Supplementary Information p47 negatively regulates IKK activation by inducing the lysosomal degradation of polyubiquitinated NEMO Yuri Shibata, Masaaki Oyama, Hiroko Kozuka-Hata, Xiao Han, Yuetsu Tanaka, Jin Gohda, and Jun-ichiro Inoue Supplementary Figures S1-S6 1
Supplementary Figure S1. p47 is mostly localized in the cytoplasm. HeLa and HCC1937 cells were washed in ice-cold PBS and resuspended in hypotonic buffer (20 mm Tris-HCl at ph 7.5, 1 mm MgCl 2, 5 mm KCl, and a protease inhibitor cocktail). After a 10-min incubation on ice, the cell suspension was homogenized with a Dounce homogenizer (20 strokes) and centrifuged at 1,000 g for 5 min at 4ºC. The pellet served as the nuclear fraction (Nuc). The supernatant was collected and further centrifuged at 22,000 g for 60 min at 4ºC to remove the plasma membrane fraction. The supernatant was used as the cytoplasmic fraction (Cyto). Both fractions were subjected to immunoblotting with anti-p47, anti-tubulin, and anti-parp-1 antibodies. Tubulin is a marker of the cytoplasmic fraction, and PARP-1 is a marker of the nuclear fraction. The depicted results are representative of three independent experiments. 2
Supplementary Figure S2. The silencing of p47 does not affect cell proliferation and induces neither apoptosis nor ER stress. (a, b) The silencing of p47 does not affect cell proliferation. HeLa cells (a) and HEK293T cells (b) were mock-transfected (None) or transfected with control or p47 sirna. After 24, 3
48, and 72 h, the cells were trypsinized, and the numbers of viable cells were counted using the trypan blue exclusion method. The results shown indicate the mean ± SD (n=3). (c, d) The silencing of p47 does not induce apoptosis. HeLa cells were transfected with control or p47 sirna (c). After 72 h, the cells were harvested, and the apoptotic cell death was determined by flow cytometry analysis of cells stained with Annexin V-FITC/propidium iodide (PI) using an Annexin V-FITC Apoptosis Detection Kit (Bio Vision) according to the manufacturer s instructions. As a positive control to induce apoptosis, HeLa cells were treated with TNF-α (20 ng/ml) in combination with 1 mm cycloheximide (CHX) for 12 h. HEK293T cells were transfected with control or p47 sirna (d). The apoptotic cell death was measured as described in c. As a positive control to induce apoptosis, HEK293T cells were transfected with an expression plasmid for Myc-tagged apoptosis signal-regulating kinase 1 (Mys-ASK1). (e-h) The silencing of p47 does not induce ER stress. HEK293T cells (e, g) and HeLa cells (f, h) were mock transfected (None) or transfected with control or p47 sirna. After 72 h incubation, total RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer s instructions, and cdna was then synthesized from 1 µg of total RNA with Prime Script II (Takara Bio). RT-PCR was performed to detect unspliced (arrow heads) and spliced (arrows) XBP1 mrna (e, f). The spliced XBP1 mrna, an indicator of ER stress, was not generated with p47 silencing. Semi-quantitative RT-PCR was performed using a 10-fold dilution series of cdna as templates to analyze the induction of BIP mrna (g, h). As a positive control to induce ER stress, cells were treated with DTT (2 mm) for 20 min. The induction of BIP mrna, an indication of ER stress, was not observed with p47 silencing. The following primers were used: XBP1 sense, 5 -GAGTTAAGACAGCGCTTGGG-3 ; XBP1 anti-sense, 5 -ACTGGGTCCAAGTTGTCCAG-3 ; BIP sense, 5 -TGCAGCAGGACATCAAGTTC-3 ; BIP anti-sense, 5 -CGCTGGTCAAAGTCTTCTCC-3 ; p47 sense, 5 -GAGGGGATGAAGACATTGTGA-3 ; p47 anti-sense, 5 -GAATGCTGCCTCTTTTCTCCT-3, ACTB sense, 5 -TTCTACAATGAGCTGCGTGTG-3 ; and ACTB anti-sense, 5 -CCTTAATGTCACGCACGATTT -3. The depicted results are representative of three independent experiments. 4
Supplementary Figure S3. p47 negatively regulates the Tax-induced phosphorylation of IκBα. HeLa cells were transfected with a control or p47 sirna. After 48 h, cells were transfected with a plasmid encoding Tax. After 24 h, cell lysates were prepared and subjected to immunoblotting with an anti-p-iκbα antibody. The depicted results are representative of three independent experiments. 5
Supplementary Figure S4. Silencing of p47 and E64D/pepstatin A treatment result in the enhanced accumulation of polyubiquitinated NEMO after TNF-α stimulation. (a) HEK293T cells were transfected with control or p47 sirna. After 24 h, the cells were transfected with an HA-ubiquitin expression plasmid. After 48 h of incubation, the cells were treated with TNF-α (10 ng/ml) for the indicated times. The cell lysates prepared in TNE buffer were boiled for 10 min in the presence of 1% SDS to dissociate proteins noncovalently attached to NEMO. The lysates were diluted in TNE buffer to reduce the SDS concentration to 0.1%. Immunoprecipitation was performed with an anti-nemo antibody followed by immunoblotting with an anti-ha antibody. (b) HEK293T cells were transfected with expression plasmid for HA-ubiquitin. After 36 h, the cells were treated with E64D (5 µg/ml)/pepstatin A (1 µg/ml) or EtOH for 12 h. The cells were then treated with TNF-α (10 ng/ml) for the indicated times. The cell lysates prepared and immunoprecipitation and subsequent immunoblotting were performed as described in a. The depicted results are representative of three independent experiments. 6
Supplementary Figure S5. Inhibition of lysosomal protein degradation by chloroquine or bafilomycin A1 results in the enhanced accumulation of polyubiquitinated NEMO after TNF-α stimulation. (a) HEK293T cells were transfected with an expression plasmid for HA-ubiquitin. After 46 h, the cells were treated with either chloroquine (100 µm, purchased from Wako) (a) or bafilomycin A1 (BFLA, 100 µm, purchased from SIGMA) (b) for 2 h. After TNF-α (10 ng/ml) stimulation for the indicated times, the cell lysates prepared in TNE buffer were boiled for 10 min in the presence of 1% SDS to dissociate proteins noncovalently attached to NEMO. The lysates were diluted in TNE buffer to reduce the SDS concentration to 0.1%. Immunoprecipitation was performed with an anti-nemo antibody followed by immunoblotting with an anti-ha antibody. The depicted results are representative of three independent experiments. 7
Supplementary Figure S6. Correlation between the reduced p47 expression and the constitutive NF-κB activation in ATL cells. (a) Reduced p47 expression in ATL patients. Statistical analysis of p47 expression in normal controls (n=21), acute type ATL patients (n=27) and chronic type ATL patients (n=19) based on a data set deposited in Gene Expression Omnibus database with the accession number GSE33615 is shown using the box and whisker plot, which is depicted with boxes showing median and 25 th and 75 th percentile, whiskers showing minimal and maximal values. (b) Expression levels of p47 protein in ATL and HTLV-1-infected cell lines. Jurkat cells, two ATL cell lines (MT-1 and TLOM-1), and three HTLV-1-infected cell lines (MT-2, MT-4, and HUT-102) were harvested, and the cell lysates were analyzed by immunoblotting with anti-p47 and anti-tubulin antibodies. The expression levels of p47 are calculated as the 8
intensity of the band detected by the anti-p47 antibody divided by that detected by the anti-tubulin antibody. The expression level of p47 in Jurkat cell is set to 100. The intensities of the bands were quantified using ImageJ software. (c) Expression level of mrnas of p47, RELB, NFKBIA in ATL and HTLV-1-infected cell lines. The expression levels of p47, RELB, NFKBIA were measured by real-time RT-PCR. The relative expression levels were calculated by dividing each expression value by that of Jurkat cells. The following primers were used: RELB sense, 5 -ATTGAGCGGAAGATTCAACT-3 ; RELB anti-sense, 5 -TGTGGATTTCTTGTCATAGA-3 ; NFKBIA sense, 5 -GCTGAAGAAGAAGCGGCTACT-3 ; NFKBIA anti-sense, 5 -TCGTACTCCTCGTCTTTCATG-3 p47 sense, 5 -GAGGGGATGAAGACATTGTGA-3 ; p47 anti-sense, 5 -GAATGCTGCCTCTTTTCTCCT-3. The results shown indicate the mean ± SD (n=3). (d) Tax disrupts the binding between p47 and NEMO. HEK293T cells were transfected with expression plasmids for HA-NEMO (1.0 µg) and Flag-p47 (0.3 µg) with or without the Tax expression plasmid (1.0 µg). After 48 h, the cell lysates were subjected to immunoprecipitation with an anti-ha antibody followed by immunoblotting with anti-flag, anti-ha, and anti-tax antibodies. The WCL was analyzed by immunoblotting with anti-flag and anti-tubulin antibodies. (e) Overexpression of p47 inhibits constitutive NF-κB activation in MT-2 cells. MT-2 cells were transfected with 0.5 µg of firefly luciferase reporter (3xκB-luc or 3xMκB-luc (MκB: a mutated NF-κB-binding sites)) and 0.5 µg of β-actin-renilla luciferase plasmid in the presence or absence of a p47 expression plasmid (3.0 µg) using DMRIE-C reagent (Life Technologies, Carlsbad, CA, USA). After 48 h, the subsequent luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega BioSciences, San Luis Obispo, CA, USA). Renilla luciferase activity was used to normalize the transfection efficiency. Fold induction was calculated by dividing each luciferase activity by that of 3xMκB-luc transfection. The results shown indicate the mean ± SD (n=3). The depicted results are representative of three independent experiments. Statistical significance was assessed using a Student s t-test. ***, P < 0.01; **, P < 0.02; *, and P < 0.05. 9