EMBO/soda-nel huggingface dataset:
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 7

Figure 7 - ISG20 overexpression together with A3A is sufficient for reduction of transcriptionally active cccDNA.

A dHepaRG-TR-A3A cells were treated with tetracycline to induce A3A expression (Tet(A3A)) or transduced with an adenoviral vector for expression of ISG20 (AdV-ISG20). After 4 days, expression of A3A and ISG20 was detected by western blot.

B, C dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and at day 7 p.i. transduced with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. Cell lysates were analyzed for cccDNA after T5 digest (B) and total intracellular HBV DNA (C) by qPCR relative to Prnp (n=5-6 biological replicates from two independent experiments).

D Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and transfected on day 3 p.i. with mRNAs of A3A, ISG20 or catalytically inactive mutants thereof as indicated. 4 days after transfection, cccDNA after T5 digest was measured by qPCR relative to Prnp (n=4 biological replicates).

E, F dHepaRG-TR-A3A cells were infected with wildtype HBV (HBVwt) or HBV∆x at an MOI of 300 virions/cell and transduced on day 7 p.i. with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. HBeAg (E) was measured by ELISA and cccDNA after T5 digest (F) by qPCR relative to Prnp (n=6 biological replicates from two independent experiments).

Data information: Data are represented as mean +/- SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant by Student's unpaired t test with Welch's correction.

Expanded View Figure legends
Alterations of redox and iron metabolism accompany development of HIV latency (2020)
Figure 1

Figure 1. Viral replication induces upregulation of antioxidant genes in vitro and in vivo.

(A,B) RNA-Seq analysis of: different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected (A) and PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART (B). Expression levels were normalized as Log2 fold change in infected vs matched mock-infected controls (A) or as enrichment score in ART-treated vs ART-naïve animals (B). For (A) data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in (A) depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes (DEGs) in the pathway; grey dots are not statistically significant; for (B) data were analyzed by Gene Set Enrichment Analysis (GSEA, number of animals =8). Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART-naïve and ART-treated time points. Red color indicates leading edge genes. All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress (184 genes; GO:0034599).
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 1

Figure 1 The non-structural protein NS1 of ZIKV enhances ZIKV-induced NLRP3 inflammasome activation.

(A) ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 hrs) THP-1 cells then infected with ZIKV (MOI=1) for 36 hrs. Uninfected cells serves as mock control.

(B) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated or primed by LPS (500 ng/ml, 3 hrs), after ZIKV infection (MOI=1) for 36 hrs.

(C) ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1 or AIM2 KO#1 THP-1 cells left untreated (UT), or pretreated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) stimulation, or stimulated with poly (dA:dT) (1mg/ml, 6 hrs), or infected with ZIKV (MOI=1, 36 hrs).

(D) ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI=1) at the indicated time points.

(E) ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for 36 hrs.

(F) ELISA of IL-1β in the supernatant of WT, NLRP3 KO or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI=1) or DENV-2 (MOI=1) infection for 36 hrs.

(G) ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 hrs or mock control. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) treatment for 6 hrs.

(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.

(I) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1 and pro-IL-1β together with NS1.

(J) Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 hrs.

(K) ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs), or pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.

(L) Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.

Data information: In (B, H-J, L), data are representative of three independent experiments. In (A, C, D-G, K), data are mean values ± SEM (n = 3). NS (non-significant), p＞0.05; *, p＜0.05; **, p＜0.01; ***, p＜0.001.
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 2

Figure 2 NS1 enhances inflammasome activation to benefit ZIKV infection.

(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.

(B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.

(C) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs by the indicated antibodies.

(D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.

(E) Plaque titration of ZIKV in supernatants of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.

(F) Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies.

(G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.

(H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points.

(I) Relative qRT-PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors.

(J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.

(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.

Data information: In (C, F), data are representative of three independent experiments. In (A, B, D, E, G-K), data are mean values ± SEM (n = 3). NS (non-significant), p＞0.05; *, p＜0.05; **, p＜0.01; ***, p＜0.001.
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 3

Figure 3 NS1 inhibits the proteasomal degradation of caspase-1.

(A) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1 and Flag-IL-1β. WCL, whole cell lysates.

(B) Co-immunoprecipitation and immunoblot analysis of extracts of NS1-inducible THP-1 cells, which expressed Flag-tagged NS1, treated with or without doxycycline (Dox) (100 ng/ml).

(C) Immunoblot analysis of extracts of THP-1 cells treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs) followed by increasing doses of Dox treatment.

(D) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-NS1. Below, caspase-1 mRNA RT-PCR; RPL13A mRNA RT-PCR serves as a loading control.

(E) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-DENV-2-NS1.

(F) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 followed by cycloheximide (CHX) treatment (100 μg/ml) for the indicated time points. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV2E.

(G) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), Lactacystin (5 μM), Carfilzomib (100 mM) or CQ (50 mM) for 6 hrs.

(H) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml).

(I) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-NS1, followed by MG132 (10 mM) treatment for 6 hrs. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV2H.

Data information: In (A-I), data are representative of three independent experiments.
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 4

Figure 4 NS1 recruits USP8 to cleave K11-linked poly-ubiquitin chains from caspase-1.

(A) Immunoblot analysis of extracts of wild type (WT) and USP8 knockout (KO) 293T cells transfected with Flag-caspase-1 together with empty vector or Myc-NS1.

(B) Immunoblot analysis of extracts of transfected with Flag-caspase-1 together with empty vector or Myc-USP8 then treated with or without MG132 (10 mM) for 6 hrs.

(C) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml) then transfected with or without poly (I:C) (2 mg/ml, 6 hrs).

(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 and Myc-USP8 together with HA-NS1 followed by MG132 treatment (10 mM) for 6 hrs. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV3D.

(E) Co-immunoprecipitation and immunoblot analysis of extracts of THP-1 cells infected with ZIKV (MOI=1) for 24 hrs. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV3E.

(F) Co-immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP-1 cells infected with ZIKV (MOI=1) for 24 hrs. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV3F.

(G) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin (Ub) or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-USP8, followed by MG132 (10 mM) treatment for 6 hrs. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV3G.

(H) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag-caspase-1, HA-K11-Ub together with empty vector or Myc-NS1, followed by two-step immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV3H.

(I) Co-immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag-caspase-1 and HA-K11-Ub together with WT or inactive mutant C786A of USP8. Intensity analysis of the bands from the three independent experiments is shown in Fig. EV3I.

Data information: In (A-I), data are representative of three independent experiments.
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 5

Figure 5 NS1 stabilizes caspase-1 through the cleavage of poly-ubiquitin chains at Lys134.

(A) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA-NS1 together with Flag-caspae-1 wild type (WT) and mutants (K37R, K44R or K134R). Lower panel shows the intensity analysis of the bands from the three independent experiments. K11R, K64R, K296R and K319R are shown in Fig EV3J.

(B) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-K11-ubiquitin (Ub) together with Flag-caspase-1 (WT, K37R, K44R, K134R).

(C) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-NS1. Lower panel shows the intensity analysis of the bands from the three independent experiments.

(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-USP8. Lower panel shows the intensity analysis of the bands from the three independent experiments.

Data information: In (A-D), data are representative of three independent experiments. In (A, C, D), data are mean values ± SEM (n = 3). NS (non-significant), p＞0.05; **, p＜0.01; ***, p＜0.001.
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 6

Figure 6. NS1-mediated NLRP3 inflammasome activation attenuates type I IFN signaling.

(A) Relative qRT-PCR analysis of IFNB, IFIT2 and IFIT1 mRNA levels in wild type (WT) or caspase-1 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.

(B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.

(C) Relative qRT-PCR analysis of IFNB mRNA levels in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.

(D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points.

(E) Relative qRT-PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.

(F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.

(G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.

(H) ELISA of IL-1β production in brain of WT or Nlrp3-/- 1-day-old neonatal mice (n=3 per group) intracerebrally injected with 1×105 plaque-forming units (PFU) of ZIKV for 5 days.

(I) Relative qRT-PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection in panel (H).

(J) ELISA of IFN-β production in brains of WT or Nlrp3-/- neonatal mice in (H).

(K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection in (H).

(L) Plaque titration of ZIKV in brain tissues of WT or Nlrp3-/- neonatal mice shown in (H).

(M) Body weight gain of WT and Nlrp3-/- neonatal mice (n=4 per group) inoculated with ZIKV (1×105 PFUs).

Data information: In (A-M), data are mean values ± SEM (n = 3). NS (non-significant), p＞0.05; **, p＜0.01; ***, p＜0.001.
Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis (2018)
Figure 7

Figure 7 Cleavage of cGAS by caspase-1 inhibits type I IFN signaling and assists ZIKV replication.

(A) Relative qRT-PCR analysis of ZIKV RNA levels in wild type (WT) or cGAS knockout (KO) THP-1 cells after ZIKV infection (MOI=1) for indicated time points.

(B) Relative qRT-PCR analysis of IFNΒ, IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP-1 cells after ZIKV infection (MOI=1) for indicated time points.

(C) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP, cGAS or NLRP3 and infected with ZIKV (MOI=1) for 36 hrs.

(D) Relative qRT-PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP-1 cells treated with or without YVAD (20 μM), followed by ZIKV infection (MOI=1) for indicated times points.

(E) Immunoblot analysis of extracts of Flag-cGAS-overexpressed THP-1 cells treated with or without YVAD (20 μM) then infected with ZIKV (MOI=5) for 24 hrs or infected with HSV-1 (MOI=1) for 24 hrs.

(F-G) Immunoblot analysis of WT, NLRP3 KO (F) and caspase-1 KO (G) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.

(H) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.

(I) Immunoblot analysis of PBMCs from Donor#1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for the indicated time points.

(J) Immunoblot analysis of extracts of 293T cells transfected with Myc-cGAS (tagged on C-terminal), GFP-ASC, Flag-caspase-1, together with empty vector or HA-NS1.

(K) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in WT, cGAS KO THP-1 cells as well as cGAS KO THP-1 cells reconstituted with WT or D140A/D157A mutant of cGAS, followed by ZIKV infection (MOI=1) for 24 hrs. Expression levels of restored cGAS WT or D140A/D157A mutant were determined by immunoblot analysis in Fig EV4F.

Data information: In (E-J), data are representative of three independent experiments. In (A-D, K), data are mean values ± SEM (n = 3). NS (non-significant), p＞0.05; **, p＜0.01; ***, p＜0.001.
XIAP inhibits autophagy via XIAP‐Mdm2‐p53 signalling (2013)
Figure 1

XIAP inhibits autophagy. (A) Representative images of GFP‐LC3 staining in HCT116 XIAP WT and XIAP KO cells with stable expression of GFP‐LC3. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments. (B) Endogenous LC3 expression in HCT116 XIAP WT and XIAP KO cells was analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1A. (C) Lysates from XIAP+/+ and XIAP−/− mouse embryonic fibroblasts were analysed by western blotting with the indicated antibodies. The data are representative of two biological replicates. (D) Autophagic vesicles in HCT116 XIAP WT and XIAP KO cells were evaluated by electron microscopy. The images are representative of three biological replicates. (E) HCT116 cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1B. (F) HCT116 XIAP KO cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. (G) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1D. (H) MEF ATG5 WT and ATG5 KO cells were individually transfected with XIAP‐specific or control siRNAs. Forty‐eight hours after transfection, cell lysates were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1E. (I) HCT116 XIAP WT and XIAP KO cells were treated with 20 μM Z‐VAD‐FMK or DMSO for 2 h. Cell lysates were then analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1F. (J) HCT116 XIAP KO cells were transfected with either Flag‐XIAP (D148A/W310A) or control plasmid. Twenty‐four hours after transfection, cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1G.
XIAP inhibits autophagy via XIAP‐Mdm2‐p53 signalling (2013)
Figure 2

XIAP inhibits autophagy via the Mdm2‐p53 pathway. (A) A549 and IMR90 cells were transfected with XIAP‐specific or control siRNAs. Cell lysates were subjected to western blot analysis with the indicated antibodies. The data are representative of two biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2A. (B) HCT116 cells expressing XIAP‐specific or control siRNAs, HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2D. (C) HCT116 XIAP WT and XIAP KO cells were individually transfected with Flag‐XIAP or control vector. Cytoplasmic/nuclear fractionation was performed to analyse cellular localization of Mdm2 and p53. PARP and GAPDH were used as nuclear (N) and cytoplasmic (C) fraction markers, respectively. The data are representative of three biological replicates. (D) HCT116 p53+/+ and p53−/− cells were individually transfected with XIAP‐specific or control siRNAs. LC3 conversion was detected by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2E. (E) HCT116 p53+/+ and p53−/− cells were individually transfected with Flag‐XIAP or control vector. LC3 conversion was detected by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2F. (F) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2H. (G) HCT116 XIAP KO cells were treated with the indicated amounts of Nutlin for 8 h. Cell lysates were analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates.