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. 2022 Oct 10;21(2):170–186. doi: 10.1158/1541-7786.MCR-22-0486

Figure 6.

Figure 6. RelB mediates TWEAK–Fn14-dependent enrichment of CSCs and elimination of non-CSCs. A, Western blot for Fn14 and RelB from cells treated with vehicle, TWEAK, or carboplatin for 72 hours. B–C, Subcutaneous tumors were generated in nude mice using OV90 cells. Tumors were allowed to grow for 21 days followed by 3 cycles of carboplatin (50 mg/kg) or vehicle and resected 12 days after the final carboplatin treatment. B, Experimental design created with BioRender.com (left). Tumor volume was measured twice weekly and graphed normalized to the volume at treatment start (center). Two-way ANOVA, Tukey post hoc test (n = 6). TWEAK concentration per tumor weight measured using ELISA (right). Unpaired t test (n = 6). C, Flow cytometry analysis of CD117 expression on excised tumors following dissociation into single-cell suspension. Unpaired t test (n = 8). D, Fixed tumor fractions were histologically stained for nuclei (DAPI) and RelB. Representative images (left) and nuclear relative to cytosolic RelB were quantified per field using CellProfiler (right). Unpaired t test (n = 3). E, Western blot of NF-κB proteins in CAOV4-RelB knockout (KO) lines with TWEAK treatment (100 ng/mL) for 48 hours. F, qRT-PCR for select genes in CAOV4-RelB KO lines treated with TWEAK (100 ng/mL) for 48 hours, relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 3). G, Spheroid efficiency of CAOV4-RelB KO lines treated with TWEAK (100 ng/mL) for 7 days, relative to vehicle. Two-way ANOVA, Tukey post hoc test (n = 6). H, EdU incorporation pulse-chase assay of CAOV4-RelB KO lines treated with TWEAK (100 ng/mL) for 60 hours, relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 6). I, Flow cytometry of CD117 expression on Fn14+ CAOV4-RelB KO lines treated with TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours, represented relative to vehicle as mean plus SD. One-way ANOVA, Tukey post hoc test (n = 3). Data, mean and SEM. *, P < 0.05.

RelB mediates TWEAK–Fn14-dependent enrichment of CSCs and elimination of non-CSCs. A, Western blot for Fn14 and RelB from cells treated with vehicle, TWEAK, or carboplatin for 72 hours. B–C, Subcutaneous tumors were generated in nude mice using OV90 cells. Tumors were allowed to grow for 21 days followed by 3 cycles of carboplatin (50 mg/kg) or vehicle and resected 12 days after the final carboplatin treatment. B, Experimental design created with BioRender.com (left). Tumor volume was measured twice weekly and graphed normalized to the volume at treatment start (center). Two-way ANOVA, Tukey post hoc test (n = 6). TWEAK concentration per tumor weight measured using ELISA (right). Unpaired t test (n = 6). C, Flow cytometry analysis of CD117 expression on excised tumors following dissociation into single-cell suspension. Unpaired t test (n = 8). D, Fixed tumor fractions were histologically stained for nuclei (DAPI) and RelB. Representative images (left) and nuclear relative to cytosolic RelB were quantified per field using CellProfiler (right). Unpaired t test (n = 3). E, Western blot of NF-κB proteins in CAOV4-RelB knockout (KO) lines with TWEAK treatment (100 ng/mL) for 48 hours. F, qRT-PCR for select genes in CAOV4-RelB KO lines treated with TWEAK (100 ng/mL) for 48 hours, relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 3). G, Spheroid efficiency of CAOV4-RelB KO lines treated with TWEAK (100 ng/mL) for 7 days, relative to vehicle. Two-way ANOVA, Tukey post hoc test (n = 6). H, EdU incorporation pulse-chase assay of CAOV4-RelB KO lines treated with TWEAK (100 ng/mL) for 60 hours, relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 6). I, Flow cytometry of CD117 expression on Fn14+ CAOV4-RelB KO lines treated with TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours, represented relative to vehicle as mean plus SD. One-way ANOVA, Tukey post hoc test (n = 3). Data, mean and SEM. *, P < 0.05.