Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: Elevated lactylation, H3K18la and IGF2BP2 levels were associated with Nrf2‐mediated ferroptosis resistance in CRC. (A) Lactylation levels in normal adjacent tissues and patients with CRC ( n = 10) were analysed. (B) H3K18la, IGF2BP2 and Nrf2 levels in normal adjacent and CRC tissues were examined by western blotting ( n = 10). (C) Kaplan–Meier survival analysis of normal adjacent tissues and patients with CRC stratified by high/low lactylation, H3K18la, IGF2BP2 and Nrf2 levels ( n = 40). (D) Lactylation, H3K18la, IGF2BP2 and Nrf2 levels in CRC tissues with different tumour stages (T stages) ( n = 40). (E) Upper panels: TSA‐amplified multiplexed immunofluorescence of normal adjacent and CRC tissues for H3K18la, IGF2BP2, Nrf2 and GPX4 expression (scale bar = 100 µm). Below panels: quantitative analysis of the fluorescence signals using ImageJ software and correlation analyses between: (left) H3K18la versus IGF2BP2 expression, (middle) IGF2BP2 versus Nrf2 expression and (right) Nrf2 versus GPX4 expression in CRC tissue samples ( n = 10). (F) Intracellular lactylation levels in the normal colon epithelial cell line NCM460, and eight CRC cell lines (LoVo, LS513, HCT116, SW480, SW620, SW1116, HT29 and Caco‐2) were measured by colorimetric assay. (G) H3K18la levels in these cells were examined using western blot. Measurement data are presented as mean ± SD. n = 3 for F and G. * p < .05, ** p < .01, *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Western Blot, Amplification, Immunofluorescence, Expressing, Fluorescence, Software, Colorimetric Assay

Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: Lactate promoted Nrf2‐mediated ferroptosis resistance in CRC cells. SW480 and Caco‐2 cells were treated with lactate (10 mM) and the Nrf2 inhibitor ML385 (5 µM) for 24 h, followed by treatment with RSL3. Experimental groups: control, RSL3, Lac+RSL3, Lac+ RSL3+ML385. (A) Cell viability was measured by CCK‐8 assay. (B) Colony formation assay was performed to evaluate cell proliferation. (C) Representative transmission electron microscopy (TEM) images of mitochondrial ultrastructure in different groups (scale bar = 500 nm). (D) Representative immunofluorescence images of cells stained with C11‐BODIPY (oxidised form, green) and DAPI (nuclei, blue) following treatments as indicated. Scale bar = 100 µm. (E) MDA, Fe 2+ and GSH levels were measured using commercial kits. (F) H3K18la, Nrf2 and GPX4 levels were examined using western blot. Measurement data are presented as mean ± SD. n = 3. * p < .05, ** p < .01, *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Control, CCK-8 Assay, Colony Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: IGF2BP2 promoted CRC cell ferroptosis resistance. (A and B) Western blotting and RT‐qPCR analysis of IGF2BP2 expression in the normal colon epithelial cell line NCM460 and CRC cell lines (LoVo, LS513, HCT116, SW480, SW620, SW1116, HT29 and Caco‐2). (C) Western blot analysis of IGF2BP2 and Nrf2 levels in SW480 and Caco‐2 cells transfected with shIGF2BP2 (IGF2BP2‐KD). IGF2BP2‐KD cells were treated with RSL3 and group as: shNC, shIGF2BP2‐1, shIGF2BP2‐2, RSL3 + shNC, RSL3 + shIGF2BP2‐1 and RSL3 + shIGF2BP2‐2. (D) Cell viability was assessed by CCK‐8 assay. (E) Colony formation measured cell proliferative capacity. (F) Representative transmission electron microscopy (TEM) images of mitochondrial ultrastructure in different groups (scale bar = 500 nm). (G) Representative immunofluorescence images of cells stained with C11‐BODIPY and DAPI. Scale bar = 100 µm. Measurement data were presented as mean ± SD. n = 3. * p < .05, ** p < .01, *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: IGF2BP2 knockout sensitised CRC cells to ferroptosis. (A) Western blot validation of IGF2BP2 knockout (KO) in SW480 and Caco‐2 cells generated by CRISPR–Cas9 using two independent sgRNAs (sgIGF2BP2‐1, sgIGF2BP2‐2). IGF2BP2‐KO SW480/Caco‐2 cells were treated with RSL3 (or DMSO control) and group as: DMSO, RSL3, RSL3 + sgNC, RSL3 + sgIGF2BP2‐1 and RSL3 + sgIGF2BP2‐2. (B) Cell viability was measured by CCK‐8 assays. (C) Representative transmission electron microscopy (TEM) images showing mitochondrial morphology (scale bar = 500 nm). (D) Lipid peroxidation was assessed by C11‐BODIPY immunofluorescence. Scale bar = 100 µm. (E) Commercial kit quantification of intracellular MDA, Fe 2+ and GSH levels. (F) Western blot analysis of IGF2BP2, Nrf2 and GPX4 expression. Measurement data were presented as mean ± SD. n = 3. * p < .05, ** p < .01, *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Knock-Out, Western Blot, Biomarker Discovery, Generated, CRISPR, Control, CCK-8 Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Expressing

Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: Lactate accelerated CRC cell proliferation and metastasis in mice by promoting Nrf2‐mediated ferroptosis resistance. Xenograft tumour growth in nude mice injected subcutaneously with SW480 and Caco‐2 cells stably overexpressing LDHA. Mice were then treated with or without RSL3 (intraperitoneal injection) and divided into four groups: OE‐NC, OE‐LDHA, RSL3 + OE‐NC and RSL3 + OE‐LDHA. (A and B) Tumour size and volume were monitored every 5 days. (C) Western blot analysis of pan‐Kla, H3K18la, LDHA, IGF2BP2, Nrf2 and GPX4 expression in tumour tissues from each group. (D) Lactate levels in tumour tissues were measured using a lactate assay kit. For the metastasis model, LDHA‐overexpressing CRC cells were injected intravenously into nude mice. Mice were then treated with or without RSL3 (intraperitoneal injection) and divided into four groups: OE‐NC, OE‐LDHA, RSL3 + OE‐NC and RSL3 + OE‐LDHA. (E) Representative bioluminescent images showed lung metastasis in nude mice. (F) H&E staining of lung tissue sections (scale bar = 100 µm). (G) IHC analysis of the proliferation marker Ki67 in metastatic nodules (scale bar = 100 µm). Measurement data are presented as mean ± SD. n = 5. * p < .05, ** p < .01, *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Injection, Stable Transfection, Western Blot, Expressing, Lactate Assay, Staining, Marker

Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: DCA inhibited CRC cell proliferation and metastasis in mice by repressing ferroptosis resistance. SW480 and Caco‐2 cells were co‐injected with or without THP‐1‐derived macrophages (MΦ) subcutaneously into nude mice pre‐treated with clodronate liposomes to deplete endogenous macrophages. Treatment groups: CRC (control), CRC + DCA, CRC + DCA + lactate, CRC + MΦ, CRC + MΦ + DCA and CRC + MΦ + DCA + lactate. DCA (dichloroacetate; 50 mg/kg) was administered intraperitoneally every other day. (A and B) Tumour size and volume were monitored every 5 days. (C and D) Histological analysis of H&E staining and Ki67 immunohistochemistry of tumour sections (scale bar = 100 µm). (E) IHC analysis of IGF2BP2, Nrf2 and GPX4 expression in tumour tissues. Measurement data are presented as mean ± SD. n = 5. *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Injection, Derivative Assay, Liposomes, Control, Staining, Immunohistochemistry, Expressing

Journal: Clinical and Translational Medicine

Article Title: Histone lactylation‐mediated up‐regulation of IGF2BP2 enhances ferroptosis resistance via Nrf2 in colorectal cancer

doi: 10.1002/ctm2.70551

Figure Lengend Snippet: IGF2BP2 knockdown inhibited Nrf2‐mediated ferroptosis resistance, as well as CRC cell proliferation and metastasis in mice. Mice were subcutaneously injected IGF2BP2 knockdown (shIGF2BP2) CRC cells to generate xenograft tumours, followed by receiving intraperitoneal injections of RSL3 and group as: shNC, shIGF2BP2, RSL3 + shNC and RSL3 + shIGF2BP2. (A and B) The tumour size and volume were monitored every 5 days. (C) Western blot of tumour lysates to analyse Nrf2 and GPX4 levels. For the metastasis model, IGF2BP2 knockdown CRC cells were injected intravenously. (D) In vivo bioluminescence images of lung metastases. (E) H&E staining of lung tissue sections (scale bar = 100 µm). (F) IHC analysis of Ki67 expression in metastatic nodules (scale bar = 100 µm). Measurement data are presented as mean ± SD. n = 5. * p < .05, ** p < .01, *** p < .001.

Article Snippet: HEK293T cells, human CRC cells (SW480, Caco‐2, LoVo, LS513, HCT116, SW620, SW1116 and HT‐29) along with NCM460 normal colonic epithelial controls from ATCC (Manassas, VA, USA) were maintained in standard conditions (DMEM with 10% FBS, 1% antibiotics, 37°C, 5% CO 2 ).

Techniques: Knockdown, Injection, Western Blot, In Vivo, Staining, Expressing

Journal: Scientific Reports

Article Title: Single-cell and bulk transcriptomics reveal Fusobacterium nucleatum promotes colorectal cancer metastasis by upregulating LAMC2 in in vitro and in vivo models

doi: 10.1038/s41598-025-26249-w

Figure Lengend Snippet: F. nucleatum promotes the migration of CRC cells in vitro. ( A ) Scanning electron microscopy and ( B ) transmission electron microscopy images show rod-shaped bacteria (arrows) penetrating and invading HCT116 cells. ( C - J ) LOVO and HCT116 cells were incubation with PBS control, E.coli , or F. nucleatum . Cell migration ability was evaluated through wound healing assays and Transwell assays ( n = 4 per group, One-way ANOVA test). In the Transwell assays, migrated cells were stained with crystal violet, and images were randomly captured under a microscope, with 5 random fields per sample counted. Statistical analysis was performed using one-way ANOVA. Data are shown as mean ± SD.* represents p < 0 0.05, ** represents p < 0 0.01, *** represents p < 0.001, and **** represents p < 0.0001, ns represents no statistical significant.

Article Snippet: Human CRC cell lines (HCT116, LOVO, HCT8, SW480 and SW620) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Migration, In Vitro, Electron Microscopy, Transmission Assay, Bacteria, Incubation, Control, Staining, Microscopy

Journal: Scientific Reports

Article Title: Single-cell and bulk transcriptomics reveal Fusobacterium nucleatum promotes colorectal cancer metastasis by upregulating LAMC2 in in vitro and in vivo models

doi: 10.1038/s41598-025-26249-w

Figure Lengend Snippet: F. nucleatum upregulates LAMC2 in CRC cells. ( A ) Volcano plot showing the expression profile of differentially expressed genes (DEGs) in LOVO cells between F. nucleatum infection and PBS control treatment ( n = 3 per group, p < 0.05 and |log2 (fold change)| ≥ 1). ( B ) Intersection genes between the up DEGs from the GSE173549 dataset and the metastasis-related genes from the CancerSEA database. ( C and D ) The expression of LAMC2 was analyzed by single-cell RNA sequencing data GSE132465 . The cells were classified into seven distinct clusters, including T, B, myeloid, mast, epithelial, endothelial, and mesenchymal cells. The statistical significance between two groups was evaluated by wilcox.test. Normal: adjacent normal tissues of CRC, Tumor: tumor tissues of CRC. ( E and F )The mRNA and protein expression of LAMC2 after F. nucleatum or PBS treatment in CRC cells ( n = 4 per group, One-way ANOVA test). ( G and H ) The mRNA expression of LAMC2 after F. nucleatum , E. coli or PBS treatment in CRC cells ( n = 4 per group, One-way ANOVA test). ( I and K ) The protein expression of LAMC2 after PBS treatment, F. nucleatum infection of MOI = 100 or 500, or E. coli infection of MOI = 100 in CRC cells (repeated three times). ( J and L ) The protein expression of LAMC2 at 0, 4, 12, 24 and 48 h after F. nucleatum infection in CRC cells ( n = 4 per group, One-way ANOVA test). Data are shown as mean ± SD.* represents p < 0 0.05, ** represents p < 0 0.01, *** represents p < 0.001, and **** represents p < 0.0001, ns represents no statistical significant.

Article Snippet: Human CRC cell lines (HCT116, LOVO, HCT8, SW480 and SW620) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Infection, Control, RNA Sequencing

Journal: Scientific Reports

Article Title: Single-cell and bulk transcriptomics reveal Fusobacterium nucleatum promotes colorectal cancer metastasis by upregulating LAMC2 in in vitro and in vivo models

doi: 10.1038/s41598-025-26249-w

Figure Lengend Snippet: F. nucleatum promotes metastasis of CRC cells in a LAMC2 dependent manner. ( A and B ) Detection of the knockdown efficiency of LAMC2 by different sequences of lentiviral vectors at the mRNA ( n = 4 per group, One-way ANOVA test) and protein (repeated three times) levels. LOVO cell were transfected with LAMC2 shRNA lentivirus or control lentivirus, and then incubated with F. nucleatum or PBS. ( C and D ) The mRNA expression and protein expression of LAMC2 were measured by qPCR ( n = 4, One-way ANOVA test) and western blot (repeated three times). ( E and G ) The motility was detected by wound-healing assay ( n = 6, One-way ANOVA test) or ( F and H ) transwell assay( n = 5, One-way ANOVA test). ( I ) LOVO cells constructed with lentivirus were incubated with F. nucleatum or PBS and then injected into the tail vein of nude mice (5 mice per group). ( J and K ) HE staining represents the images and the number of metastatic lesions (Student’s t-test) in each group. Data are shown as mean ± SD. * represents p < 0 0.05, ** represents p < 0 0.01, *** represents p < 0.001, and **** represents p < 0.0001, ns represents no statistical significant.

Article Snippet: Human CRC cell lines (HCT116, LOVO, HCT8, SW480 and SW620) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Knockdown, Transfection, shRNA, Control, Incubation, Expressing, Western Blot, Wound Healing Assay, Transwell Assay, Construct, Injection, Staining

Journal: Scientific Reports

Article Title: Single-cell and bulk transcriptomics reveal Fusobacterium nucleatum promotes colorectal cancer metastasis by upregulating LAMC2 in in vitro and in vivo models

doi: 10.1038/s41598-025-26249-w

Figure Lengend Snippet: F.nucleatum upregulates the expression of LAMC2 through hsa-miRNA-7977. ( A ) western blot (repeated three times) was used to detect the overexpression efficiency of LAMC2 . HCT116 cell were transfected with LAMC2 oe-RNA lentivirus or control lentivirus, and then incubated with F. nucleatum or PBS. ( B and C ) The protein expression and mRNA expression of LAMC2 were measured by western blot (repeated three times) and qPCR ( n = 4, One-way ANOVA test). ( D and F ) The motility was detected by wound-healing assay ( n = 6, One-way ANOVA test) or ( E and G ) transwell assay ( n = 5, One-way ANOVA test). ( H ) Prediction of miRNA binding to LAMC2 using public databases, with a Venn diagram showing overlapping genes from the MiRDB, MirDIP, and Targetscan databases. ( I ) A Venn diagram showing candidate miRNAs binding to LAMC2 predicted by three public databases (miRNA- LAMC2 ) and downregulated miRNAs after F. nucleatum infection in CRC cells (Fn-miRNA). ( J ) Detection of miR-7977 expression using qPCR in CRC cells after F. nucleatum infection ( n = 4, One-way ANOVA test). ( K )Prediction of the binding site of has-miR-7977 to the 3’-UTR of LAMC2 using the Targetscan database.

Article Snippet: Human CRC cell lines (HCT116, LOVO, HCT8, SW480 and SW620) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Western Blot, Over Expression, Transfection, Control, Incubation, Wound Healing Assay, Transwell Assay, Binding Assay, Infection