: Cells were transfected with guide strands (1–3), a non-targeting control, or the KRT14 overexpression construct KRT14 OE (Origene, Rockville, United States of America#RC214907) using Lipofectamine ® 2000 Transfection Reagent (Invitrogen, Carlsbad, United States of America #11668019) in DMEM as per the manufacturer’s protocol.

Techniques: Derivative Assay, Imaging, Staining, Mass Spectrometry, Quantitative RT-PCR

Journal: Cancers

Article Title: Keratin-14 (KRT14) Positive Leader Cells Mediate Mesothelial Clearance and Invasion by Ovarian Cancer Cells

doi: 10.3390/cancers11091228

Figure Lengend Snippet: ( A ) Western blot analyses demonstrating the endogenous expression of MALDI identified candidates FNDC3B, HNRN, CDCA8, or KRT14 in ovarian cancer cell lines or the mesothelium LP-9. ( B ) RTCA proliferation assays. Representative results from OVCAR4 real-time proliferation assays conducted in complete culture medium. Data are mean impedance readings from triplicate wells taken every 15 min over a 30 h assay period with lower standard deviation shown, where the non-targeting control is NT and individual CRISPR gene knock-outs are indicated. ( C ) RTCA invasion assays. Representative results from OVCAR4 real-time invasion assays. Data are mean impedance readings from duplicate wells taken every 15 min over a 26 h assay period with lower standard deviation shown. Individual CRISPR gene knock-outs are indicated on the graph. ( D ) Wound-healing assays. Wounded OVCAR4 cell lines (UT = untreated, NT= non-targeting and KRT14 KO = KRT14 CRISPR knockout) were cultured in complete medium. Cells were imaged at regular intervals ranging from 0 to 48 h with the original wound area indicated by dotted lines to aid in the assessment of wound closure. Images presented are representative of the observations of three separate experiments at assay commencement and 48 h, the assay was completed on a minimum of three separate occasions.

Article Snippet: Cells were transfected with guide strands (1–3), a non-targeting control, or the KRT14 overexpression construct KRT14 OE (Origene, Rockville, United States of America#RC214907) using Lipofectamine ® 2000 Transfection Reagent (Invitrogen, Carlsbad, United States of America #11668019) in DMEM as per the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Standard Deviation, Control, CRISPR, Knock-Out, Cell Culture

Journal: Cancers

Article Title: Keratin-14 (KRT14) Positive Leader Cells Mediate Mesothelial Clearance and Invasion by Ovarian Cancer Cells

doi: 10.3390/cancers11091228

Figure Lengend Snippet: ( A ) Representative qRT-PCR of KRT14 expression in LP9 cells, whole normal ovary, benign fibroma, primary ovarian tumour, or ascites-derived cells; and migratory leader cell populations ( n = 3 separate patient isolations where individual data points represent individual patient samples). ( B ) KRT14 expression in cultured cells by immunofluorescence. KRT14 expression was detected as a granular cytoplasmic stain expressed by a small proportion of ovarian cancer cells in the OVCAR4 line cultured as a two-dimensional (2D) monolayer. Scale bar = 100 µm. ( C ) KRT14 expression was detected at the periphery of OVCAR4 ovarian cancer cells cultured as three-dimensional (3D) spheroids with no signal detected in the core of the spheroid. Scale bar = 100 µm. ( D ) Localisation of KRT14 at the periphery of ovarian cancer spheroids by comparison to the intracellular staining observed for N-cadherin. Scale bar = 100 µm. ( E ) Vector control (NT), KRT14 KO , and KRT14 OE cells were suspended in SFM/0.25% methylcellulose in U-bottom 96-well plates and imaged at regular intervals (0–48 h) to observe spheroid aggregation. Images are representative of multiple wells observed at the 13-hour and 48-hour time points. Scale bar = 100 µm. ( F ) Spheroid attachment to the target mesothelium. Vector control (NT) and KRT14 KO spheroids were co-cultured with the target mesothelium, and attachment was measured 6 h post-addition. The results presented are from one representative assay where data points are the attachment counts of three individual wells per cell line.

Article Snippet: Cells were transfected with guide strands (1–3), a non-targeting control, or the KRT14 overexpression construct KRT14 OE (Origene, Rockville, United States of America#RC214907) using Lipofectamine ® 2000 Transfection Reagent (Invitrogen, Carlsbad, United States of America #11668019) in DMEM as per the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Cell Culture, Immunofluorescence, Staining, Comparison, Plasmid Preparation, Control

Journal: Cancers

Article Title: Keratin-14 (KRT14) Positive Leader Cells Mediate Mesothelial Clearance and Invasion by Ovarian Cancer Cells

doi: 10.3390/cancers11091228

Figure Lengend Snippet: ( A ) Mesothelial displacement assays. Ovarian cancer spheroids were generated from wild-type (WT), KRT14 KO and KRT14 OE lines and overlaid onto a confluent layer of mesothelial cells and imaged by light microscope. Representative images following overnight and 48-h co-culture are presented where dotted lines indicate spheroid outgrowth and mesothelial displacement. ( B , C ) Embedding and outgrowth assays. OVCAR4 ovarian cancer spheroids were formed and overlaid into ( B ) Collagen I (24 h) or ( C ) Matrigel (48 h) samples, and subsequently fixed and stained for KRT14 expression. ( D ) Wound-healing assays OVCAR4 cells were grown as a monolayer, cells were wounded, fixed, and stained for KRT14 expression at time-points preceding total wound closure. Images from a representative well are shown with scale bars = 100 µm.

Article Snippet: Cells were transfected with guide strands (1–3), a non-targeting control, or the KRT14 overexpression construct KRT14 OE (Origene, Rockville, United States of America#RC214907) using Lipofectamine ® 2000 Transfection Reagent (Invitrogen, Carlsbad, United States of America #11668019) in DMEM as per the manufacturer’s protocol.

Techniques: Generated, Light Microscopy, Co-Culture Assay, Staining, Expressing

Journal: Cancers

Article Title: Keratin-14 (KRT14) Positive Leader Cells Mediate Mesothelial Clearance and Invasion by Ovarian Cancer Cells

doi: 10.3390/cancers11091228

Figure Lengend Snippet: ( A ) Table of the immunohistochemical assessment of KRT14 expression in ovarian cancer tissue microarrays. Staining intensity was scored according to 0 (no stain), 1 (low), 2 (medium), or 3 (high). ( B ) Representative immunohistochemical staining of KRT14 in the various ovarian cancer subtypes (as labelled in figure) from TMA assessments. Scale bar = 200 µm. ( C – F ) Association of KRT14 expression with progression-free survival (PFS) according to ( C ) overall PFS (HR 1.17; 95% CI 1.03–1.33 p < 0.015; B); ( D ) early stage (I/II) diagnosis (HR 1.96; 95% CI 1.08–3.56 p < 0.025); ( E ) following platinum and taxol-based chemotherapy (HR 1.27; 95% CI 1.07–1.51 p < 0.006); and ( F ) following optimal debulk (HR 1.24; 95% CI 1.03–1.5 p < 0.026).

Article Snippet: Cells were transfected with guide strands (1–3), a non-targeting control, or the KRT14 overexpression construct KRT14 OE (Origene, Rockville, United States of America#RC214907) using Lipofectamine ® 2000 Transfection Reagent (Invitrogen, Carlsbad, United States of America #11668019) in DMEM as per the manufacturer’s protocol.

Techniques: Immunohistochemical staining, Expressing, Staining, Biomarker Discovery

Journal: Genes

Article Title: Identification of Key Proteins Related to Cashmere Fiber Diameter by Integrated Proteomics and Bioinformatic Analyses in the Alpas and Alxa Goat Breeds

doi: 10.3390/genes15091154

Figure Lengend Snippet: Western blot profiles of KRT14 . ( A ) Three biological replicates from the cashmere fiber of Alxa and Alpas. ( B ) Relative intensity of protein is expressed as the mean ± standard deviation. The asterisk represents levels of significance ( t -test: * p < 0.05).

Article Snippet: The source proteins obtained above were incubated with corresponding antibodies ( KRT14 ) and Protein A/G Magnetic Beads (MCE) at 4 °C overnight.

Techniques: Western Blot, Standard Deviation

Journal: Genes

Article Title: Identification of Key Proteins Related to Cashmere Fiber Diameter by Integrated Proteomics and Bioinformatic Analyses in the Alpas and Alxa Goat Breeds

doi: 10.3390/genes15091154

Figure Lengend Snippet: The interacting protein with KRT14 obtained through IP.

Article Snippet: The source proteins obtained above were incubated with corresponding antibodies ( KRT14 ) and Protein A/G Magnetic Beads (MCE) at 4 °C overnight.

Techniques:

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: Oncogene

Article Title: YEATS2 promotes DNA repair and induces anoikis resistance by enhancing chromatin accessibility to drive prostate cancer metastasis

doi: 10.1038/s41388-026-03696-x

Figure Lengend Snippet: A The Cistrome Data Browser (Cistrome DB) database identifies enrichment of H3K9ac and H3K14ac in the RAD50 promoter region. B Relative mRNA levels of RAD50 in PCa cells after treatment with different concentrations of Trichostatin A. C Protein levels of H3K9ac, H3K14ac, and RAD50 in PCa cells treated with different concentrations of Trichostatin A. β-actin and Histone H3 were used as internal controls. D Protein levels of H3K9ac and H3K14ac in anoikis-resistant and metastatic PCa cells. Histone H3 was used as an internal control. E Protein levels of H3K9ac and H3K14ac in PCa cells with YEATS2 overexpression. Histone H3 was used as an internal control. F Protein levels of H3K9ac and H3K14ac in PCa cells with YEATS2 knockdown. Histone H3 was used as an internal control. G Protein levels of histone benzoylation in the indicated groups. Histone H3 was used as an internal control. H Relative mRNA levels of RAD50 in PCa cells after treatment with different concentrations of NaBz. I Protein levels of RAD50 in PCa cells treated with different concentrations of NaBz. β-actin was used as internal controls. J Protein levels of histone crotonylation in the indicated groups. Histone H3 was used as an internal control. K Relative mRNA levels of RAD50 in PCa cells after treatment with different concentrations of crotonic acid. L Protein levels of RAD50 in PCa cells treated with different concentrations of crotonic acid. β-actin was used as an internal control. M The enrichment of H3K27ac in the RAD50 promoter region. N Protein levels of H3K27ac in anoikis-resistant and metastatic PCa cells. Histone H3 was used as an internal control. Statistical analysis is shown on the bar graphs. Data represent mean ± SD from n = 3 biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were: SRPRB (A10591, Abclonal, China)), YEATS2 (24717-1-AP, Proteintech), FAM126A (26243-1-AP, Proteintech), OSR2 (A18178, Abclonal), γH2AX (AP0687, Abclonal), RAD50 (29390-1-AP, Proteintech), VAV3 (A15385, Abclonal), BCL2 (A0208, Abclonal), BRCA1 (A0212, Abclonal), PARP1 (A27791, Abclonal), NR2C2 (ab109301, Abcam, UK), Rabbit control IgG (AC005, Abclonal), Histone H3 (68345-1-Ig, Proteintech), H3K9ac (A7255, Abclonal), H3K14ac (A7254, Abclonal), H3K27ac (A22264, Abclonal), H3K27cr (PTM-545RM, PTM BIO, China), anti-pan Kbz (PTM-762, PTM BIO), anti-Flag (66008-4-Ig, Proteintech), anti-GST (AE077, Abclonal), MRE11(A2559, Abclonal), β-actin (66009-1-Ig, Proteintech), GAPDH (60004-1-Ig, Proteintech).

Techniques: Control, Over Expression, Knockdown

Journal: Oncogene

Article Title: YEATS2 promotes DNA repair and induces anoikis resistance by enhancing chromatin accessibility to drive prostate cancer metastasis

doi: 10.1038/s41388-026-03696-x

Figure Lengend Snippet: A Schematic diagram of the YEATS domain structure in YEATS2. B Pull-down assay showing that the YEATS domain binds to the H3K27ac peptide. C Co-immunoprecipitation assay demonstrating the interaction between YEATS2 and H3K27ac in PCa cells. D Western blot analysis illustrating the effect of the H3K27ac-specific inhibitor A485 on YEATS2-mediated regulation of RAD50 expression. β-actin and Histone H3 were used as internal controls. E Protein levels of RAD50 in the indicated groups. β-actin was used as an internal control. F Fluorescent images of lung tissues in nude mice acquired using the In-Vivo FX PRO Imaging System (Bruker Corporation, USA). Mirin concentrations: #1, 50 mg/kg; #2, 100 mg/kg. G Schematic diagram showing YEATS2-mediated DNA damage repair and promotion of anoikis resistance after ECM detachment in PCa cells. YEATS2 is upregulated after ECM detachment in PCa cells. 1. YEATS2 recognizes H3K27ac via its YEATS domain, upregulates H3K9ac and H3K14ac, and increases chromatin accessibility at the RAD50 promoter. It also recruits NR2C2 to promote RAD50 transcription. 2. Upregulation of RAD50 enhances DNA damage repair capacity, promotes PCa cell survival under detachment conditions, thereby inducing anoikis resistance, and this process can be blocked by the MRN complex inhibitor Mirin. Image created with BioRender.com, with permission. Data represent mean ± SD from n = 3 biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were: SRPRB (A10591, Abclonal, China)), YEATS2 (24717-1-AP, Proteintech), FAM126A (26243-1-AP, Proteintech), OSR2 (A18178, Abclonal), γH2AX (AP0687, Abclonal), RAD50 (29390-1-AP, Proteintech), VAV3 (A15385, Abclonal), BCL2 (A0208, Abclonal), BRCA1 (A0212, Abclonal), PARP1 (A27791, Abclonal), NR2C2 (ab109301, Abcam, UK), Rabbit control IgG (AC005, Abclonal), Histone H3 (68345-1-Ig, Proteintech), H3K9ac (A7255, Abclonal), H3K14ac (A7254, Abclonal), H3K27ac (A22264, Abclonal), H3K27cr (PTM-545RM, PTM BIO, China), anti-pan Kbz (PTM-762, PTM BIO), anti-Flag (66008-4-Ig, Proteintech), anti-GST (AE077, Abclonal), MRE11(A2559, Abclonal), β-actin (66009-1-Ig, Proteintech), GAPDH (60004-1-Ig, Proteintech).

Techniques: Pull Down Assay, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, In Vivo, Imaging

Journal: iScience

Article Title: Unveiling critical signaling pathways in the murine salivary gland and the role of midkine

doi: 10.1016/j.isci.2026.114852

Figure Lengend Snippet: Branching morphogenesis of embryonic E13.5 SMGs treated with midkine inhibitors is reduced in ex vivo culture (A) Upper panel are light micrographs of wild type C57BL/6J SMGs harvested at E13.5 and cultured for 72 h in the presence of midkine inhibitor (iMDK) or DMSO control. Lower panel shows whole-mount immunofluorescence staining of DMSO control and iMDK treated glands shows expression of Keratin 5 (K5) and neuronal tubulin expressing nerves (Tubb3). Scale bars, 100 μm, ( n = 3). (B) Spooner’s ratio quantification of the number of end buds (expressed as a ratio of the number of end buds at 72 h/number at 1 h) of DMSO control ( n = 10) and iMDK treated glands ( n = 10). (C) Immunofluorescence staining of paraffin embedded DMSO control and iMDK treated glands reveal decreased expression of ΔNp63 and reduced expression of K14 expressing basal stem/progenitor cells. Alterations to the acinar cell differentiation program were also observed in the iMDK treated glands as evident by reduced expression of Mist1, Nkcc1, and Aqp5. Ductal cell differentiation was also altered as depicted by decreased K7 expression in the iMDK treated glands compared to DMSO controls. Scale bars, 20 μm. Lower panel shows quantification of the p63 + K14 + /p63 + basal cells, p63 + Sma + /p63 + MECs, and Nkcc1 + Mist1 + /Nkcc1 + acinar cell population as mean ± SD ( n = 3). (D) Quantitative RT-PCR validation of select genes in the DMSO control and iMDK treated glands. Values were normalized to the housekeeping gene Hprt . Data are represented as means ± SD ( n = 3). ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies used at the indicated dilutions include Midkine (R&D Systems, 1:100), Midkine (ProteinTech, 1:100), K14 (Rizzo et al., 2016, 1:100), Sma (Sigma 1A4, 1:200), Nkcc1 (Santa Cruz Biotechnology, 1:100), ΔNp63 (Cell Signaling Technology, 1:50), ΔNp63 ( ,1:50) Mist1 (Abcam, 1:100), Aqp5 (Alomone Labs, 1:100), Tubb3 (R&D Systems, 1:100), K7 (Abcam, 1:50), K5 (gift from Dr. Julie Segre, 1:100), Troma-III/K19 (Development Studies Hybridoma Bank, 1:50), and Rock1 (Cell Signaling Technology, 1:25).

Techniques: Ex Vivo, Cell Culture, Control, Immunofluorescence, Staining, Expressing, Cell Differentiation, Quantitative RT-PCR, Biomarker Discovery