Journal: International Journal of Molecular Sciences

Article Title: SLC25A5 Suppresses Colorectal Cancer Growth and Metastasis Through Regulation of the EIF3A/PI3K/AKT Axis

doi: 10.3390/ijms27104334

Figure Lengend Snippet: SLC25A5 inhibits CRC progression by suppressing proliferation and promoting apoptosis: ( A , B ) Western blot ( A ) and RT-qPCR ( B ) analysis confirming the successful overexpression of SLC25A5 in HCT-8 and HT-29 cell lines. ( C , D ) CCK-8 proliferation assays showing the growth rates of HCT-8 ( C ) and HT-29 ( D ) cells transfected with either empty vector or SLC25A5 expression plasmid. ( E – H ) Representative images ( E , F ) and quantitative analysis ( G , H ) of EdU incorporation assays in HCT-8 and HT-29 cells (Nuclei were stained with DAPI (blue), and proliferating cells were stained with EdU (red). Scale bars: 200 μm). ( I ) Representative images and statistical analysis of colony formation assays. ( J ) Flow cytometry analysis and quantification of apoptotic cells in the indicated groups. ( K ) Western blot analysis of Bcl-2, Bax, and Cleaved-caspase3 (C-caspase3) protein levels in HCT-8 and HT-29 cells. ( L – N ) Representative images of xenograft tumors ( L ), tumor growth curves ( M ), and final tumor weights ( N ) from nude mice injected with the indicated HCT-8 cells (n = 6 per group). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human CRC cell lines HCT-8 and HT-29 were obtained from ATCC and cultured in RPMI-1640 or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Western Blot, Quantitative RT-PCR, Over Expression, CCK-8 Assay, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Injection

Journal: International Journal of Molecular Sciences

Article Title: SLC25A5 Suppresses Colorectal Cancer Growth and Metastasis Through Regulation of the EIF3A/PI3K/AKT Axis

doi: 10.3390/ijms27104334

Figure Lengend Snippet: SLC25A5 inhibits CRC metastasis by suppressing EMT and cell motility: ( A , B ) Representative images ( A ) and statistical analysis ( B ) of wound healing assays in HCT-8 and HT-29 cells at 0, 24, and 48 h (Scale bars: 500 μm). ( C , D ) Representative images ( C ) and quantitative analysis ( D ) of Transwell migration and Matrigel invasion assays (Scale bars: 100 μm). ( E ) Immunofluorescence staining of E-cadherin (orange) and N-cadherin (red) in the indicated CRC cells; nuclei were stained with DAPI (blue) (Scale bars: 50 μm). ( F ) Western blot analysis of E-cadherin, N-cadherin, and Vimentin protein levels in HCT-8 and HT-29 cells. ( G ) Representative images of livers and H&E-stained liver sections from the in vivo metastasis model. ( H , I ) Quantification of the number of metastatic nodules per area ( H ) and the percentage of tumor/liver area ( I ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human CRC cell lines HCT-8 and HT-29 were obtained from ATCC and cultured in RPMI-1640 or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Migration, Immunofluorescence, Staining, Western Blot, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: SLC25A5 Suppresses Colorectal Cancer Growth and Metastasis Through Regulation of the EIF3A/PI3K/AKT Axis

doi: 10.3390/ijms27104334

Figure Lengend Snippet: SLC25A5 Modulates the Proteasomal Degradation of EIF3A: ( A ) Silver staining showing protein bands pulled down by SLC25A5 in HCT-8 cells; arrows indicate EIF3A and SLC25A5. ( B ) Endogenous Co-IP analysis demonstrating the interaction between SLC25A5 and EIF3A in CRC cells. ( C ) Immunofluorescence images showing the co-localization of EIF3A (red) and SLC25A5 (green) in HCT-8 and HT-29 cells (Nuclei were counterstained with DAPI (blue). The yellow color in the merged images indicates the co-localization of EIF3A and SLC25A5 proteins. Scale bars: 10 μm). ( D , E ) RT-qPCR ( D ) and Western blot ( E ) analysis of EIF3A expression in CRC cells overexpressing SLC25A5. ( F ) Western blot analysis of EIF3A in SLC25A5-overexpressing cells treated with MG132 (10 μM) or CQ (50 μM). ( G , H ) Representative Western blot ( G ) and quantitative degradation curves ( H ) of EIF3A in HCT-8 cells treated with CHX (100 μg/mL) over a 24-h time course. ( I ) Ubiquitination assay showing the effect of SLC25A5 overexpression on the poly-ubiquitination of EIF3A in HCT-8 cells. ( J , K ) Representative Western blot ( J ) and quantitative degradation curves ( K ) of EIF3A in HT-29 cells treated with CHX (100 μg/mL). ( L ) Ubiquitination assay showing SLC25A5-mediated EIF3A poly-ubiquitination in HT-29 cells. ( M ) Representative IHC images showing the expression of SLC25A5 and EIF3A in two clinical CRC cases (Scale bars: 100 μm). ( N ) Pearson correlation analysis between SLC25A5 and EIF3A IHC scores in CRC specimens (n = 30). *** p < 0.001, ns, non-significant.

Article Snippet: Human CRC cell lines HCT-8 and HT-29 were obtained from ATCC and cultured in RPMI-1640 or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Silver Staining, Co-Immunoprecipitation Assay, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Ubiquitin Proteomics, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: SLC25A5 Suppresses Colorectal Cancer Growth and Metastasis Through Regulation of the EIF3A/PI3K/AKT Axis

doi: 10.3390/ijms27104334

Figure Lengend Snippet: SLC25A5 Suppresses CRC Progression in Association with EIF3A: ( A – D ) Representative images ( A , C ) and quantitative analysis ( B , D ) of EdU incorporation assays in HCT-8 and HT-29 cells transfected with the indicated plasmids (Nuclei were stained with DAPI (blue), and proliferating cells were stained with EdU (red). Scale bars: 200 μm). ( E , F ) Colony formation assays of CRC cells after the indicated treatments. ( G ) Western blot analysis of Bcl-2, Bax, and cleaved-caspase3 protein levels in rescue experiments. ( H – J ) Representative images of xenograft tumors ( H ), tumor growth curves ( I ), and final tumor weights ( J ) from the indicated groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human CRC cell lines HCT-8 and HT-29 were obtained from ATCC and cultured in RPMI-1640 or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Transfection, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SLC25A5 Suppresses Colorectal Cancer Growth and Metastasis Through Regulation of the EIF3A/PI3K/AKT Axis

doi: 10.3390/ijms27104334

Figure Lengend Snippet: SLC25A5 Suppresses CRC Metastasis and EMT in Association with EIF3A: ( A – C ) Representative images ( A ) and quantification ( B , C ) of Transwell invasion assays in CRC cells after the indicated treatments (Scale bars: 100 μm). ( D – F ) Representative images ( D ) and quantitative analysis ( E , F ) of wound healing assays. ( G , H ) Immunofluorescence staining of E-cadherin (orange) and N-cadherin (red) in HCT-8 ( G ) and HT-29 ( H ) cells (Nuclei were counterstained with DAPI (blue). Scale bars: 50 μm). ( I ) Western blot analysis of EIF3A, E-cadherin, N-cadherin, and Vimentin protein levels in rescue groups. ( J ) Representative images of liver metastases and H&E staining from the indicated groups. ( K ) Statistical analysis of the number of metastatic nodules and tumor/liver area ratio. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human CRC cell lines HCT-8 and HT-29 were obtained from ATCC and cultured in RPMI-1640 or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Immunofluorescence, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SLC25A5 Suppresses Colorectal Cancer Growth and Metastasis Through Regulation of the EIF3A/PI3K/AKT Axis

doi: 10.3390/ijms27104334

Figure Lengend Snippet: SLC25A5 Suppresses CRC Progression in Association with the EIF3A–PI3K–AKT Axis: ( A ) Volcano plot identifying differentially expressed genes (DEGs) after SLC25A5 overexpression in HCT-8 cells. ( B ) Top 10 enriched KEGG pathways based on the DEGs. ( C ) Immunofluorescence staining of E-cadherin and N-cadherin in cells treated with the AKT activator SC79 (Nuclei were counterstained with DAPI (blue). E-cadherin and N-cadherin were labeled with orange and red fluorescence, respectively. Scale bars: 50 μm). ( D ) Western blot analysis of SLC25A5, EIF3A, P-AKT, AKT, and EMT markers in the indicated groups. ( E ) Transwell invasion assays of CRC cells treated with DMSO or SC79 (Scale bars: 100 μm). ( F – H ) Representative liver images ( F ) and quantification of liver metastatic nodules ( G ) and tumor/liver area ratio ( H ) in the indicated groups. ** p < 0.01, **** p < 0.0001.

Article Snippet: Human CRC cell lines HCT-8 and HT-29 were obtained from ATCC and cultured in RPMI-1640 or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO 2 .

Techniques: Over Expression, Immunofluorescence, Staining, Labeling, Fluorescence, Western Blot

Journal: Frontiers in Oncology

Article Title: Efficacy of an XPO1 inhibitor in combination with irinotecan in a preclinical colorectal cancer model

doi: 10.3389/fonc.2026.1721685

Figure Lengend Snippet: In vitro evaluation of sequential dosing of SN38 and/or eltanexor in CRC cell lines (HCT8, HCT116, LS1034, and HCT15). (A) Dosing strategies for in vitro viability assay. Cells were incubated with SN38 [10 nM] for the first 24 hours. Cells were then washed with PBS and incubated with eltanexor [100 nM] for an additional 48 hours. (B) Cell viability % measured by CellTiter Glo 2.0 and (C) heatmaps of the 4 CRC cell lines treated sequentially with SN38 [0 - 30 nM] followed by eltanexor [0 -100 μM]. Bliss synergy score was analyzed using SynergyFinder.

Article Snippet: Human CRC cell lines HCT8 (RRID: CVCL_2478), HCT15 (RRID: CVCL_0292), HCT116 (RRID: CVCL_0291), and LS1034 (RRID: CVCL_1382) were purchased from American Type Culture Collection (ATCC) (Manassas, VA; ).

Techniques: In Vitro, Viability Assay, Incubation

Journal: Frontiers in Oncology

Article Title: Efficacy of an XPO1 inhibitor in combination with irinotecan in a preclinical colorectal cancer model

doi: 10.3389/fonc.2026.1721685

Figure Lengend Snippet: (A) A Western blot analysis of RAD51, p53, p-H2A.X and MSH2 in CRC cell lines HCT8, HCT116, LS1034, and HCT15 following sequential treatment of SN38 and/or eltanexor. (B) Densitometry analysis total cell protein. Cells were exposed to SN38 (10 nM) or vehicle for 6hr. (C) Cells were then washed with PBS and incubated with or without the presence of eltanexor (1 uM) for an additional 24 hr. Nuclear/Cytoplasmic. (D) Densitometry analysis of nuclear/cytoplasmic proteins.

Article Snippet: Human CRC cell lines HCT8 (RRID: CVCL_2478), HCT15 (RRID: CVCL_0292), HCT116 (RRID: CVCL_0291), and LS1034 (RRID: CVCL_1382) were purchased from American Type Culture Collection (ATCC) (Manassas, VA; ).

Techniques: Western Blot, Incubation

Journal: Frontiers in Oncology

Article Title: Efficacy of an XPO1 inhibitor in combination with irinotecan in a preclinical colorectal cancer model

doi: 10.3389/fonc.2026.1721685

Figure Lengend Snippet: Immunocytochemistry in HCT8 and HCT 116 cell lines. Cells were first treated with SN38 (10 nM) in time series (0 hr, 2 hr, 4 hr, and 6 hr). Cells were washed with PBS and then treated with eltanexor (1 uM) for 48 hours. Cells were fixed and then stained with p53 and p21 for cell cycle arrest or stained with P-H2A.X for double stained DNA breaks. (A) Schematic illustration of dosing strategy, (B) immunostaining in HCT8 cell line and (C) immunostaining in HCT116 cell line.

Article Snippet: Human CRC cell lines HCT8 (RRID: CVCL_2478), HCT15 (RRID: CVCL_0292), HCT116 (RRID: CVCL_0291), and LS1034 (RRID: CVCL_1382) were purchased from American Type Culture Collection (ATCC) (Manassas, VA; ).

Techniques: Immunocytochemistry, Staining, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: α-hederin Targets USP5 to Inhibit Colorectal Tumorigenesis by Disrupting STAT3 Deubiquitination

doi: 10.7150/ijbs.119868

Figure Lengend Snippet: Identification of α-hederin binding proteins. (A) Chemical structure of α-hederin and Bio-α-hederin. (B) Schematic showing steps for identifying α-hederin binding proteins using microarrays fabricated with recombinant human proteins. (C) Representative image of protein array showing positive (red arrow), negative control (blue arrow) spots, and spots for USP5 (yellow arrow). The left panel shows the microarray scan of Bio-α-hederin, the right panel shows the microarray scan of D-biotin, and the center panel shows the partial enlargement of the microarray of Bio-α-hederin (top) and D-biotin (bottom), respectively. (D) The top 3 potential binding targets of α-hederin were identified by proteome microarray. (E) Magnified image of Bio-α-hederin and D-biotin binding to USP5 spot on the protein array. The values of NOR-SNR and FC were showed. (F) Three- and two-dimensional docking of α-hederin and USP5. (G) CETSA was performed to assess the effect of α-hederin on the thermal stability of USP5 in HCT116 cells, n = 3. (H) DARTS assay measured the impact of α-hederin on the proteolytic stability of USP5 in HCT116 cells, n = 3. (I) Streptavidin‒biotin pull-down assay was performed on the protein mixture extracted from HCT116 cells and colon tissues of CRC-mice. (J) Pathway analysis of potential proteins in the KEGG database. (K, L) The effects of α-hederin treatment on cell proliferation, apoptosis, and related processes through in vivo (K) and in vitro (L) experiments. (M) GO enrichment analysis of the proteins interacting with α-hederin based on the GO database. Data are presented as means ± SD. * p < 0.05, ** p < 0.01 versus DMSO group or control group, # p < 0.05, ## p < 0.01 versus pronase E group or model group.

Article Snippet: Five human CRC cell lines (Lovo, Caco-2, RKO, HCT8, and HCT116) and three normal human IECs (FHC, FHs 74 Int, and NCM460) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Recombinant, Protein Array, Negative Control, Microarray, Pull Down Assay, In Vivo, In Vitro, Control

Journal: International Journal of Biological Sciences

Article Title: α-hederin Targets USP5 to Inhibit Colorectal Tumorigenesis by Disrupting STAT3 Deubiquitination

doi: 10.7150/ijbs.119868

Figure Lengend Snippet: USP5 acted as a pro-cancer factor in CRC. (A) Differential gene expression of USP5 between tumor and adjacent normal tissues from TIMER database. (B) The bar plot of USP5 gene expression profile across all tumor samples and paired normal tissues from GEPIA database. (C) The gene expression of USP5 was analysed using the GEPIA database in datasets: COAD and READ. (D) The expression status of USP5, STAT3 and p-STAT3 (Tyr705) was simultaneously detected in paired CRC tumor tissues (T) and adjacent normal tissues (N) by immunoblotting, n = 7. (E, F) The protein and gene expression of USP5 in colon tissues of mice in control group and AOM/DSS group were detected by western blotting (E), n = 3, and qPCR (F), n = 5. (G, H) The expression of USP5 was examined in normal human IECs and CRC cell lines by qPCR (G) and western blotting (H), n = 3. (I) The expression of USP5 in different CRC cell lines in the CCLE database. (J, K) The qPCR (J) and western blotting (K) analysis confirmed the knockdown or overexpression efficacy of USP5 in HCT116 cell lines, n = 3. (L) Cell viability was determined by CCK8 assay after USP5 knockdown or overexpression, n = 3. (M) The colony formation ability was evaluated after USP5 knockdown or overexpression, n = 3. (N, O) The growth ability (N) and colony formation ability (O) of USP5 overexpression cells was evaluated after treatment with STAT3 inhibitor Stattic, n = 3. (P, Q) The colony formation ability (P) and growth ability (Q) of USP5 knockdown cells was evaluated after treatment with STAT3 activator Colivelin, n = 3. Data are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Five human CRC cell lines (Lovo, Caco-2, RKO, HCT8, and HCT116) and three normal human IECs (FHC, FHs 74 Int, and NCM460) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Western Blot, Control, Knockdown, Over Expression, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: α-hederin Targets USP5 to Inhibit Colorectal Tumorigenesis by Disrupting STAT3 Deubiquitination

doi: 10.7150/ijbs.119868

Figure Lengend Snippet: α-hederin diminished the protein stability of STAT3 mediated by USP5. (A, B) Using qPCR and western blotting methods to assess STAT3 mRNA (A) and protein (B) levels in HCT116 cells treated with various concentrations of α-hederin for 24 h, n = 3. (C) IB assay was performed to detect STAT3 expression in HCT116 cells treated with either DMSO or 10 µM α-hederin alongside 30 µM CHX at the indicated times, n = 3. (D) The STAT3 mRNA level in HCT116 cells after treatment with 10 µM α-hederin for the indicated times, n = 3. (E, F) α-hederin-induced ubiquitination of STAT3 was analyzed by Co-IP with an anti-STAT3 antibody followed by western blotting with antibodies against the Ubiquitin protein after treatment with different concentrations of α-hederin (0, 2.5, 5, 10, 15, and 20 μmol·L -1 ) (E) or different times (0, 2, 4, 6, 8, and 12 h) (F) in HCT116 cells. (G) The ubiquitination of STAT3 was analyzed in colon tissues of model and α-hederin H groups of CRC-mice by Co-IP. (H) The protein expression of STAT3 in HCT116 cells treated with a combination of α-hederin (10 µM) and the proteasome inhibitor MG132 (10 µM) using IB assay, n = 3. (I) Co-localization of USP5 with STAT3 in HCT116 cells treated with or without α-hederin using confocal observation, and the qualitative (J) and quantitative (K) analysis of co-localization were performed with Image J, n = 5. Scale bar = 20 μm. (L) Co-localization of USP5 with STAT3 in colon tissue of mice in control group. Scale bar = 50 μm. (M, N) The Co-IP experiments were performed to validate α-hederin inhibition of USP5 binding to STAT3 in colon tissues of CRC-mice (M) and HCT116 cells (N).

Article Snippet: Five human CRC cell lines (Lovo, Caco-2, RKO, HCT8, and HCT116) and three normal human IECs (FHC, FHs 74 Int, and NCM460) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Control, Inhibition, Binding Assay