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

Figure 5.

Figure 5. TWEAK receptor Fn14 is required for TWEAK-mediated stem-like features. A, Western blot for Fn14 from cells grown in standard adherent conditions or in spheroid conditions. B, Western blot for Fn14 from cells grown in standard monolayer conditions or in spheroid suspension conditions with or without FGF. C, Western blot of Fn14 and NF-κB proteins in siNeg or siFN14 HGSOC cells treated with vehicle or TWEAK (100 ng/mL) for 72 hours. Representative image (top) and quantification of band intensities (bottom). One-way ANOVA, Tukey post hoc test (n = 3). D, Nuclear and cytosolic western blots for indicated proteins in CAOV4 cells treated with TWEAK (100 ng/mL), or TWEAK plus FN14 inhibitor (L542-0366, 6.25–50 μmol/L). Quantification values normalized to Lamin A/C for nuclear fraction and GAPDH for a cytosolic fraction. E, Luciferase reporter assay showing NF-κB activity is lost in response to TWEAK (100 ng/mL) plus FN14i (L542-0366, 50 μmol/L) in CAOV4 cells (n = 4). F, Spheroid efficiency of siNeg or siFN14 HGSOC cells treated with vehicle or TWEAK (100 ng/mL) for 4 days, represented relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 4). G, qRT-PCR for SOX2 or NIK in siNeg or siFN14 HGSOC cells treated with TWEAK (100 ng/mL) for 48 hours, relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 3). H, qRT-PCR for SOX2 or NIK in CAOV4 cells treated with FN14 inhibitor (L542–0366, 50 μmol/L) for 72 hours in combination with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin conditions. Unpaired t test for condition versus condition plus FN14 inhibitor (n = 3). Data represent mean and SEM. * P < 0.05.

TWEAK receptor Fn14 is required for TWEAK-mediated stem-like features. A, Western blot for Fn14 from cells grown in standard adherent conditions or in spheroid conditions. B, Western blot for Fn14 from cells grown in standard monolayer conditions or in spheroid suspension conditions with or without FGF. C, Western blot of Fn14 and NF-κB proteins in siNeg or siFN14 HGSOC cells treated with vehicle or TWEAK (100 ng/mL) for 72 hours. Representative image (top) and quantification of band intensities (bottom). One-way ANOVA, Tukey post hoc test (n = 3). D, Nuclear and cytosolic western blots for indicated proteins in CAOV4 cells treated with TWEAK (100 ng/mL), or TWEAK plus FN14 inhibitor (L542-0366, 6.25–50 μmol/L). Quantification values normalized to Lamin A/C for nuclear fraction and GAPDH for a cytosolic fraction. E, Luciferase reporter assay showing NF-κB activity is lost in response to TWEAK (100 ng/mL) plus FN14i (L542-0366, 50 μmol/L) in CAOV4 cells (n = 4). F, Spheroid efficiency of siNeg or siFN14 HGSOC cells treated with vehicle or TWEAK (100 ng/mL) for 4 days, represented relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 4). G, qRT-PCR for SOX2 or NIK in siNeg or siFN14 HGSOC cells treated with TWEAK (100 ng/mL) for 48 hours, relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 3). H, qRT-PCR for SOX2 or NIK in CAOV4 cells treated with FN14 inhibitor (L542–0366, 50 μmol/L) for 72 hours in combination with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin conditions. Unpaired t test for condition versus condition plus FN14 inhibitor (n = 3). Data represent mean and SEM. * P < 0.05.