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