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Reagents

Thalidomide (Tokyo Chemical Industry), pomalidomide (Sigma–Aldrich), lenalidomide (FujiFilm Wako), 5-hydroxythalidomide (5-HT, synthesized as previously reported45), MG132 (Peptide Institute), MLN4924 (Chemscene), cycloheximide (Merck Millipore), and zVAD-FMK (Peptide Institute) were dissolved in DMSO (FujiFilm Wako) at 2–100 mM and stored at −20 °C as stock solutions. All drugs were diluted 1000-fold for in vivo experiments or 200-fold for in vitro experiments. 2′,3′-cGAMP (InvivoGen) was dissolved in endotoxin-free water. Recombinant human TNF-α (R&D Systems) was reconstituted according to the manufacturer’s protocol.

Antibodies

The following horseradish peroxidase (HRP)-conjugated antibodies were used in this study: FLAG (Sigma–Aldrich, A8592, 1:5,000), AGIA19 (produced in our laboratory, 1:10,000), α-tubulin (MBL, PM054-7, 1:10,000), and biotin (Cell Signaling Technology, #7075, 1:5,000). The following primary antibodies were used in this study: CRBN (#71810, 1:1000), IκBα (#4814, 1:1000), phospho-IκBα (#9246, 1:1000), NF-κB1 p105/p50 (#12540, 1:1000), p65 (RelA) (#8242, 1:1000), phospho-p65 (RelA) (#3033, 1:1000), CYLD (#8462, 1:1000), RIP1 (#3493, 1:1000), caspase-8 (#9746, 1:1000), caspase-3 (#9662, 1:1000), PARP (#9542, 1:1000), FADD (#2782, 1:1000), phospho-TBK1 (#5483, 1:1000), and TBK1 (#3504, 1:1000) (all from Cell Signaling Technology); and lamin B (#sc-6217, 1:500) (from Santa Cruz Biotechnology). Anti-rabbit IgG (HRP-conjugated, Cell Signaling Technology, #7074, 1:10,000), anti-mouse IgG (HRP-conjugated, Cell Signaling Technology, #7076, 1:10,000), and anti-goat IgG (HRP-conjugated, Invitrogen/Thermo Fisher Scientific, #81-1620, 1:10,000) were used as secondary antibodies.

Immunoblot analysis

Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked using 5% skim milk (Megmilk Snow Brand) in TBST [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween20] at room temperature for 1 h, and then treated with the appropriate antibodies. Immobilon (Millipore) or Immuno-Star LD (FujiFilm Wako Pure Chemical Corporation) was used as a substrate for HRP, and the luminescent signal was detected on an ImageQuant LAS 4000 mini (GE Healthcare). In some blots, the membrane was stripped with Stripping Solution (FujiFilm Wako Pure Chemical Corporation) and reprobed with other antibodies.

Cell culture and transfection

HEK293T or HeLa cells were cultured in low-glucose DMEM (FujiFilm Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FujiFilm Wako Pure Chemical Corporation), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco/Thermo Fisher Scientific) at 37 °C under 5% CO2. HEK293T or HeLa cells were transfected using TransIT-LT1 transfection reagent (Mirus Bio) or Polyethyleneimine (PEI) Max (MW 40,000) (PolyScience, Inc.).

Construction of CRBN-KO HEK293T cells

CRBN-KO HEK293T cells were generated by genome editing using CRISPR/CAS9 system. The guide nucleotide sequence 5´-ACTCCGGGCGGTTACCAGGC- 3´ in the human CRBN gene was inserted into the Guide-It plasmid vector (Takara Bio). HEK293T cells were cultured in six-well plates and transfected with the plasmid for 2 days. Then, GFP-positive cells were sorted on a FACSAria (BD Biosciences), and cell clones were obtained by limiting dilution. Genomic DNA was then isolated, and the mutation was confirmed by sequencing after TA cloning (Toyobo).

Plasmids

The pDONR221, pcDNA3.1(+), and pCAGGS plasmids were purchased from Invitrogen or Riken, and the pEU vector for the wheat cell-free system was constructed in our laboratory, as previously described46. The pcDNA3.1(+)-FLAG-GW, pcDNA3.1(+)-AGIA-MCS, pEU-bls-GW, pEU-FLAG-GST-MCS, pCAGGS-MCS-S4D-AGIA, pCAGGS-MCS-(IKZF3 degron)-AGIA, and pCAGGS-S4D-MCS-AGIA plasmids were constructed by PCR and by using the In-Fusion system (Takara Bio) or PCR and restriction enzymes. The SALL4 gene sequence was purchased from the Kazusa DNA Research Institute. The Venus19 and SALL4 sequences were amplified, and restriction enzyme sites were added by PCR and cloned into pEU-FLAG-GST-MCS. The pEU-FLAG-GST-Venus-m1, -m2, and -m3 plasmids were constructed by inverse PCR from the pEU-FLAG-GST-Venus-SALL4 plasmid. Venus-m1, -m2, and -m3 were digested by restriction enzymes from pEU-FLAG-GST-Venus-m1, -m2, and -m3, and cloned into pcDNA3.1(+)-AGIA-MCS. Venus was also cloned into the pCAGGS-MCS-S4D-AGIA, pCAGGS-S4D-MCS-AGIA, and pCAGGS-MCS-IKZF3 degron (I3D)-AGIA plasmids using restriction enzymes. The open reading frames (ORFs) of CRBN, RELA (RelA), NFKBIA (IκBα), DRD1, and TP53 (p53) were purchased from the Mammalian Gene Collection (MGC)47, while the ORF of STING was purchased from Promega (Flexi Clone). The ORF of MAVS was provided by Dr. Youichi Suzuki. CRBN was amplified, and the BP reaction sequence (attB and attP) was added by PCR and cloned into pDONR221 using BP recombination (Invitrogen/Thermo Fisher Scientific). Then, pDONR221-CRBN was recombined into pEU-bls-GW or pcDNA3.1(+)-FLAG-GW using LR recombination (attL and attR). RelA, IκBα, and sfGFP were amplified, and restriction enzyme sites were added by PCR. Then, RelA-sfGFP and IκBα-sfGFP were cloned into pcDNA3.1(+)-AGIA-MCS, and the S4D tag was added to the plasmids pcDNA3.1(+)-AGIA-RelA-sfGFP and pcDNA3.1(+)-AGIA-IκBα-sfGFP using inverse PCR. p53, DRD1, STING, and MAVS were amplified, and restriction enzyme sites were added by PCR. They were then cloned into pCAGGS-MCS-AGIA, pCAGGS-MCS-S4D-AGIA, or pCAGGS-S4D-MCS-AGIA. Firefly luciferase (FLuc) was amplified by PCR using pGL4.32[luc2P/NF-kB-RE/Hygro] as a template and cloned into pCAGGS-MCS-AGIA or pCAGGS-MCS-S4D-AGIA using restriction enzymes.

For the generation of KI cells, genomic DNA was isolated from HeLa cells, locus-specific 5′ and 3′ homology arms were amplified by PCR, and restriction enzyme sites were added. These homology arms and protospacer adjacent motif (PAM) sequences were designed to remove the stop codon from the gene ORF, and to attach sfGFP and the S4D tag. Then, these fragments were cloned into the LITMUS29 vector48 using the In-Fusion system (Takara Bio). The sgRNA vector (Addgene) was used to produce KI cells. The guide nucleotide sequences were as follows: 5′-AGTCAGATCAGCTCCTAAGG-3′ (RelA) and 5′-GCAAAGGGGCTGAAAGAACA- 3′ (IκBα). The target sequences were cloned into the sgRNA vectors using Gibson assembly (New England Biolabs).

For cells stably expressing DRD1 or FLuc, pCSII-CMV-DRD1-S4D-AGIA, FLuc-AGIA, -FLuc-S4D-AGIA, or -FLuc-I3D-AGIA was constructed by using PCR and restriction enzyme.

Produce of lentivirus

Lentivirus for expressing DRD1-S4D-AGIA, S4D-FLuc-AGIA, FLuc-S4D-AGIA, or FLuc-I3D-AGIA were produced in HEK293T cells by transfection of pCSII-CMV-DRD1 or -FLuc expression vector together with pCMV-VSV-G-RSV-Rev and pCAG-HIVgp. Culture medium was exchanged after 24 h transfection, and the cells were cultured for 48 h. Then, the lentiviruses were concentrated by using Lenti-X concentrator (Takara Bio).

Generation of stably HEK293T or HeLa cells lines

HEK293T or HeLa cells supplemented with 5 µg/ml Polybrene (Nacalai Tesque) were infected by the appropriate lentivirus, and culture medium was exchanged after 24 h infection. 5 µg/ml blasticidin S (InvivoGen) selection was started after 24 h from exchange of culture medium.

Synthesis of recombinant protein using the wheat cell-free system

In vitro transcription and wheat cell-free protein synthesis were performed using the WEPRO1240 expression kit (Cell-Free Sciences). Transcription was performed using SP6 RNA polymerase with the plasmids or DNA fragments described above as templates. The translation reaction was performed in bilayer mode using the WEPRO1240 expression kit (Cell-Free Sciences), according to the manufacturer’s protocol. For biotin labeling, cell-free synthesized crude biotin ligase (BirA) produced using the wheat cell-free expression system was added to the bottom layer, and 0.5 µM (final concentration) of d-biotin (Nacalai Tesque) was added to both the upper and lower layers, as described previously49.

AlphaScreen-based interaction assay of CRBN-Venus-S4D

IMiD at a final concentration of 50 µM and 0.5 µl biotinylated CRBN were mixed in 15 µl AlphaScreen buffer containing 100 mM Tris (pH 8.0), 0.01% Tween20, 100 mM NaCl, and 1 mg/ml BSA. Then, 5 µl of a mixture containing 0.8 µl FLAG-GST-Venus-m1, -m2, or -m3 in AlphaScreen buffer was added, and the reaction mixtures were incubated at 26 °C for 1 h in a 384-well AlphaPlate (PerkinElmer). Subsequently, 5 µl of a detection mixture containing 0.2 µg/ml anti-DYKDDDDK mouse mAb (Wako), 0.08 µl streptavidin-coated donor beads, and 0.08 µl Protein A-coated acceptor beads (PerkinElmer) in AlphaScreen buffer was added to each well. After incubation at 26 °C for 1 h, luminescence was read on an EnVision plate reader (PerkinElmer).

In-cell IMiD-dependent degradation of S4D-tagged proteins

To confirm IMiD-dependent degradation of Venus-tagged proteins, HEK293T-CRBN−/− cells were cultured in 24-well plates and transfected with 400 ng pcDNA3.1(+)-FLAG-CRBN-WT together with 50 ng pcDNA3.1(+)-AGIA-Venus-m1, -m2, -m3, or -m2 variant (G416A). After the cells were transfected for 6 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

For degradation Venus-S4D by endogenous CRBN, HEK293T cells were cultured in 24-well plates and transfected with 5 ng pcDNA3.1(+)-AGIA-Venus-S4D. After the cells were transfected for 6 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

For degradation of S4D-Venus, Venus-S4D, and Venus-I3D, HEK293T cells were cultured in 48-well plates and transfected with 10 ng pCAGGS-S4D-Venus-AGIA, pCAGGS-Venus-S4D-AGIA, or pCAGGS-Venus-I3D-AGIA. After the cells were transfected for 6 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

For degradation of FLuc-S4D, HEK293T cells were cultured in 48-well plates and transfected with 2 ng pCAGGS-FLuc-AGIA or 25 ng pCAGGS-FLuc-S4D-AGIA. After the cells were transfected for 6 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

To examine the degradation of proteins with various subcellular localizations, we cultured HEK293T cells, HeLa cells, or HEK293T-CRBN−/− cells in 48-well plates and transfected with 200 ng pcDNA3.1(+)-FLAG-CRBN-WT or 200 ng pcDNA3.1(+)-empty together with 30 ng pCAGGS-p53-S4D-AGIA, 20 ng pCAGGS-STING-S4D-AGIA, 10 ng pCAGGS-DRD1-S4D-AGIA, 100 ng pCAGGS-MAVS-S4D-AGIA, 200 ng pCAGGS-GM130-S4D-AGIA. After the cells were transfected for 8 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

For degradation of DRD1-S4D-AGIA in stable cell line, HeLa cells expressing DRD1-S4D-AGIA were cultured in 24-well plates. Then, the cells were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

To examine the degradation of S4D-tagged RelA or IκBα, HeLa cells or HEK293T-CRBN−/− cells were cultured in 48-well plates and transfected with 200 ng pcDNA3.1(+)-FLAG-CRBN-WT or 200 ng pcDNA3.1(+)-FLAG-CRBN-YW/AA together with 15 ng pCDNA3.1(+)-AGIA-sfGFP-RelA or 15 ng pCDNA3.1(+)-AGIA-sfGFP-IκBα. After the cells were transfected for 8 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

To examine the degradation of endogenous S4D-tagged RelA or IκBα, parental, heterogeneous, or homogeneous KI HeLa cells were cultured in 48-well plates. Then, the cells were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

To show that IMiD-dependent degradation of S4D-tagged proteins is dependent on CRL and the 26 S proteasome, the cells were treated with 2 µM MLN4924 and 10 µM MG132 (0.2% DMSO) for the times indicated in each figure.

For protein degradation by 5-HT, HEK293T cells expressing S4D-FLuc-AGIA, FLuc-S4D-AGIA, or FLuc-I3D-AGIA or KI HeLa cells were culture in 48-well plates. Then, the cells were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure.

The cells were lysed by boiling at 95 °C for 5 min in 1× sample buffer containing 5% 2-mercaptoethanol, and the lysates were analyzed by immunoblot. In the case of cells expressing transmembrane protein, the cells were lysed by boiling at 50 °C for 10 min in 1× sample buffer containing 5% 2-mercaptoethanol, and the lysates were analyzed by immunoblot after sonication.

Quantitative evaluation using Firefly luciferase activity

HEK293T cells were cultured in 96-well plates and transfected with 0.1 ng pCAGGS-FLuc-AGIA or 1 ng pCAGGS-FLuc-S4D-AGIA. After the cells were transfected for 6 h, they were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure. In the case of stable cell lines expressing S4D-FLuc-AGIA, FLuc-S4D-AGIA, or FLuc-I3D-AGIA, the cells were cultured in 96-well plates and were treated with IMiD or DMSO (0.1%) in culture medium, at the times and concentrations indicated in each figure. The cells were lysed with 45 µl 1× Passive Lysis Buffer (Promega), and the lysates were diluted 200-fold with 1× Passive Lysis Buffer. Then, FLuc luciferase activity was measured on a Glomax luminometer (Promega) using the luciferase assay system (Promega).

Immunofluorescence staining

HeLa cells were cultured on 12 mm poly-L-lysine-coated glass slides (Sigma–Aldrich) in 24-well plates and transfected with 200 ng pCAGGS-p53-S4D-AGIA, 100 ng pCAGGS-STING-S4D-AGIA, 400 ng pCAGGS-DRD1-S4D-AGIA, 500 ng pCAGGS-MAVS-S4D-AGIA, or 300 ng pCAGGS-GM130-S4D-AGIA. After transfection for 24 h, the cells were fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 min, and permeabilization was performed with 0.1% Triton X-100 in PBS (for p53, STING, MAVS, and GM130) or 0.01% digitonin in PBS (for DRD1) at room temperature for 15 min. Then, the cells were incubated with 0.5% cattle serum (CS) in TBST at 4 °C for 1 h, and incubated with an anti-AGIA antibody (produced by our laboratory) at 4 °C for 16 h. After washing with TBST at room temperature for 15 min, the cells were incubated with an Alexa Flour 488-conjugated anti-rabbit secondary antibody at room temperature for 1 h. The nucleus, ER, and mitochondria were stained with 4′,6-diamidino-2-phenylindole (DAPI), ER-ID (Enzo), or Mito-ID (Enzo), respectively, according to the manufacturer’s protocol. After washing with TBST at room temperature for 15 min, the stained cells were mounted with anti-fade (Invitrogen/Thermo Fisher Scientific) and observed under a BZ-X810 Microscope (Keyence).

For immunofluorescence staining of GM130, the cells were incubated with an anti-AGIA antibody (for GM130-S4D-AGIA) together with an anti-GM130 antibody (for staining Golgi apparatus) (MBL, M179-3MS, 1:200) at 4 °C for 16 h. After washing with TBST at room temperature for 15 min, the cells were incubated with an Alexa Flour 488-conjugated anti-rabbit secondary antibody and Alexa Flour 555-conjugated anti-mouse secondary antibody at room temperature for 1 h.

For immunofluorescence staining of stably expressed DRD1, HeLa cells expressing DRD1-S4D-AGIA were incubated with an anti-AGIA antibody19 (produced in our laboratory, 1:1000) at 4 °C for 16 h. After washing with TBST at room temperature for 15 min, the cells were incubated with an Alexa Flour 488-conjugated anti-rabbit secondary antibody at room temperature for 1 h.

cGAMP-induced ISRE luciferase reporter assay

HEK293T cells were cultured in 96-well plates and transfected with 1 ng empty vector, 0.1 ng pCAGGS-STING-AGIA, or 1 ng pCAGGS-STING-S4D-AGIA together with 4 ng pGL4-ISRE-promoter-Luc plasmid (Promega) and 1 ng pRL-TK-Renilla-Luc plasmid (Promega). After the cells were transfected for 6 h, they were pretreated with DMSO (0.1%) or pomalidomide in culture medium for 16 h. The cells were stimulated with 2 µg/ml cGAMP in permeabilization buffer [50 mM HEPES (pH 7.2–7.5), 100 mM KCl, 3 mM MgCl2, 0.1 mM dithiothreitol (DTT), 85 mM sucrose, 1 mM ATP, 0.2% BSA, 10 µg/ml digitonin) for 3 h, and then lysed with 45 µl 1× Passive Lysis Buffer, and the Firefly and Renilla luciferase activity was measured on a Glomax luminometer using the dual-luciferase reporter assay system (Promega).

Generation of KI cells expressing RelA- or IκBα-sfGFP-S4D

HeLa cells were cultured in six-well plates for 24 h and we made RelA-sfGFP-S4D and IkBa-sfGFP-S4D-KI cells as described previously48 by using TransIT-LT1 transfection reagent (Mirus Bio). After 2 days of transfection, the cells were passaged and cultured in 10 cm dishes. After 2 days of culture, the GFP-positive cells were isolated by sorting on a FACSAria (BD Biosciences). The insertion of sfGFP-S4D into RelA or IκBα gene loci was confirmed by a band shift on immunoblot analysis.

Reversibility of the S4D system

To examine the reversibility of the S4D system, KI (RelA-sfGFP-S4D or IκBα-sfGFP-S4D) HeLa cells were cultured in 12-well plates. The cells were treated with DMSO or 10 µM pomalidomide for 6 h, and then the DMSO or pomalidomide was removed by washing with culture medium. Cells were harvested at the times indicated in Fig. 4g and lysed with RIPA buffer containing protease inhibitor cocktail (Sigma–Aldrich). The protein concentrations of the lysates were quantitated by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), and the expression of RelA-sfGFP-S4D or IκBα-sfGFP-S4D was analyzed by immunoblot.

Quantitative RT-PCR

For analysis of cGAMP-induced genes, HEK293T cells were cultured in 96-well plates and transfected with 1 ng pCAGGS-STING-S4D-AGIA. After the cells were transfected for 6 h, they were pretreated with DMSO (0.1%) or 10 µM pomalidomide in culture medium for 16 h. Then, the cells were stimulated with 2 µg/ml cGAMP in permeabilization buffer. After 3 h, total RNA was isolated from the cells using the SuperPrep cell lysis kit (Toyobo), and cDNA was synthesized using the SuperPrep RT kit (Toyobo), according to the manufacturer’s protocol. RT-PCR was performed using KOD SYBR qPCR Mix (Toyobo), and data were normalized against the GAPDH mRNA levels. PCR primers are as follows50,51: IFN-β sense 5′-GGACCATAGTCAGAGTGGAAATCCTAAG-3′, IFN-β anti-sense 5′-CACTTAAACAGCATCTGCTGGTTGAAG-3′, CXCL10 sense 5′-AGCAGAGGAACCTCCAGTCT-3′, CXCL10 anti-sense 5′-AGGTACTCCTTGAATGCCACT-3′, CCL5 sense 5′-CTGCTTTGCCTACATTGCCC-3′, CCL5 anti-sense 5′-TCGGGTGACAAAGACGACTG-3′, ISG56 sense 5′- CAAAGGGCAAAACGAGGCAG-3′, ISG56 anti-sense 5′-CCCAG GCATAGTTTCCCCAG-3′, GAPDH sense 5′-AGCAACAGGGTGGTGGAC-3′, and GAPDH anti-sense 5′-GTGTGGTGGGGGACTGAG-3′.

For analysis of TNF-α-induced genes, parental or KI (RelA-sfGFP-S4D) HeLa cells were cultured in 96-well plates and pretreated with DMSO (0.1%) or 10 µM pomalidomide. After 12 h of pomalidomide pretreatment, the cells were treated with 20 ng/ml TNF-α for 2 h. Total RNA was isolated from the cells using the SuperPrep cell lysis kit (Toyobo), and cDNA was synthesized using the SuperPrep RT kit (Toyobo), according to the manufacturer’s protocols. RT-PCR was performed using KOD SYBR qPCR Mix (Toyobo), and data were normalized against GAPDH mRNA levels. PCR primers are as follows37: IκBα sense 5′-CGGGCTGAAGAAGGAGCGGC-3′, IκBα anti-sense 5′-ACGAGTCCCCGTCCTCGGTG-3′, IL-6 sense 5′-AGCCACTCACCTCTTCAGAAC-3′, and IL-6 anti-sense 5′-GCCTCTTTGCTGCTTTCACAC-3′.

Extraction of nuclei and cytoplasm

Parental or KI HeLa cells were cultured in 6-well or 12-well plates and pretreated with DMSO (0.1%) or pomalidomide in culture medium for the times indicated in each figure. Then, the cells were stimulated with 20 ng/ml TNF-α for the indicated times and harvested using TrypLE (Gibco/Thermo Fisher Scientific). Nuclear and cytoplasmic fractions were extracted using the NE-PER Nuclear and Cytoplasmic Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. The protein fractions were separated by SDS-PAGE and analyzed by immunoblot using specific antibodies.

Cell viability assay

For combination treatment with TNF-α and cycloheximide (CHX), parental or KI (RelA-sfGFP-S4D) HeLa cells were cultured in 96-well plates and treated with 20 ng/ml TNF-α and 10 µg/ml CHX for 24 h. For combination treatment with TNF-α and pomalidomide, parental or KI (RelA-sfGFP-S4D) HeLa cells were cultured in 96-well plates and pretreated with DMSO (0.1%) or pomalidomide in culture medium. After 12 h of pomalidomide treatment, the cells were treated with 20 ng/ml TNF-α for 12 h. Then, the number of viable cells was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega) and a SpecraMax M3 microplate reader (Molecular Devices).

Analysis of TNF-α-induced cell death by immunoblot

Parental or KI (RelA-sfGFP-S4D) HeLa cells were cultured in six-well plates and pretreated with pomalidomide at the concentrations indicated in each figure. After 12 h of pomalidomide pretreatment, cells were treated with 50 ng/ml TNF-α for the times indicated in each figure. Then, the cells were harvested using TrypLE, and the number of dead cells was counted by trypan blue staining using a Countess Automated Cell Counter (Invitrogen/Thermo Fisher Scientific). The cells were lysed with RIPA buffer containing protease inhibitor cocktail (Sigma–Aldrich), and protein concentrations were measured by BCA assay (Thermo Scientific). Then, the lysates were analyzed by immunoblot with specific antibodies. For experiments with zVAD-FMK, the cells were treated with DMSO (0.1%) or 10 µM zVAD-FMK for 2 h before stimulation with TNF-α.

Statistics and reproducibility

All data were analyzed from at least three technical repeats (n = 3–5) and were presented as the means ± standard deviation (SD). Significant changes were analyzed by one-way analysis of variants (ANOVA) followed by Tukey’s tests using GraphPad Prism (version 8) software (GraphPad, Inc.). Western blots and immunofluorescence images were repeated more than three times with similar results.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.



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