Fig. 5: FSL0717 suppressed NF-κB activation by inhibiting the HOIL-1L NZF, whereas FSL0720 increased cell death by inhibiting both the HOIL-1L NZF and SHARPIN NZF.

A Chemical structures of the identified compounds. B, D, E WT MEFs were stimulated with TNF-α (1 ng/mL) (B) or TNF-α (2.5 ng/mL) plus CHX (20 μg/mL) (D) for the indicated times after treatment with DMSO or the indicated compounds (10 μM) for 1 h and analyzed by immunoblotting with the indicated antibodies. Quantitative analysis of cleaved caspase-3 shown in D was performed at 6 h after stimulation (E). Data are representative of three (B) or five (D) independent experiments and are shown as the mean ± s.d. (E). P values were obtained using a one-sample t test. C HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL) for the indicated time after treatment with DMSO or the indicated compounds (10 μM), and expression of NF-κB target genes was measured by quantitative PCR. All gene expression levels are normalized against the corresponding levels of ACTB and expressed as fold changes relative to DMSO-treated cells at 0 h. Data are shown as the mean ± s.d. of three independent experiments. P values were calculated by a one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ****P < 0.0001. F JR-GFP cells were treated with DMSO or the indicated compounds (10 μM) for 1 h and stimulated with TNF-α (5 ng/mL) plus CHX (20 μg/mL) for 0 or 6 h. Samples were analyzed by flow cytometry after Annexin V and TO-PRO-3 staining. Data are displayed as the frequencies of Annexin V-positive cells (left) and TO-PRO-3-positive cells (right). Data are shown as the mean ± s.d. of three independent experiments. P values were calculated by a one-way ANOVA with Tukey’s post hoc test. G HEK293T cells were transiently transfected with Sm-di-ubiquitin (GA mutant) and Lg-HOIL-1L NZF (WT or TR-AA mutant), cultured for 24 h, and then treated with DMSO or the indicated compounds (10 μM) for 1 h. Luminescence intensity was measured using a NanoBiT system. Data are shown as the mean ± s.d. of five independent experiments. P values were obtained using a one-sample t test (Lg-HOIL-1L NZF WT) or a two-tailed Student’s t test (Lg-HOIL-1L TR-AA mutant). H His-tagged di-ubiquitin, GST-tagged SHARPIN NZF, and the indicated compounds were mixed and incubated for 1 h. Fluorescence intensity was measured using a FRET assay. Data are shown as the mean ± s.d. of three independent experiments. P values are from a one-sample t test. I, J HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL) for the indicated times after treatment with DMSO or the indicated compounds (10 μM) for 1 h, and analyzed by immunoblotting with the indicated antibodies (I). Quantitative analysis of IκBα was performed, with all data normalized against the corresponding values at 0 min (J). Data are representative of four independent experiments (I) and shown as the mean ± s.d. (J). P values were obtained using a one-way ANOVA with Tukey’s post hoc test. *P < 0.05. K, L HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (2.5 ng/mL) plus CHX (20 μg/mL) for the indicated times after treatment with DMSO or the indicated compounds (10 μM) for 1 h and analyzed by immunoblotting with the indicated antibodies. Quantitative analysis of cleaved caspase-3 was performed at 6 h after stimulation (L). Data are representative of three independent experiments (K) and shown as the mean ± s.d. (L). P values were obtained using a one-sample t test. Uncropped western blots are available for this figure in the Supplementary Material.