human crc cell line hct116  (ATCC)

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: ICE analysis of CRISPR/Cas9-mediated CLDN2 -KO efficiency in HCT116 cells. ICE software analysis confirmed successful editing of the CLDN2 locus with an efficiency of ~91%. The editing score reflects the proportion of indels detected in the cell population. ICE, Inference of CRISPR Edits; CLDN2 , claudin-2; Wt, wild-type; KO, knockout; indel, insertion-deletion.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: CRISPR, Software, Knock-Out

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: Wound healing assay assessing cell migration in Wt and CLDN2 -KO HCT116 cells. (A) Representative images of wound closure at 0 and 24 h post-scratch (scale bar, 100 µm). (B) Quantification of wound closure percentage. Wt cells achieved ~96% closure, while CLDN2 -KO cells demonstrated 41% closure after 24 h (P=0.0027; unpaired two-tailed t-test; n=3). Data are presented as the mean ± SEM. **P<0.01. Wt, wild-type; CLDN2 -KO, claudin-2 knock out.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: Wound Healing Assay, Migration, Two Tailed Test, Knock-Out

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: Gene expression analysis of invasion- and metastasis-related genes in CLDN2 -KO vs. Wt HCT116 cells. Reverse transcription-quantitative PCR exhibited significant downregulation of multiple target genes, including ZONAB, NDRG1, CLDN14, CLDN23, Bcl-2, p53 and Bcl-6. Gene expression levels were normalized to GAPDH. Data are represented as mean ± SEM (n=3). Statistical comparisons were made using unpaired two-tailed t-tests. *P<0.05. ns, not significant; Wt, wild-type; CLDN2 -KO, claudin-2 knock out; ZO-1, zonula occludens-1; VDR , vitamin D receptor; ZONAB, ZO-1 -associated nucleic acid binding protein; NDRG1, N-Myc downstream-regulated gene 1; APC, adenomatous polyposis coli; AF-6/AFDN , Afadin; TJP1, tight junction protein 1; YBX3, Y-box binding protein 3; PTMS, parathymosin; TCN-1, transcobalamin 1.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Two Tailed Test, Knock-Out, Binding Assay

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: Relative expression levels of metastasis-associated genes in Wt and CLDN2 -KO HCT116 cells. Gene expression was quantified using reverse transcription-quantitative PCR. Each bar represents the mean ± SEM of three independent experiments. Values were calculated using the 2 −ΔΔCq method and normalized to GAPDH. Expression in Wt cells was set to 1.0 and knock out values were expressed relative to this baseline. Each bar corresponds to a specific gene and the height of the bars indicates the magnitude of the fold-change observed in the CLDN2 -KO samples compared with the Wt samples. The highest bars on the figure represent the gene with the lowest degree of expression variation. IL-6 exhibited the lowest degree of downregulation (fold-change, 0.718), whereas AF-6 demonstrated the most pronounced reduction (fold-change, 0.008). Wt, wild type; ZO-1 , zonula occludens-1; VDR , vitamin D receptor; ZONAB , ZO-1-associated nucleic acid binding protein; NDRG1, N-Myc downstream-regulated gene 1; APC, adenomatous polyposis coli; AF-6 , Afadin; PTMS, parathymosin; TCN-1, transcobalamin 1; CLDN2 -KO, claudin-2 knock out; AU, arbitrary Units.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Knock-Out, Binding Assay

Journal: Materials Today Bio

Article Title: Primary tissue metabolic fingerprinting for efficient diagnosis of lymph node metastasis and metabolic reprogramming mechanisms in colorectal cancer

doi: 10.1016/j.mtbio.2025.102712

Figure Lengend Snippet: Biological function validation of metabolic biomarkers in CRC lymph node metastasis. Relative abundance of (A) γ-glutamylcysteine (γ-Glu-Cys) and (B) glutathione (GSH) in LNM-versus LNM + CRC tissues. (C) Representative images of immunohistochemical (IHC) staining of GCLC, GCLM and GSS in CRC tissues (n = 10). Scale bar: 200 μm. (D) Quantitative IHC score analysis for GCLC, GCLM and GSS, showing significant upregulation of GCLC in LNM-positive samples (mean ± SEM, Mann-Whitney U test). (E) Glutamate-cysteine ligase (GCL) activity in CRC tissues (n = 6, mean ± SEM, Mann-Whitney U test)). (F) GCLC protein level was determined through western blot analysis following the transfection of GCLC and control siRNAs. (G) Cell migration ability was estimated using a scratch wound healing assay (n = 3, mean ± SEM, Mann-Whitney U test). (H) Transwell migration ability of HCT116 cells after GCLC and control siRNAs transfection (n = 3, mean ± SEM, Mann-Whitney U test). (I) Protein expression analysis of GCLC in CRC samples from The Cancer Genome Atlas (TCGA) database, confirming upregulation in LNM + cases (Mann-Whitney U test, p < 0.001). (J) Schematic illustration depicting the metabolic reprogramming mechanism in primary CRC leading to LNM, highlighting cysteine depletion, GCLC-driven GSH accumulation, and enhanced tumor migration.

Article Snippet: The HCT116 human CRC cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Immunohistochemical staining, Immunohistochemistry, MANN-WHITNEY, Activity Assay, Western Blot, Transfection, Control, Migration, Wound Healing Assay, Expressing

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF knockdown inhibits CRC cell proliferation and cell-cycle progression ( A ) Correlation analysis between NSMF expression and replication stress score in CRC cell line from CCLE dataset. ( B ) Cell growth curves of HCT116 and SW480 cells following transfection with control or NSMF siRNAs over 5 days, measured by direct cell counting ( n = 3). Data represent the mean ± SD. **** P < .0001, two-way ANOVA with repeated measures, followed by Tukey’s multiple comparisons test. ( C ) Colony formation assay showing the effect of NSMF knockdown in HCT116 and SW480 cells. Representative images (upper panel) and quantification of colonies area (lower panel) are shown. Data represent mean ± SD. *** P < .001, unpaired two-tailed Student’s t -test (HCT116, n = 2; SW480, n = 3). ( D ) Relative cell number of five CRC cell lines (HCT116, SW480, SW620, SNU-407, and RKO) measured 3 days after NSMF knockdown compared to control. Data represent the mean ± SEM. ( n = 3) *** P < .001, **** P < .0001, unpaired two-tailed Student’s t -test. ( E ) Cell-cycle analysis by flow cytometry in HCT116 cells transfected with siCtrl or siNSMF#1. Representative flow cytometry plots (left) and quantification of cell cycle distribution in G1, S, and G2/M phases with percentages indicated in bar graph (right). Data are presented as the mean ± SD ( n = 3). All experiments were independently performed at least three times, and representative results are shown.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Knockdown, Expressing, Transfection, Control, Cell Counting, Colony Assay, Two Tailed Test, Cell Cycle Assay, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF depletion affects DNA replication dynamics and induces replication stress. (A, B) DNA fiber analysis examining the effect of NSMF on replication dynamics in HCT116 cells. For normal conditions, cells were sequentially labeled with CldU and IdU for 30 min each ( A ). For replication stress conditions, cells were treated with 2 mM hydroxyurea (HU) for 2 h between CldU and IdU pulses ( B ). Representative DNA fiber images (upper) and quantification of IdU tract lengths (lower). The median value is indicated, derived from the analysis of 200 or more IdU and CldU tracts per experimental condition. **** P < .0001, two-tailed Mann–Whitney test. ( C, D ) Quantification of newly fired origins ( C ), IdU-only fibers) and stalled forks ( D ), CldU-only fibers) in HCT116 cells transfected with siCtrl or siNSMF#1. A total of 300–350 fibers from 7 to 18 randomly selected non-overlapping images per condition were analyzed. Data represent mean ± SEM. * P < .05, **** P < .0001. Statistical significance was assessed using a Mann–Whitney test. Results are representative of three independent experiments. ( E ) BrdU pulse-chase analysis of cell-cycle kinetics in HCT116 cells transfected with siCtrl or siNSMF#1 following release from a HU block (2 mM, 12 h). Representative results from three independent experiments are shown. ( F ) Immunofluorescence analysis of phospho-RPA2 foci in control (shCtrl) and NSMF-depleted (shNSMF#1 and #2) HCT116 cells under normal (NT) or HU treatment (2 mM, 16 h) conditions. The number of phospho-RPA2 foci per cell was quantified from at least 54 cells across two independent experiments. Scale bar, 10 μm. Data are presented as median. * P < .05, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. ( G ) γH2AX staining for control (shCtrl) or NSMF-depleted (shNSMF#2) HCT116 cells treated with HU treatment (2 mM, 16 h), and directly fixed or allowed to grow in the complete medium for 5 h in the absence of HU. The number of γH2AX foci per cell was quantified from at least 106 cells across three independent experiments. Scale bar, 10 μm. Data are presented as median. **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Labeling, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Transfection, Pulse Chase, Blocking Assay, Immunofluorescence, Control, Staining

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF mitigates replication stress and prevents oncogene-induced senescence. ( A ) SA-β-galactosidase (SA-β-Gal) staining in stable NSMF knockdown (shNSMF #1 and #2) or control (shCtrl) HCT116 cells. Representative images (upper) and quantification of SA-β-gal positive cells (lower). Scale bar, 50 μm. Data are presented as mean ± SEM from 150 cells across seven images obtained from three independent experiments. ** P < .01, *** P < .001, one-way ANOVA followed by Dunnett’s multiple comparisons test. ( B ) GSEA plot showing enrichment of cellular senescence-related genes in RNA-seq data from Nsmf +/+ ; Apc Min/+ and Nsmf −/− ; Apc Min/+ intestinal tumor (upper). Heatmap visualization of differentially expressed senescence- and SASP-related genes between genotypes (lower). ( C ) qRT-PCR analysis of senescence-associated genes in intestinal tumors from Nsmf +/+ ; Apc Min/+ ( n = 4) and Nsmf −/− ; Apc Min/+ ( n = 3) mice. Data represent the mean ± SEM. * P < .05, ** P < .01, *** P < .001, unpaired two-tailed t -test with Holm–Sidak correction for multiple comparisons. ( D ) Western blot analysis of p16INK4A and p21CIP1 in intestinal tumor tissues from Nsmf +/+ ; Apc Min/+ and Nsmf −/− ; Apc Min/+ mice. GAPDH served as a loading control. ( E ) Schematic representation of the experimental design for the oncogene-induced senescence model. IMR-90 cells were transduced with lentiviruses encoding either GFP-vector or GFP-NSMF. Following selection, senescence was induced by expression of oncogenic Ras G12V . ( F ) Western blot analysis of the indicated proteins on day 4 after induction of oncogenic Ras G12V expression. α-Tubulin was used as a loading control. ( G ) SA-β-Gal staining in IMR-90 cells 8 days post-transduction. Representative images (left) and quantification of SA-β-Gal positive cells (right). Scale bar, 20um. Data are presented as mean ± SEM ( n = 4–6 independent images per sample). *** P < .001, n.s., not significant, one-way ANOVA followed by Tukey’s HSD test. ( H ) Immunofluorescence analysis of γH2AX in GFP-vector or GFP-NSMF expressing IMR-90 cells with or without Ras G12V . Quantification of γH2AX foci per GFP-positive cell was performed in at least 42 cells per group. Scale bar, 20 μm. Data are presented as median. ** P < .01, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. All experiments were independently performed at least three times, and representative results are shown. ( I ) Correlation of NSMF expression with genomic instability in pan-cancer analysis. Genomic instability was assessed across 4315 pan-cancer samples from TCGA using multiple genomic instability features, including frequency of LOH, HRD-related LOH frequency, telomeric allelic imbalance, large-scale transitions, mutation burden per sample, and weighted genome integrity index. Tumors were categorized based on genomic instability scores as low (<25%, below first quartile), medium (25%–75%, between first and third quartile), or high (>75%, above third quartile). Statistical comparisons of NSMF expression levels across groups were performed using Wilcoxon rank-sum test. ( J ) Hypothetical model illustrating the role of NSMF in regulating replication stress, highlighting its critical function in alleviating excessive replication stress and preventing cytotoxic DNA damage. This regulatory activity supports a controlled level of genomic instability, thereby promoting CRC progression. Figure was created using BioRender.com.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Staining, Knockdown, Control, RNA Sequencing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transduction, Plasmid Preparation, Selection, Expressing, Immunofluorescence, Mutagenesis, Activity Assay