crc cell lines sw480  (ATCC)

Journal: International Journal of Oncology

Article Title: lncRNA AL445238.2 -USP4 axis regulates cell survival and stemness in colon cancer

doi: 10.3892/ijo.2026.5858

Figure Lengend Snippet: AL445238.2 regulates the survival of colon cancer cells. (A) Validation of gene manipulation efficiency in SW480 and DLD1 cell lines with stable overexpression and knockdown of AL445238.2 . (B) Cell Counting Kit-8 assay of SW480 and DLD1 stable cell lines. (C) Proportion of apoptotic SW480 and DLD1 stable cell lines (percentage of supernatant LDH/total LDH). (D) Statistical analysis of the Annexin V and PI staining assay(Proportion percentage of Apoptotic cells). (E) Annexin V and PI staining of SW480 and DLD1 stable cell lines. ** P<0.01, *** P<0.001, **** P<0.0001. NC, negative control; sh, short hairpin; OE, overexpression; LDH, lactate dehydrogenase; ns, not significant.

Article Snippet: The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection.

Techniques: Biomarker Discovery, Over Expression, Knockdown, Cell Counting, Stable Transfection, Staining, Negative Control

Journal: International Journal of Oncology

Article Title: lncRNA AL445238.2 -USP4 axis regulates cell survival and stemness in colon cancer

doi: 10.3892/ijo.2026.5858

Figure Lengend Snippet: AL445238.2 promotes the stemness and mitochondrial stability of colon cancer cells. (A) The expression of CD133 in SW480 and DLD1 stable cell lines was detected with flow cytometry. (B) The statistical analysis of CD133 MFI. (C) TMRM staining of SW480 and DLD1 stable cell lines. (D) The statistical analysis of TMRM MFI. (E) Tumor spheroid formation assay of SW480 and DLD1 stable cell lines. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. MFI, mean fluorescence intensity; TMRM, tetramethylrhodamine; NC, negative control; sh, short hairpin; OE, overexpression.

Article Snippet: The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection.

Techniques: Expressing, Stable Transfection, Flow Cytometry, Staining, Tube Formation Assay, Fluorescence, Negative Control, Over Expression

Journal: International Journal of Oncology

Article Title: lncRNA AL445238.2 -USP4 axis regulates cell survival and stemness in colon cancer

doi: 10.3892/ijo.2026.5858

Figure Lengend Snippet: AL445238.2 binding to USP4 stabilizes Bcl2 to regulate colon cancer cell apoptosis and stemness. (A) Pulldown assay with AL445238.2 and western blot detection of USP4. (B) Gene colocalization was detected by immunofluorescence (magnification, ×200). (C) Validation of gene manipulation efficiency in SW480 and DLD1 cell lines with stable overexpression and knockdown of USP4. (D) Western blotting was used to evaluate the expression of BAX, Bcl2 and C-CASP3 in stable SW480 cells; BAX and Bcl2 were normalized against GAPDH, C-CASP3 was normalized against total CASP3. Western blotting was used to evaluate the expression of ALDH1A1, Bmi1 and EpCAM in stable (E) DLD1 and (F) SW480 cells. (G) Co-immunoprecipitation experiments were used to verify the interaction between USP4 and Bcl2. (H) Western blotting was used to evaluate the effects of USP4 knockdown and MG132 on Bcl2 protein expression. (I) Protein half-life experiments were used to evaluate the effects of USP4 and AL445238.2 on the stability of Bcl2 protein. ** P<0.01, *** P<0.001, **** P<0.0001. USP4, ubiquitin specific peptidase 4; RIP, RNA immunoprecipitation; Ctrl, control; ALDH1A1, aldehyde dehydrogenase 1 family member A1; Bmi1, B lymphoma Mo-MLV insertion region 1; EpCAM, epithelial cell adhesion molecule; CHX, cycloheximide; OE, overexpression; sh, short hairpin; NC, negative control; ns, not significant; C-CASP3, cleaved caspase-3.

Article Snippet: The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection.

Techniques: Binding Assay, Western Blot, Immunofluorescence, Biomarker Discovery, Over Expression, Knockdown, Expressing, Immunoprecipitation, Ubiquitin Proteomics, RNA Immunoprecipitation, Control, Negative Control

Journal: International Journal of Oncology

Article Title: lncRNA AL445238.2 -USP4 axis regulates cell survival and stemness in colon cancer

doi: 10.3892/ijo.2026.5858

Figure Lengend Snippet: USP4 regulates the survival of colon cancer cells. (A) Cell Counting Kit-8 Assay of SW480 and DLD1 stable cell lines. (B) Proportion of apoptotic SW480 and DLD1 stable cell lines (percentage of supernatant LDH/total LDH). (C) Statistical analysis of the Annexin V and PI staining assay (percentage of apoptotic cells). (D) Annexin V and PI staining of SW480 and DLD1 stable cell lines. ** P<0.01, *** P<0.001, **** P<0.0001. USP4, ubiquitin specific peptidase 4; OE, overexpression; sh, short hairpin; NC, negative control; ns, not significant; LDH, lactate dehydrogenase.

Article Snippet: The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection.

Techniques: Cell Counting, Stable Transfection, Staining, Ubiquitin Proteomics, Over Expression, Negative Control

Journal: International Journal of Oncology

Article Title: lncRNA AL445238.2 -USP4 axis regulates cell survival and stemness in colon cancer

doi: 10.3892/ijo.2026.5858

Figure Lengend Snippet: USP4 promotes stemness and mitochondrial stability of colon cancer cells. (A) Western blotting was used to evaluate the expression of BAX, Bcl2 and C-CASP3 in stable SW480 cells; BAX and Bcl2 were normalized against GAPDH, C-CASP3 was normalized against total CASP3. (B) The expression of CD133 in the SW480 and DLD1 stable cell lines was detected with flow cytometry. (C) Statistical analysis of the CD133 MFI. (D) Statistical analysis of the TMRM MFI. (E) TMRM staining of SW480 and DLD1 stable cell lines. (F) Tumor spheroid formation assay of SW480 and DLD1 stable cell lines. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. MFI, mean fluorescence intensity; TMRM, tetramethylrhodamine; C-CASP3, cleaved caspase-3; NC, negative control; USP4, ubiquitin specific peptidase 4; sh, short hairpin; OE, overexpression.

Article Snippet: The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection.

Techniques: Western Blot, Expressing, Stable Transfection, Flow Cytometry, Staining, Tube Formation Assay, Fluorescence, Negative Control, Ubiquitin Proteomics, Over Expression

Journal: International Journal of Oncology

Article Title: lncRNA AL445238.2 -USP4 axis regulates cell survival and stemness in colon cancer

doi: 10.3892/ijo.2026.5858

Figure Lengend Snippet: AL445238.2 controlled USP4 promotes colon cancer cell migration. Migration assay of DLD1 and SW480 cells treated with shNC, sh- AL445238.2 , Vector or OE- AL445238.2 . (A) Representative images show migrated cells stained with crystal violet. (B) Quantitative analysis of migrated cell numbers in DLD1 (upper panel) and SW480 (lower panel) cells following AL445238.2 knockdown or overexpression. Migration assay of DLD1 and SW480 cells treated with shNC, sh-USP4, Vector or OE-USP4. (C) Representative images show migrated cells stained with crystal violet. (D) Quantitative analysis of migrated cell numbers in DLD1 (upper panel) and SW480 (lower panel) cells following USP4 knockdown or overexpression. Migration assay of DLD1 and SW480 cells with combined treatments: NC, OE- AL445238.2 , OE- AL445238.2 + OE-USP4 or OE- AL445238.2 + sh-USP4. (E) Representative images show migrated cells. (F) Quantitative analysis of migrated cell numbers in DLD1 (left panel) and SW480 (right panel) cells under combined treatment conditions. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. sh, short hairpin; NC, negative control; USP4, ubiquitin specific peptidase 4; OE, overexpression.

Article Snippet: The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection.

Techniques: Migration, Plasmid Preparation, Staining, Knockdown, Over Expression, Negative Control, Ubiquitin Proteomics

Journal: Translational Oncology

Article Title: The VAX2-SERPINE1 axis modulates colorectal cancer cell proliferation and apoptosis through WNT/beta-catenin signaling

doi: 10.1016/j.tranon.2026.102703

Figure Lengend Snippet: VAX2 is upregulated and correlates with malignant progression in CRC. (A) Expression of VAX2 in the indicated CRC cell lines was determined by western blotting normalized to colon epithelial cell line FHC as a control. (B) Expression of VAX2 was determined in 13 pairs of CRC and normal tissues by western blotting using Wilcoxon signed-rank test. **, P < 0.05. (C) Expression of VAX2 was determined in 13 pairs of CRC and normal tissues by IHC. (D) Expression of VAX2 was determined in 79 pairs of CRC and normal tissues by IHC. (E) Quantitative expression of VAX2 in 79 pairs of CRC compared with normal tissues using Wilcoxon signed-rank test. ****, P < 0.001. (F) Quantitative expression of VAX2 in 99 CRC compared with 79 normal tissues using Mann–Whitney U test. ****, P < 0.001. (G) VAX2 expression levels in different subgroups stratified based on T stage. Mann–Whitney U test, **, P < 0.05. (H) VAX2 expression levels in different subgroups stratified based on AJCC stage. Mann–Whitney U test, **, P < 0.05. (I) Kaplan-Meier analyses of the overall survival probability in the TMA consisting of 99 patients based on VAX2 expression. Scale bars in C&D represent 100 μm.

Article Snippet: Normal colonic epithelial cell line FHC, human CRC cell lines (RKO, SW1116, HCT116 and SW620) and human GC cell lines (AGS) were purchased from the American Type Culture Collection (ATCC, USA; Catalog Numbers: FHC CRL-1831, RKO CRL-2577, SW1116 CCL-233, HCT116 CCL-247, SW620 CCL-227, AGS CRL-1739).

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY

Journal: Translational Oncology

Article Title: The VAX2-SERPINE1 axis modulates colorectal cancer cell proliferation and apoptosis through WNT/beta-catenin signaling

doi: 10.1016/j.tranon.2026.102703

Figure Lengend Snippet: VAX2 facilitates CRC cell malignant behavior in vitro and in vivo. (A1/2) Expression of VAX2 in transfected LoVo, HCT116 and SW480 cell lines were analyzed by western blotting. (B1/2) Effects of VAX2 overexpression (B1) or knockdown (B2) on the proliferation of CRC cell lines, as determined by colony formation assay. ***, P < 0.01, Vector vs. VAX2; ***, P < 0.01, ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. (C1/2) Representative graphs of EdU positivity in cells transfected with Vector and VAX2(C1) or Scr-siRNA and VAX2-siRNAp (C2). ***, P < 0.01, Vector vs. VAX2; ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. (D1/2) Representative graphs of apoptosis assays in cells transfected with Vector and VAX2(D1) or Scr-siRNA and VAX2-siRNAp (D2). ****, P < 0.001, Vector vs. VAX2; ***, P < 0.01, ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. (E1/2) Representative graphs of Transwell assays in cells transfected with Vector and VAX2(E1) or Scr-siRNA and VAX2-siRNAp (E2). ***, P < 0.01, Vector vs. VAX2; **, P < 0.05, ***, P < 0.01, Scr-siRNA vs. VAX2-siRNAp.

Article Snippet: Normal colonic epithelial cell line FHC, human CRC cell lines (RKO, SW1116, HCT116 and SW620) and human GC cell lines (AGS) were purchased from the American Type Culture Collection (ATCC, USA; Catalog Numbers: FHC CRL-1831, RKO CRL-2577, SW1116 CCL-233, HCT116 CCL-247, SW620 CCL-227, AGS CRL-1739).

Techniques: In Vitro, In Vivo, Expressing, Transfection, Western Blot, Over Expression, Knockdown, Colony Assay, Plasmid Preparation

Journal: Translational Oncology

Article Title: The VAX2-SERPINE1 axis modulates colorectal cancer cell proliferation and apoptosis through WNT/beta-catenin signaling

doi: 10.1016/j.tranon.2026.102703

Figure Lengend Snippet: VAX2 involves in WNT/beta-catenin signaling by promoting beta-catenin nucleus accumulation. (A&B) Kyoto Encyclopedia of Genes and Genomes (KEGG) (A) and Gene Set Enrichment Analysis (GSEA) (B) were conducted with differentially-expressed genes between Scr-siRNA and VAX2-siRNAp. (C) Western blotting of cytoplasmic and nuclear fractions of beta-catenin in VAX2 knockdown HCT116 cells. (D) Immunofluorescence of beta-catenin in VAX2 knockdown HCT116 and SW480 cells. (E) Co-immunoprecipitation of enrichment of beta-catenin binding to LEF1 in VAX2 knockdown HCT116 and SW480 cells. (F) qRT-PCR of abundance of beta-catenin-TCF/LEF-1 targets (MMP7, Cyclin D1 and c-jun) in VAX2 knockdown HCT116 and SW480 cells. **, P < 0.05, ***, P < 0.01, ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. (G) VAX2-modulated CRC cells were transfected with the TOP/FOPflash reporter plasmid, and the reporter activities were determined 48 hours later ( n = 3). ***, P < 0.01, ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. Yellow scale bar in D, 50 μm; green scale bar in D, 20 μm.

Article Snippet: Normal colonic epithelial cell line FHC, human CRC cell lines (RKO, SW1116, HCT116 and SW620) and human GC cell lines (AGS) were purchased from the American Type Culture Collection (ATCC, USA; Catalog Numbers: FHC CRL-1831, RKO CRL-2577, SW1116 CCL-233, HCT116 CCL-247, SW620 CCL-227, AGS CRL-1739).

Techniques: Western Blot, Knockdown, Immunofluorescence, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Translational Oncology

Article Title: The VAX2-SERPINE1 axis modulates colorectal cancer cell proliferation and apoptosis through WNT/beta-catenin signaling

doi: 10.1016/j.tranon.2026.102703

Figure Lengend Snippet: VAX2 transcriptionally activates SERPINE1 in gastrointestinal cancers. (A) Heatmap of top 10 upregulated and downregulated genes between Scr-siRNA and VAX2-siRNAp. (B) Volcano plot of differentially-expressed genes between Scr-siRNA and VAX2-siRNAp. (C) SERPINE1 abundance was detected between Scr-siRNA and VAX2-siRNAp in CRC cell lines via qRT-PCR. ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. (D) SERPINE1 abundance was detected between Vector and VAX2 in LoVo via qRT-PCR. ***, P < 0.01, Vector vs. VAX2. (E1/2) SERPINE1 abundance was detected between Vector and VAX2 in AGS (E1) and HGC-27 (E2) cells via qRT-PCR. **, P < 0.05, ****, P < 0.001, Vector vs. VAX2. (F) Enrichment site and binding motif of VAX2 on SERPINE1 promotor predicted by Jaspar database. (G) Chromatin immunoprecipitation (ChIP) was performed in CRC cell lines HCT116 and SW480 and GC cell lines AGS. (H) Wildtype (WT) or mutation (MUT) SERPINE1 promotor was cloned into luciferase plasmids. (I) The WT or MUT SERPINE1 promoter construct was co-transfected with VAX2-siRNAp or Scr-siRNA in HCT116 and SW480 cells, and the relative luciferase activity to corresponding control was determined ( n = 3). *, P >0.05, ***, P < 0.01, ****, P < 0.001, Scr-siRNA vs. VAX2-siRNAp. (J) The WT or MUT SERPINE1 promoter construct was co-transfected with VAX2 or Vector in AGS cells, and the relative luciferase activity to corresponding control was determined ( n = 3). *, P >0.05, ***, P < 0.01, ****, P < 0.001, Vector vs. VAX2.

Article Snippet: Normal colonic epithelial cell line FHC, human CRC cell lines (RKO, SW1116, HCT116 and SW620) and human GC cell lines (AGS) were purchased from the American Type Culture Collection (ATCC, USA; Catalog Numbers: FHC CRL-1831, RKO CRL-2577, SW1116 CCL-233, HCT116 CCL-247, SW620 CCL-227, AGS CRL-1739).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Mutagenesis, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Control

Journal: Translational Oncology

Article Title: The VAX2-SERPINE1 axis modulates colorectal cancer cell proliferation and apoptosis through WNT/beta-catenin signaling

doi: 10.1016/j.tranon.2026.102703

Figure Lengend Snippet: VAX2 mediates CRC proliferation by transactivating SERPINE1 expression in vitro. (A) Proliferation of transfected CRC cell lines, as determined by colony formation assays. ****, P < 0.001. (B) Proliferation of transfected CRC cell lines, as determined by EdU assays. ****, P < 0.001. (C) Apoptosis of transfected CRC cell lines, as determined by apoptpsis assays using flow cytometry. (D) Co-immunoprecipitation of enrichment of beta-catenin binding to LEF1 in transfected HCT116 and SW480. (E) qRT-PCR of abundance of beta-catenin-TCF/LEF-1 targets (MMP7, Cyclin D1 and c-jun) in transfected HCT116 and SW480. **, P < 0.05, ***, P < 0.01, ****, P < 0.001.

Article Snippet: Normal colonic epithelial cell line FHC, human CRC cell lines (RKO, SW1116, HCT116 and SW620) and human GC cell lines (AGS) were purchased from the American Type Culture Collection (ATCC, USA; Catalog Numbers: FHC CRL-1831, RKO CRL-2577, SW1116 CCL-233, HCT116 CCL-247, SW620 CCL-227, AGS CRL-1739).

Techniques: Expressing, In Vitro, Transfection, Flow Cytometry, Immunoprecipitation, Binding Assay, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: The VAX2-SERPINE1 axis modulates colorectal cancer cell proliferation and apoptosis through WNT/beta-catenin signaling

doi: 10.1016/j.tranon.2026.102703

Figure Lengend Snippet: VAX2 is correlated with SERPINE1 in human CRC tissues. (A) Expression of VAX2 was determined in 71 pairs of CRC and NT by qRT-PCR using waterfall plot. (B) Expression of SERPINE1 was determined in 71 pairs of CRC and NT by qRT-PCR using waterfall plot. (C) Expression of VAX2 was determined in 71 pairs of CRC and NT by qRT-PCR using box plot. ****, P < 0.001. (D) Expression of SERPINE1 was determined in 71 pairs of CRC and NT by qRT-PCR using box plot. **, P < 0.05. (E) Correlation between VAX2 and SERPINE1 in 71 pairs CRC tissues. (F) Correlation between VAX2 and SERPINE1 in TCGA-CRC. (G) VAX2 and SERPINE1 expression in CRC tissues were detected using HE and IHC. ( H) The signal transduction mechanism of VAX2. Scale bar in G, 100 μm.

Article Snippet: Normal colonic epithelial cell line FHC, human CRC cell lines (RKO, SW1116, HCT116 and SW620) and human GC cell lines (AGS) were purchased from the American Type Culture Collection (ATCC, USA; Catalog Numbers: FHC CRL-1831, RKO CRL-2577, SW1116 CCL-233, HCT116 CCL-247, SW620 CCL-227, AGS CRL-1739).

Techniques: Expressing, Quantitative RT-PCR, Transduction

Journal: iScience

Article Title: Circ_0003323 binds METTL1 to mediate KLK6 m7G methylation modification and promote the progression of colorectal cancer

doi: 10.1016/j.isci.2026.114999

Figure Lengend Snippet: Identification and expression detection of hsa_circ_0003323 in CRC tissues and cells (A) Venn diagram showing overlapping differentially expressed genes in the GSE126094 and GSE138589 datasets. (B) Heatmap displaying the fold change (logFC) of the overlapping genes. (C) Schematic diagram illustrating the formation of hsa_circ_0003323 through reverse splicing, confirmed by Sanger sequencing. (D) RT-PCR using divergent and convergent primers to amplify hsa_circ_0003323 from cDNA or gDNA extracted from HCT116 cells. (E) RT-qPCR analysis of hsa_circ_0003323 and APP mRNA expression levels in HCT116 cells with or without RNase R treatment. GAPDH was used as a negative control. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the mock group. (F) RT-qPCR showing the expression level of hsa_circ_0003323 in CRC tissues and paracancerous (PC) tissues ( n = 30), PC tissues were collected at least 5 cm away from the edge of the primary tumor lesion. ∗∗ p < 0.01 compared to the PC group. (G) RT-qPCR demonstrating the expression of hsa_circ_0003323 in CRC cells. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to NCM460 group. (H) RT-qPCR detecting the expression levels of hsa_circ_0003323 in CRC cells after hsa_circ_0003323 knockdown. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the shNC group. (I) CCK8 assay, (J and K) transwell assay, and (L) colony formation assay were performed to evaluate the effect of knockdown of hsa_circ_0003323 expression on the proliferation, migration, invasion, and colony formation ability of HCT116 and SW480 cells. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the shNC group. Scale bars represent 100 μm.

Article Snippet: And CRC cell lines (HCT116 and SW480; ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO 2 .

Techniques: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Knockdown, CCK-8 Assay, Transwell Assay, Colony Assay, Migration

Journal: iScience

Article Title: Circ_0003323 binds METTL1 to mediate KLK6 m7G methylation modification and promote the progression of colorectal cancer

doi: 10.1016/j.isci.2026.114999

Figure Lengend Snippet: hsa_circ_0003323 regulates CRC m7G methylation by binding to METTL1 (A) Peptide segments of METTL1 pulled down by the hsa_circ_0003323 probe. (B) FISH combined with immunofluorescence experiment to observe the co-localization of hsa_circ_0003323 and METTL1 in the nucleus of CRC cells. Scale bars represent 20 μm. (C) RNA pulldown experiment using biotin-labeled antisense or sense probes against hsa_circ_0003323 in HCT116 cell lysates, followed by western blot to detect METTL1 in the pulled-down products. (D) RIP experiment using METTL1 antibody and RT-qPCR to detect hsa_circ_0003323 . The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the IgG group. (E) IHC experiment was used to measure the overall m7G modification levels in CRC tissues and PC tissues. Scale bars represent 100 μm. Dot blot analysis using an m7G antibody to detect the overall m7G modification levels in CRC tissues (F) and CRC cells (G). (H) IHC was used to detect the expression levels of METTL1 in CRC tissues and PC tissues. Scale bars represent 100 μm. (I) Western blot assays were used to detect the expression levels of METTL1 in CRC tissues and PC tissues. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the PC group. (J and K) Western blot assays were used to detect the expression levels of METTL1 in CRC cells. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to NCM460 group. (L) RT-qPCR assays were used to detect the expression levels of METTL1 in CRC cells after knockdown of METTL1. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the shNC group. Dot blot analysis to measure the changes in m7G modification levels of CRC cells after knockdown of METTL1 (M and N) or knockdown of hsa_circ_0003323 (O).

Article Snippet: And CRC cell lines (HCT116 and SW480; ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO 2 .

Techniques: Methylation, Binding Assay, Immunofluorescence, Labeling, Western Blot, Quantitative RT-PCR, Modification, Dot Blot, Expressing, Knockdown

Journal: iScience

Article Title: Circ_0003323 binds METTL1 to mediate KLK6 m7G methylation modification and promote the progression of colorectal cancer

doi: 10.1016/j.isci.2026.114999

Figure Lengend Snippet: METTL1 reverses the regulatory effects of hsa_circ_0003323 on the proliferation, migration, and invasion capacity of CRC cells (A) CCK8 assay, (B and C) transwell assay, and (D) colony formation assay were performed to evaluate the effect of knockdown of METTL1 expression on the proliferation, migration, invasion, and colony formation ability of HCT116 and SW480 cells. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the shNC group. Scale bars represent 100 μm. (E) CCK8 assay, (F and G) transwell assay, and (H) colony formation assay were performed to evaluate the effect of proliferation, migration, invasion, and colony formation ability of HCT116 cells. The values are presented as mean ± SD ( n = 3), ns, no significant, ∗∗ p < 0.01 compared to the control group. Scale bars represent 100 μm.

Article Snippet: And CRC cell lines (HCT116 and SW480; ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO 2 .

Techniques: Migration, CCK-8 Assay, Transwell Assay, Colony Assay, Knockdown, Expressing, Control

Journal: iScience

Article Title: Circ_0003323 binds METTL1 to mediate KLK6 m7G methylation modification and promote the progression of colorectal cancer

doi: 10.1016/j.isci.2026.114999

Figure Lengend Snippet: hsa_circ_0003323 mediates m7G methylation of KLK6 (A) Heatmap showing RNA-seq results upon knockdown of METTL1. (B) Knockdown of METTL1 was followed by RNA-seq combined with the TCGA database to analyze shared differential genes. (C) Potential m7G modification sites in KLK6 mRNA. (D) IHC experiment to examine the expression levels of KLK6 in CRC tissues and PC tissues. Scale bars represent 100 μm. (E) RT-qPCR assays were used to detect the mRNA levels of KLK6 in CRC cells after METTL1 knockdown. (F and G) Western blot assays were used to detect the protein levels of KLK6 in CRC cells after METTL1 knockdown. MeRIP-qPCR assay to detect the m7G modification level of KLK6 mRNA (H) and the change of m7G modification level of KLK6 mRNA after knockdown of METTL1 (I). (J) The nascent RNA assay detects changes in the nascent RNA level of KLK6 after knockdown of METTL1. (K and L) The RNA half-life assay detects changes in the mRNA stability level of KLK6 after knockdown of METTL1. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the shNC group. (M) m7G-RIP-qPCR experiment to measure the changes in m7G modification levels of KLK6 mRNA in HCT116 cells with hsa_circ_0003323 overexpression. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the vector group. (N) A schematic representation of KLK6-5′ UTR of luciferase reporters. R-Luc is an internal control reporter vector. F-Luc: Firefly luciferase; R-Luc: Renilla luciferase. (O) The mRNA abundancy of KLK6 -5′ UTR of luciferase reporters in METTL deficiency cells. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the shNC group.

Article Snippet: And CRC cell lines (HCT116 and SW480; ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO 2 .

Techniques: Methylation, RNA Sequencing, Knockdown, Modification, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Plasmid Preparation, Luciferase, Control

Journal: iScience

Article Title: Circ_0003323 binds METTL1 to mediate KLK6 m7G methylation modification and promote the progression of colorectal cancer

doi: 10.1016/j.isci.2026.114999

Figure Lengend Snippet: KLK6 reverses the regulatory effects of METTL1 on the proliferation, migration, and invasion capacity of CRC cells (A) CCK8 assay, (B and C) transwell assay, and (D) colony formation assay were performed to evaluate the effect of knockdown of KLK6 expression on the proliferation, migration, invasion, and colony formation ability of HCT116 cells. The values are presented as mean ± SD ( n = 3), ∗∗ p < 0.01 compared to the siNC group. Scale bars represent 100 μm. (E) CCK8 assay, (F and G) transwell assay, and (H) colony formation assay were performed to evaluate the effect of proliferation, migration, invasion, and colony formation ability of HCT116 cells. The values are presented as mean ± SD ( n = 3), ns, no significant, ∗∗ p < 0.01 compared to the control group. Scale bars represent 100 μm.

Article Snippet: And CRC cell lines (HCT116 and SW480; ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO 2 .

Techniques: Migration, CCK-8 Assay, Transwell Assay, Colony Assay, Knockdown, Expressing, Control