human crc cell lines  (ATCC)

Journal: Genes & Diseases

Article Title: Targeting PAK1 suppresses tumor progression by promoting mRNA decay of oncogenic factors and enhancing chemotherapeutic efficacy in colorectal cancer

doi: 10.1016/j.gendis.2025.101683

Figure Lengend Snippet: PAK1 deficiency inhibits CRC progression. (A) Relative mRNA expression levels of PAK1 in CRC cell lines (CACO2, DLD1, HT29, LoVo, SW480, and HCT116) compared with normal intestinal epithelial cells (HCoEpiC and NCM460). (B) Western blotting analysis of PAK1 protein levels in CRC cell lines and normal intestinal epithelial cells (HCoEpiC and NCM460). (C) Immunohistochemical staining of PAK1 in CRC tissues from the Human Protein Atlas (HPA) database. (D) Proliferation assays of DLD1 and HT29 cells with PAK1 knockout (KO) compared with wild-type (WT) cells. Cell viability was measured using the CCK8 assay at different time points (0, 24, 48, and 72 h). (E) Colony formation assays of DLD1 and HT29 cells with PAK1 KO compared with WT cells. (F) Migration and invasion assays of DLD1 and HT29 cells with PAK1 KO compared with WT cells. The data were presented as mean ± standard deviation, with statistical significance indicated (ns, no significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: We acquired six human CRC cell lines (CACO2, DLD1, HT29, SW480, HCT116, and LoVo) from the American Type Culture Collection, along with the human normal colon epithelial cell lines HCoEpiC and NCM460.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Knock-Out, CCK-8 Assay, Migration, Standard Deviation

Journal: Genes & Diseases

Article Title: Targeting PAK1 suppresses tumor progression by promoting mRNA decay of oncogenic factors and enhancing chemotherapeutic efficacy in colorectal cancer

doi: 10.1016/j.gendis.2025.101683

Figure Lengend Snippet: PAK1 activates the mTOR-S6K pathway in CRC . (A) GSEA results showed significant enrichment in pathways related to PI3K/AKT/mTOR signaling and mTORC1 signaling in PAK1 high-expression samples. (B) SUnSET assay showed the translation rates in wild-type (WT) and PAK1 knockout (KO) DLD1 (left) and HT29 (right) cell lines after 15 min and 30 min of puromycin treatment. Puromycin incorporation was detected via immunoblotting. (C) Western blotting analysis showed that PAK1 KO in DLD1 cells resulted in a significant decrease in the expression of mTOR, p70 S6K, and EIF4G1 proteins. Re-expressing PAK1 in KO cells restored the expression of mTOR, p70 S6K, and EIF4G1 to levels similar to those observed in WT cells. (D) Western blotting analysis showed that PAK1 OE in SW480 cells led to increased expression of mTOR, p70 S6K, and EIF4G1 proteins. Knocking down PAK1 in OE cells using shRNA reduced the expression of these proteins to levels similar to those observed in NC cells. (E, F) Quantitative real-time PCR results showed the relative expression of MTOR, RPS6KB1, and EIF4G1 in DLD1 and SW480 cells. The data were presented as mean ± standard deviation, with statistical significance indicated (ns, no significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: We acquired six human CRC cell lines (CACO2, DLD1, HT29, SW480, HCT116, and LoVo) from the American Type Culture Collection, along with the human normal colon epithelial cell lines HCoEpiC and NCM460.

Techniques: Expressing, Knock-Out, Western Blot, shRNA, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Genes & Diseases

Article Title: Targeting PAK1 suppresses tumor progression by promoting mRNA decay of oncogenic factors and enhancing chemotherapeutic efficacy in colorectal cancer

doi: 10.1016/j.gendis.2025.101683

Figure Lengend Snippet: PAK1 inhibits mRNA decay of multiple oncogenic factors in CRC cells. (A – H) Relative mRNA expression levels of MTOR (A), CD44 (B), SAA1 (C), U6 (D), PAK2 (E), PAK4 (F), PAK5 (G), and PAK6 (H) in wild-type (WT) and knockout (KO) HT29 and DLD1 cells at 1, 3, and 6 h after actinomycin D treatment. The data were presented as mean ± standard deviation, with statistical significance indicated (ns, no significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: We acquired six human CRC cell lines (CACO2, DLD1, HT29, SW480, HCT116, and LoVo) from the American Type Culture Collection, along with the human normal colon epithelial cell lines HCoEpiC and NCM460.

Techniques: Expressing, Knock-Out, Standard Deviation

Journal: Genes & Diseases

Article Title: Targeting PAK1 suppresses tumor progression by promoting mRNA decay of oncogenic factors and enhancing chemotherapeutic efficacy in colorectal cancer

doi: 10.1016/j.gendis.2025.101683

Figure Lengend Snippet: Efficacy of PF3758309 (PF-309) in CRC cell lines and its impact on signaling pathways. (A, B) Impact of PAK1 knockout (KO) on the sensitivity to PF-309 in DLD1 (A) and HT29 (B) cells. PAK1 KO led to a significant increase in IC 50 compared with wild-type (WT) cells. (C) Dose–response curves for PF-309 in HCT116 cells expressing Control, Mock, and mut-K299R construct. The mut-K299R construct showed a significantly higher IC 50 compared with other groups, indicating reduced sensitivity to PF-309 when PAK1 kinase activity is disrupted. (D) Based on the binding mode of PF-309 and PAK1 obtained by docking, the left picture is the overall view, and the right picture is the local view. In the figure, the yellow stick is a small molecule, the cyan cartoon is a protein, the blue line represents hydrogen bonding, and the gray dashed line represents hydrophobic action. (E, F) Western blotting analysis of HCT116 cells treated with PF-309 explored its impact on various signaling pathways. Treatment for 3 h (E) and 24 h (F) with PF-309 resulted in the inhibition of several key pathways, including mTOR, p70 S6K, EIF4G1, NF-κB, IKB-α, c-Myc, WNT3A, and β-catenin. (G) Western blotting analysis of HCT116 cells treated with DMSO or PF-309, comparing Control, Mock, and mut-K299R groups. The mut-K299R group displayed minimal changes in key signaling pathways, including mTOR, p70 S6K, p-p70 S6K, NF-κB, IKB-α, c-Myc, CD44, and SAA1, following PF-309 treatment, highlighting the importance of intact PAK1 function for PF-309's efficacy.

Article Snippet: We acquired six human CRC cell lines (CACO2, DLD1, HT29, SW480, HCT116, and LoVo) from the American Type Culture Collection, along with the human normal colon epithelial cell lines HCoEpiC and NCM460.

Techniques: Protein-Protein interactions, Knock-Out, Expressing, Control, Construct, Activity Assay, Binding Assay, Western Blot, Inhibition

Journal: Genes & Diseases

Article Title: Targeting PAK1 suppresses tumor progression by promoting mRNA decay of oncogenic factors and enhancing chemotherapeutic efficacy in colorectal cancer

doi: 10.1016/j.gendis.2025.101683

Figure Lengend Snippet: Synergistic effects of PF3758309 (PF-309) and oxaliplatin (OXA) in HCT116 cells. (A) Synergy analysis of PF-309 and OXA combination using SynergyFinder software in HCT116 cells. The HSA synergy score of 7.069 indicates a moderate level of synergy. (B) Determination of optimal concentrations for OXA (3125 nM) and PF-309 (1.53 nM) based on SynergyFinder analysis. (C) CCK-8 proliferation assay results show the cell viability of HCT116 cells treated with optimal concentrations of OXA, PF-309, and their combination over three days, measured by absorbance at 450 nm. The combination treatment significantly reduced cell proliferation. (D) Colony formation assay demonstrated that the combination treatment significantly decreased the number of colonies formed in HCT116 cells. (E) Transwell assay results indicate that the combination of PF-309 and OXA could significantly inhibit the migration and invasion of HCT116 cells. (F) Effect of PF-309 and OXA combination on CRC organoids. Treatment with PF-309, OXA, or their combination was applied to two CRC organoid models (P01 and P02). The combination treatment significantly reduced organoid size and viability, as indicated by ATP levels, compared with each drug alone. The scale bar represents 100 μm. The data were presented as mean ± standard deviation, with statistical significance indicated (ns, no significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: We acquired six human CRC cell lines (CACO2, DLD1, HT29, SW480, HCT116, and LoVo) from the American Type Culture Collection, along with the human normal colon epithelial cell lines HCoEpiC and NCM460.

Techniques: Software, CCK-8 Assay, Proliferation Assay, Colony Assay, Transwell Assay, Migration, Standard Deviation

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