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: 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: SW480 cell line , ATCC , CCL-228.

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: 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: SW480 cell line , ATCC , CCL-228.

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: Combined application of hsa_circ_0003323 and METTL1 deficiency achieved better tumor inhibitory effect in vivo (A and B) The effect of hsa_circ_0003323 and METTL1 deficiency on tumor formation in HCT116 and SW480 cells was examined by subcutaneous graft assay in nude mice and the tumor volume. The values are presented as mean ± SD ( n = 6), ∗∗ p < 0.01 compared to the control group, ## p < 0.01 compared to the sh hsa_circ_0003323 group; (H) a schematic diagram.

Article Snippet: SW480 cell line , ATCC , CCL-228.

Techniques: In Vivo, Control