Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: DNA 6 mA levels gradually decreased as CRC development. ( A ) Schematic diagram of DSS/AOM induced CRC model in C57BL/6J mice. ( B-C ) DNA 6 mA levels in DSS/AOM CRC model were determined by dot blot ( B ) and ELISA assays ( C ). ( D ) Schematic diagram of transgenic APC min/+ mice induced CRC. ( E-F ) Dot blot (E) and ELISA assays ( F ) detected DNA 6 mA levels in transgenic APC min/+ CRC model. ( G ) Schematic diagram of DNA 6 mA levels in orthotopic xenograft CRC model. ( H-I ) Dot blot ( H ) and ELISA assays ( I ) of DNA 6 mA levels derived from orthotopic xenograft CRC model. ( J ) The DNA 6 mA levels in the NCM460 cell line and CRC cell lines (HT29, HCT116, SW480, HCT15, and RKO) were determined by dot blot (up panel) followed by relative quantitative analysis (down panel). ( K ) ELISA assays indicated DNA 6 mA levels in NCM460 cell line and CRC cell lines (HT29, HCT116, SW480, HCT15, and RKO). ( L ) Representative IHC of 6 mA modification levels in human adjacent tissue and CRC tissues at different stages. ( M-P ) The histograms of 6 mA IHC scores at different AJCC stages ( M ), T stages(N), N stages ( O ), and M stages ( P ) Data are represented as mean ± SEM. * P < 0.05

Article Snippet: Consistently, the Human Protein Atlas (HPA) public immunostaining dataset demonstrated elevated ALKBH1 protein expression in primary CRC tissues (Fig. E).

Techniques: Dot Blot, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Derivative Assay, Modification

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: Extracellular matrix stiffness was associated with a reduction of 6 mA level. ( A ) Dot blot (left panel) and relative quantitative statistics (right panel) showed DNA 6 mA level of HCT116 cells (up panel) and RKO cells (down panel) stimulated by soft and stiff substrate. ( B-C ) ELISA assay ( B ) and IF staining ( C ) indicated DNA 6 mA levels of HCT116 cells (up panel) and RKO cells (down panel) stimulated by soft and stiff substrate. ( D-E ) Representative IHC ( D ) and Spearman’s correlation analysis ( E ) of collagen I and 6 mA in human CRC tissues. ( F-G ) Histogram ( F ) and heat map ( G ) of elastic modulus among normal colorectal tissues, AOM-induced neoplasia tissues, and AOM-induced CRC tissues. ( H ) Spearman’s correlation analysis of 6 mA IHC scores and elastic modulus in neoplasia tissues and CRC tissues of DSS/AOM-induced mice. ( I ) The BAPN treatment process for orthotopic xenograft mice model. ( J ) Histogram of elastic modulus in PBS-treated xenografts and BAPN-treated xenografts. ( K ) Western blot of vimentin, E-cadherin, CDKN1A in PBS-treated xenografts and BAPN-treated xenografts, using GAPDH as a control. ( L ) Representative IHC (left panel) and relative quantitative statistics (right panel) of 6 mA modification levels in PBS-treated xenografts and BAPN-treated xenografts Data are represented as mean ± SEM. * P < 0.05

Article Snippet: Consistently, the Human Protein Atlas (HPA) public immunostaining dataset demonstrated elevated ALKBH1 protein expression in primary CRC tissues (Fig. E).

Techniques: Dot Blot, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Control, Modification

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: Substrate stiffness upregulates ALKBH1. ( A ) Histograms of mRNA expression of ALKBH1, METTL4 and N6AMT1 in HCT116 (left panel) and RKO cells (right panel) stimulated by soft and stiff substrate. ( B ) Western blot of ALKBH1, METTL4 and N6AMT1 in HCT116 (left panel) and RKO cells (right panel) stimulated by soft and stiff substrate, using GAPDH as a control. ( C-D ) IF staining indicated ALKBH1 levels of HCT116 cells ( C ) and RKO cells ( D ) stimulated by soft and stiff substrate. ( E-F ) Western blot ( E ) and qRT-PCR (F) of ALKBH1, METTL4 and N6AMT1 in orthotopic xenograft model treated by PBS and BAPN, using GAPDH as a control. ( G-H ) Western blot ( G ) and qRT-PCR (H) of ALKBH1, METTL4 and N6AMT1 in DSS/AOM-induced CRC model treated by PBS and BAPN, using GAPDH as a control. ( I-J ) Representative IHC (left panel) and relative quantitative statistics (right panel) of ALKBH1 in orthotopic xenograft model ( I ) and DSS/AOM-induced CRC model ( J ) treated by PBS and BAPN. ( K ) Representative IHC of ALKBH1 in low collagen I group and high collagen I group of human CRC tissues. ( L ) Spearman’s correlation analysis of ALKBH1 IHC scores and collagen I IHC scores in human CRC tissues. ( M ) Representative IHC of ALKBH1 in low stiffness group and high stiffness group of DSS/AOM induced CRC mice. ( N ) Spearman’s correlation analysis of ALKBH1 IHC scores and elastic modulus in AOM/DSS-induced CRC tissues Data are represented as mean ± SEM. * P < 0.05; ns, not significant

Article Snippet: Consistently, the Human Protein Atlas (HPA) public immunostaining dataset demonstrated elevated ALKBH1 protein expression in primary CRC tissues (Fig. E).

Techniques: Expressing, Western Blot, Control, Staining, Quantitative RT-PCR

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: Demethylase ALKBH1 was responsible for 6 mA downregulation stimulated by stiffness. ( A ) Western blot of ALKBH1 distribution in HCT116 and RKO cells, using GAPDH as a cytoplasmic control and Lamin B1 as a nuclear control. ( B ) Representative confocal microscopy images of ALKBH1 and 6 mA in HCT116 and RKO cells. ( C-D ) Spearman’s correlation analysis of 6 mA and ALKBH1 IHC scores in human CRC ( C ) and AOM/DSS-induced neoplasia and CRC tissues ( D ). ( E-F ) Elisa assays of 6 mA levels in HCT116 (up panel) and RKO cells (down panel) transfecting ALKBH1 plasmids ( E ) or shALKBH1 plasmids ( F ). ( G-H ) Dot blot of 6 mA levels in HCT116 ( G ) and RKO cells ( H ) transfecting shALKBH1 plasmids (up panel) or ALKBH1 plasmids (down panel). ( I ) Diagram of 6 mA PGL4 luciferase reporter containing 3 X GGAGG motif. ( J-K ) Histograms of luciferase activity of 6 mA in HCT116 (up panel) and RKO (down panel) with ALKBH1 downregulation ( J ) and ALKBH1 overexpression ( K ). ( L ) Diagram of mutant 6 mA PGL4 luciferase reporter containing 3 X GGTGG motif. ( M ) Histograms of luciferase activity of 6 mA and mutant 6 mA in HCT116 (up panel) and RKO (down panel) transfecting ALKBH1 plasmid and plasmid vector. ( N ) Histograms of 6 mA luciferase activity of HCT116 (left panel) and RKO cells (right panel) stimulated by soft and stiff substrate. ( O-P ) Dot blot ( O ) and ELISA ( P ) showed DNA 6 mA levels of HCT116 cells with knockdown ALKBH1 and control after soft and stiff substrate stimulation. Data are represented as mean ± SEM. * P < 0.05; ns, not significant; Nuc: nucleus; Cyto: cytoplasm

Article Snippet: Consistently, the Human Protein Atlas (HPA) public immunostaining dataset demonstrated elevated ALKBH1 protein expression in primary CRC tissues (Fig. E).

Techniques: Western Blot, Control, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Dot Blot, Luciferase, Activity Assay, Over Expression, Mutagenesis, Plasmid Preparation, Knockdown

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: Demethylase ALKBH1 was associated with poor prognosis in CRC. ( A ) Boxplot presented ALKBH1 expression in CRC and adjacent normal tissues using the TCGA combined with GTEx datasets. ( B ) Scatter plot of ALKBH1 expression using TCGA paired datasets. ( C-D ) Boxplot presented ALKBH1 expression in CRC and adjacent normal tissues from GSE25071 ( C ) and GSE18105 datasets ( D ). ( E ) Representative IHC (left panel) and IHC (right panel) analysis of ALKBH1 protein expression in normal tissues and CRC tissues from HPA immunostaining dataset. ( F-G ) The protein ( F ) and mRNA levels ( G ) of ALKBH1 in CRC cells lines (HT29, HCT116, SW480, RKO and HCT15) and normal colon endothelial cell line (NCM460), using GAPDH as a control. ( H ) Representative IHC of ALKBH1 in human adjacent normal tissues and CRC tissues under different stages. ( I-L ) The histograms of ALKBH1 scores at different AJCC stages ( I ), T stages( J ), N stages ( K ), and M stages ( L ). ( M ) Boxplot presented ALKBH1 expression in primary CRC tissues and metastasis tissues from GSE77953 dataset. ( N ) Heat map of ALKBH1 expression in CRC tissues of different stages from TCGA, GSE20970 , GSE77955 , GSE103512 , GSE128449 , GSE156451 , and GSE211831 datasets. ( O-P ) Kaplan-Meier plots of overall survival of CRC patients from TCGA ( O ) and Kaplan-Meier Plotter datasets ( P ), stratified according to mean ALKBH1 expression. Data are represented as mean ± SEM. * P < 0.05; ns, not significant

Article Snippet: Consistently, the Human Protein Atlas (HPA) public immunostaining dataset demonstrated elevated ALKBH1 protein expression in primary CRC tissues (Fig. E).

Techniques: Expressing, Immunostaining, Control

Journal: iScience

Article Title: ExIR enables prioritizing driver and biomarker genes from omics data in a reference free manner

doi: 10.1016/j.isci.2026.116303

Figure Lengend Snippet: Differential immunoreactivity of known and novel ExIR-predicted LUAD biomarkers between normal and cancer samples (A–E) Immunohistochemical (IHC) data from the Human Protein Atlas database in LUAD and normal lung tissue for top five known LUAD biomarkers. (A) SFTPC–ExIR rank #1, LUAD (negative intensity; patient ID: 1847) and normal pneumocytes (quantity: 75%–25%; strong intensity; patient ID: 2268). (B) SPP1—ExIR rank #117, LUAD (quantity: >75%; moderate intensity; patient ID: 537) and normal pneumocytes (not detected; patient ID: 2268). (C) CBLC—ExIR rank #140, LUAD (quantity: >75%; moderate intensity; patient ID: 1847) and normal pneumocytes (not detected; patient ID: 2417). (D) MDK—ExIR rank #247, LUAD (quantity: 75%–25%; strong intensity; patient ID: 1847) and normal pneumocytes (not detected; patient ID: 2222). (E) MRC1 – ExIR rank #471, LUAD (undetected; patient ID: 1932) and normal macrophages (quantity: 75%–25%; strong intensity; patient ID: 2208). (F–I) IHC data of top five ExIR-predicted LUAD biomarkers (excluding SFTPC rank #1 already in a). (F) AGER – ExIR rank #2, LUAD (not detected; patient ID: 3144) and normal pneumocytes (quantity: >75%; strong intensity; patient ID: 4840). (G) EMP2 – ExIR rank #3, LUAD (not detected; patient ID: 1847) and normal pneumocytes (quantity: >75%; strong intensity; patient ID: 2101). (H) CAV1 – ExIR rank #4, LUAD (not detected; patient ID: 1249) and normal pneumocytes (quantity: >75%; strong intensity; patient ID: 2208). i, RTKN2 – ExIR rank 5, LUAD (not detected; patient ID: 3003) and normal pneumocytes (quantity: <25%; moderate intensity; patient ID: 2268).(J–N) IHC data of top six ExIR-predicted LUAD up-regulated biomarkers (excluding FAM83A rank #2, for which no IHC data was available). (J) PYCR1 – ExIR rank #1, LUAD (quantity: >75%; moderate intensity; patient ID: 2777) and normal pneumocytes (not detected; patient ID: 2208). (K) TOP2A – ExIR rank #3, LUAD (quantity: 75%–25%; strong intensity; patient ID: 3003) and normal pneumocytes (quantity: 75%–25%; weak intensity; patient ID: 2101). (L) MMP11 – ExIR rank #4, LUAD (quantity: >75%; weak intensity; patient ID: 1847) and normal pneumocytes (not detected; patient ID: 2438). (M) TEDC2– ExIR rank 5, LUAD (quantity: 75%–25%; moderate intensity; patient ID: 4208) and normal pneumocytes (not detected; patient ID: 1470). (N) IQGAP3– ExIR rank 6, LUAD (quantity: >75%; strong intensity; patient ID: 3048) and normal pneumocytes (not detected; patient ID: 1470). Per the Human Protein Atlas database usage guidelines, the link to the immunostaining images of all of the selected proteins in normal pneumocytes and LUAD samples are included as hyperlinks within the figure legend. Ab: antibody; LUAD: lung adenocarcinoma.

Article Snippet: Examination of public immunohistochemical data for top-ranked ExIR biomarkers in LUAD from the Human Protein Atlas revealed differential immunoreactivity of all previously established LUAD biomarkers between normal and cancer samples ( A–4E).

Techniques: Immunohistochemical staining, Immunostaining