Journal: STAR Protocols

Article Title: Protocol for developing a mucus-producing gut-on-a-chip model from Caco-2 and HT29-MTX-E12 cells

doi: 10.1016/j.xpro.2025.104321

Figure Lengend Snippet: Representative examples of Caco-2/HT29-MTX-E12 cell seeding densities in the GoC platform (A) Empty chip with porous membrane. (B) Example of evenly seeded Caco-2/HT29-MTX-E12 (7:3) cells in the top channel of the chip. (C) Example of low density of Caco-2/HT29-MTX-E12 (7:3) cells in the top channel of the chip. (D) Example of high density of Caco-2/HT29-MTX-E12 (7:3) cells in the top channel of the chip. Scale bar - 0.2mm.

Article Snippet: Caco-2 , ATCC , HTB-37.

Techniques: Membrane

Journal: STAR Protocols

Article Title: Protocol for developing a mucus-producing gut-on-a-chip model from Caco-2 and HT29-MTX-E12 cells

doi: 10.1016/j.xpro.2025.104321

Figure Lengend Snippet: GoC characterization with brightfield and confocal microscopy (A) Caco-2:HT29-MTX brightfield microscopy at ratio 9:1 and 7:3. (B) Confocal microscopy for co-culture 9:1 and 7:3 with cell markers DAPI, Villin1, Muc5AC 3D imaging. (C) Immunofluorescence and confocal microscopy for co-culture 9:1 and 7:3 with cell markers DAPI, Phalloidin, Villin, Muc5AC and ZO1 2D imaging. Scale bar= 75 μm. 2D maximum intensity projections from corresponding Z-stacks were subsequently processed and exported using Leica Application Suite X software.

Article Snippet: Caco-2 , ATCC , HTB-37.

Techniques: Confocal Microscopy, Microscopy, Co-Culture Assay, Imaging, Immunofluorescence, Software

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