Fig 2: M1 ubiquitin chains stimulate STING-mediated NFκB-related immune signaling.(A) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKOpool cells prepared following treatment with 1 µM diABZI for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (B) Quantification of pIκBαS32 in (A). Values represent the relative intensity of corresponding bands to untreated bands per cell line. Replicates for each condition from two independent experiments are shown. (C) Quantification of pIκBαS32 in (D). Values represent the relative intensity of bands to WT-treated bands. Error bars represent the mean ± s.d. of three independent experiments. (D) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT, HOIPKOpool, and HOIPKOpool stably expressing untagged HOIP prepared following treatment with 1 µM diABZI for 4 h. Immunoblotting was replicated in three independent experiments. (E, F) Relative expression changes of indicated NFκB- (E) and IRF3/interferon-related (F) genes detected by quantitative RT-PCR in WT and HOIPKOpool THP1 cells treated with 1 µM diABZI for 1, 2, 4, and 8 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2−ΔΔCt values. Mean ± s.d. n = 3 * < 0.05, ***<0.001, ****<0.0001 (TNF 6 h p = <0.0001, 8 h p = 0.0006; TNFAIP3 6 h p = 0.0001, 8 h p = <0.0001; IL6 6 h p = 0.0117, 8 h p = <0.0001; IFNB1 2 h p = 0.0135, 4 h p = <0.0001, 6 h p = <0.0001). (G, H) Relative expression changes of indicated NFκB- (G) and IRF3/interferon-related (H) genes detected by quantitative RT-PCR in THP1 WT, HOIPKOpool, and HOIPKOpool stably expressing untagged HOIP cells treated with 1 µM diABZI for 4 h. Quantification of relative expression is from four independent experiments analyzed at the same time. A two-way ANOVA with Tukey’s multiple comparisons test was performed on 2−ΔΔCt values. Mean ± s.d. n = 4 *<0.05, **<0.01, ****<0.0001 (TNF WTvHOIPKOpool p = 0.0019, HOIPKOpoolvHOIPKOpool + HOIP p = <0.0001; TNFAIP3 WTvHOIPKOpool p = 0.0333, HOIPKOpoolvHOIPKOpool + HOIP p = 0.0014; IL6 WTvHOIPKOpool p = 0.0349, HOIPKOpoolvHOIPKOpool + HOIP p = <0.0001; IFNB1 WTvHOIPKOpool p = 0.05, HOIPKOpoolvHOIPKOpool + HOIP p = <0.0001; IFIT3 WTvHOIPKOpool p = <0.0001, HOIPKOpoolvHOIPKOpool + HOIP p = <0.0001; ISG15 HOIPKOpoolvHOIPKOpool + HOIP p = <0.0001). Source data are available online for this figure.

Fig 3: HOIP is important for NFκB signaling following STING activation by cGAMP and in mouse bone marrow-derived macrophages.(A) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKOpool cells prepared following treatment with 120 µg/mL cGAMP for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (B, C) Relative expression changes of indicated NFκB- (B) and IRF3/interferon-related (C) genes detected by quantitative RT-PCR in WT and HOIPKOpool THP1 cells treated with 120 µg/mL cGAMP for 1, 2, 4, and 8 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2−ΔΔCt values. Mean ± s.d. n = 3 *<0.05, **<0.01, ***<0.001, ****<0.0001 (TNF 6 h p = <0.0001, 8 h p = <0.0001; TNFAIP3 4 h p = 0.0084, 6 h p = <0.0001, 8 h p = <0.0001; IL6 6 h p = <0.0001, 8 h p = <0.0001; IFNB1 2 h p = 0.0019, 4 h p = 0.0005, 8 h p = 0.0281). (D, E) Relative expression changes of indicated NFκB- (D) and IRF3/interferon-related (E) genes detected by quantitative RT-PCR in WT and shCtrl and shHOIP iBMDM cells treated with 0.2 µM diABZI for 1, 2, and 4 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2−ΔΔCt values. Mean ± s.d. n = 3 **<0.01, ***<0.001, ****<0.0001 (Tnf 2 h p = <0.0001, 4 h p = 0.0005; Tnfaip3 2 h p = <0.0001, 4 h p = <0.0001; Il6 4 h p = <0.0001; Ifnb1 2 h p = 0.0011, 4 h p = 0.0031; Ifit3 4 h p = <0.0001; Isg15 4 h p = <0.0001).

Fig 4: CYPJ limits LUBAC-induced NF-κB activation through impedes HOIP-SHARPIN complex.A, GST and GST-SHARPIN-UBL protein agarose was incubated with GFP-HOIP-UBA lysates from transfected 293T cells in the absence/presence of 4 μg His-CYPJ to perform pull-down assays. GFP-HOIP-UBA pull-down with GST-SHARPIN-UBL agarose was measured with an anti-GFP antibody in the IB analysis. B, GST and GST-HOIP-NZF2 proteins were incubated with 1 μg His-SHARPIN-UBL in the absence/presence of 4 μg His-CYPJ to perform pull-down assays. His-SHARPIN-UBL pull-down with GST-HOIP-NZF2 agarose was monitored by IB analysis using an anti-His antibody. C, HA-CYPJ alone was transfected into 293T cells (upper panel), HCT116 (middle panel), and HCT15 cells (lower panel) to estimate whether CYPJ affects the binding between endogenous HOIP and endogenous SHARPIN; this was assessed with a co-IP assay using 1 μg anti-SHARPIN antibody and IB analysis using an anti-HOIP antibody. D, Flag-HOIP, Flag-HOIL-1L, and Flag-SHARPIN plasmids were transfected at a ratio of 2:1:1 with or without cotransfection of HA-CYPJ/CYPA into 293T cells. The endogenous linear ubiquitin signal was detected by an antilinear ubiquitin antibody. E, WT and KO bone marrow-derived macrophages (BMDMs) were isolated from WT and KO mice, and WT CYPJ and two CYPJ-deficiency monoclones includes, KO-1 and KO-2 of 293T cells were prepared for the detection of endogenous linear ubiquitin signals. F, effects of HA-CYPJ on NF-κB reporter induced by cotransfection of HOIP-HOIL-1L or HOIP-SHARPIN in 293T cells. G, Flag-HOIP and Flag-SHARPIN expression plasmids with or without HA-tagged CYPJ plasmid were transfected into 293T cells, and the transcription level of TNF and IL-8 was assessed by qRT-PCR. H, Effects of CYPJ re-expression on the activity of the NF-κB reporter induced by HOIP-HOIL-1L or HOIP-SHARPIN in CRISPR-Cas9-mediated CYPJ-KO-1 monoclonal 293T cells. I, Effects of CYPJ re-expression on the activity of NF-κB reporter induced by HOIP-HOIL-1L or HOIP-SHARPIN in CRISPR-Cas9-mediated CYPJ-KO-2 monoclonal 293T cells. J, GFP-CYPJ and its truncations, covering FL, Δ1, Δ2, and Δ3 of CYPJ, were transfected with or without Flag-HOIP and Flag-SHARPIN into KO-1 (upper panel) or KO-2 (lower panel) monoclonal 293T cells. Twenty-fourhours later, the downstream phosphorylation of IκBα and the expression of total IκBα and the indicated proteins were measured by IB analysis. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001. Error bars represent the standard deviation. co-IP, co-immunoprecipitation; CYPA/J, cyclophilin A/J; FL, full length; HA, hemagglutinin; HOIL-1L, heme-oxidized iron-regulatory protein 2 ubiquitin ligase 1; HOIP, HOIL-1-interacting protein; IB, immunoblotting; LUBAC, linear ubiquitin chain assembly complex; NF-κB, nuclear factor kappa-B; NZF, Npl4 zinc finger;qRT-PCR, quantitative real-time PCR; SHARPIN, Shank-associated RH domain-interacting protein; TNF, tumor necrosis factor; UBA, Ub-associated domain; UBL, ubiquitin-like.

Fig 5: STING activation by multiple agonists induces HOIP-mediated M1 ubiquitin chain formation, which is not required for STING degradation or LC3B lipidation in HeLa or THP1 cells.(A) Representative Airyscan-processed confocal images of HeLaSTING cells stably expressing mScarletI-LC3B and mEGFP-HOIP treated with 120 µg/mL of cGAMP for 8 h with no saponin extraction prior to PFA-fixation. Scale bar = 10 µm. Corresponding to representative images in Fig. 2A. Imaging was replicated in three independent experiments. (B) Representative spinning disk confocal images of HeLaSTING; mEGFP-HOIP (green); mScarletI-LC3B (magenta) cells treated with 120 µg/mL of cGAMP for 8 h prior to prior to saponin extraction and PFA-fixation. Scale bar = 20 µm. Corresponding to quantification in Fig. 2B. (C, D) Representative immunoblots of indicated proteins detected in cell lysates from HeLaSTING WT and HOIPKO cells (C) or HeLaSTING and HeLaSTING stably expressing mEGFP-OTULIN (D) prepared following treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (E, F) Representative immunoblots of indicated proteins detected in HeLaSTING cell lysates from WT and HOIPKO cells prepared following treatment with 120 µg/mL cGAMP (E) or 1 µM diABZI (F) for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (G) Representative immunoblots of endogenous STING were detected in cell lysates from WT and HOIPKO HeLa cells without stable overexpression of STING. Cells were treated with 15 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (H) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKOpool cells prepared following treatment with 120 µg/mL cGAMP for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments.