Fig 1: Fap1-inhibition increased Fas or oxaliplatin induced apoptosis in CD133+ colon cancer cells(A) Fap1-blocking SLV peptide increases Fas-induced apoptosis. Total SW480 or SW620 cells were treated with SLV peptide (or VLS control), with or without Fas-agonist antibody, and analyzed for apoptosis by Annexin V staining. Significant differences indicated by *, **, or *** (p<0.01, n=4 for all comparisons). (B) Fap1-inhibition increases Fas or oxaliplatin induced apoptosis in CD133+ SW620 cells. CD133+ SW620 cells were analyzed for apoptosis by Annexin V staining, with or without Fas-agonist antibody. Some cells were treated with SLV peptide (or VLS control) or transduced with a vector to express Fap1 specific shRNAs (or scrambled control). Significant differences indicated by *, **, ***, or # (p<0.001, n=4 for all comparisons). (C) Fap1-inhibition increases Fas induced caspase 8 cleavage in CD133+ SW620 cells. Cells were analyzed by ELISA for cleavage of caspase 8 (death receptor induced apoptosis). Statistically significant differences indicated by * or ** (p<0.001, n=4). (D) Oxaliplatin treatment increases caspase 9 cleavage in CD133+ SW620 cells. Cells were analyzed by ELISA for cleavage of caspase 9 (intrinsic apoptosis). Statistically significant differences indicated by *, **, ***, or # (p<0.001, n=4). (E) Plasma membrane expression of Fas was not altered by SLV peptide or oxaliplatin treatment of CD133+ SW620 cells. Western blots of cell lysates were probed for Fas or Na+/K+ ATPase (as a loading control). (F) Fap1 inhibition increases Fas or oxaliplatin induced apoptosis in CD133+ SW480 cells. Similar experiments were performed with the SW480 cell line. Statistically significant differences are indicated by * or ** (p<0.001, n=6 for both comparisons).
Fig 2: Fap1-inhibition with SLV peptide increases Fas and Gsk3β phosphorylation in CD133+ cells in a murine xenograft modelSW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were >2,000 mm3. (A) SLV peptide increases Fas phosphorylation in CD133+ xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Fas or CD133 was performed with DAPI staining of nuclei. (B) SLV peptide increases Gsk3β phosphorylation in CD133+ xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Gsk3β or CD133 was performed with DAPI staining of nuclei.
Fig 3: Fap1-inhibition with SLV peptide increases phosphorylation of Fap1-substrates Fas and Gsk3β in a murine xenograft modelSW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were >2,000 mm3. (A) SLV peptide increases gland formation in xenograft tumors with or without oxaliplatin. Histology was analyzed by hematoxylin/ eosin staining. Fap1 expression was determined by immunofluorescence. Relative fluorescent intensity (RFI) of Fap1 staining is indicated below relevant panels. (B) SLV peptide increases Fas-phosphorylation in xenograft tumors with or without by oxaliplatin. Immunofluorescent detection of total versus phospho-Fas was performed with DAPI staining of nuclei. Areas without gland formation were selected for this study. (C) SLV peptide increases Gsk3β-phosphorylation with or without oxaliplatin. Immunofluorescent detection of total versus phospho- Gsk3β was performed with DAPI staining of nuclei. Areas without gland formation were selected for this study.
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