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arsenite for 0, 20 and 30 minutes, then stained with anti-G3BP1 antibodies and. Hoechst33258, similar to that described in Figure 5A, to measure SG formation.

Supplemental data

HTLV-1 Tax oncoprotein stimulates ROS production and apoptosis in T-cells by interacting with USP10

Masahiko Takahashi, Masaya Higuchi, Grace Naswa Makokha, Hideaki Matsuki, Manami Yoshita, Yuetsu Tanaka and Masahiro Fujii

Supplemental Methods Cell lines and culture conditions 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 mM of L-glutamine, 50 units/ml of penicillin and 50 g/ml of streptomycin. Jurkat, HUT78 and MOLT-4 are HTLV-1-negative human T-cell lines. While HUT-102, SLB-1, C5/MJ and MT-4 are HTLV-1-transformed human T-cell lines, MT-1, TL-OmI, ED509823 and KK1 are human T-cell lines originating from the leukemic cells of ATL patients 1. These T-cell lines were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FBS, 4 mM of L-glutamine, 50 units/ml of penicillin and 50 g/ml of streptomycin (RPMI/10% FBS). Recombinant human IL-2 (Takeda Chemical Industries) was added at 0.5 nM to cultures of ED509823 and KK1 cells. Ramos, Daudi and BJAB are B-cell lines derived from Burkitt lymphoma (BL). NB4 and THP-1 are myeloid cell lines derived from myeloid leukemia (ML). These cells were cultured in RPMI/10% FBS.

Plasmid constructs

To construct expression vector encoding human USP10, human USP10 gene was amplified by polymerase chain reaction (PCR) and inserted into the BglII and XhoI sites of pCMV-HA, a mammalian expression vector encoding a protein with an N-terminal hemagglutinin (HA) epitope tag (Clontech). To construct expression vectors encoding USP10 mutants (USP101-116, USP1096-798, USP101-726 and USP101-439), USP10 mutant genes were amplified by PCR and inserted into the BglII and XhoI sites of pCMV-HA. The expression vectors encoding Tax, Tax∆C, TaxM22 and Tax703 have been previously described

2,3

. To construct expression vectors encoding other Tax mutants

(Tax62-353, TaxSH-1 and TaxSH-2 4), the respective tax genes were amplified by PCR and inserted into the HindIII and BamHI sites of pHPr.1-neo, a mammalian expression vector which has a -actin promoter for protein expression 5. To construct lentiviral expression vectors encoding DsRed, Tax, Tax∆C, TaxM22, Tax703, TaxSH-1 and TaxSH-2, these genes were amplified by PCR, cloned into the entry vector pENTRTM2B (Invitrogen), and transferred to the lentiviral vector plasmid CSII-EF-RfA by an LR recombination reaction using LR Clonase (Invitrogen). CSII-EF-RfA was kindly provided by Dr. H. Miyoshi (RIKEN Tsukuba Institute, Japan). Lentiviral expression plasmids encoding human USP10 shRNA (sh-USP10-1 and sh-USP10-3) with a puromycin-resistant gene were purchased from Sigma-Aldrich.

Establishment of stable USP10-knockdown cell lines using lentiviral transduction VSV-G-pseudotyped HIV-1-based viruses were produced by the cotransfection of three plasmids (lentiviral plasmid encoding human USP10 shRNA: 4.28 μg; pCAG-HIVgp: 2.86 μg; pCMV-VSV-G-RSV-Rev: 2.86 μg) into 293T cells (2.0×106) on 100-mm dishes using FuGENE HD reagent according to the manufacturer’s instructions (Roche).

Seventy-two hours after transfection, the culture supernatants were harvested and infected into Jurkat and MT-4 cells in the presence of 8 μg/μl of polybrene. The infected cells were cultured in a selection medium containing 0.5 μg/ml of puromycin for 10 days.

Supplemental References 1.

Yamada Y, Nagata Y, Kamihira S, et al. IL-2-dependent ATL cell lines with phenotypes

differing

from

the

original

leukemia

cells.

Leuk

Res.

1991;15(7):619-625. 2.

Iwanaga Y, Tsukahara T, Ohashi T, et al. Human T-cell leukemia virus type 1 tax protein abrogates interleukin-2 dependence in a mouse T-cell line. J Virol. 1999;73(2):1271-1277.

3.

Hirata A, Higuchi M, Niinuma A, et al. PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein augments the transforming activity in a rat fibroblast cell line. Virology. 2004;318(1):327-336.

4.

Matsumoto K, Akashi K, Shibata H, Yutsudo M, Hakura A. Single amino acid substitution (58Pro-->Ser) in HTLV-I tax results in loss of ras cooperative focus formation in rat embryo fibroblasts. Virology. 1994;200(2):813-815.

5.

Matsumoto K, Shibata H, Fujisawa JI, et al. Human T-cell leukemia virus type 1 Tax protein transforms rat fibroblasts via two distinct pathways. J Virol. 1997;71(6):4445-4451.

Supplemental Figure Legends Supplemental Figure 1. Tax induces arsenite-induced apoptosis in 293T cells. 293T

cells were transfected with the indicated plasmids (Tax, Tax62-353, TaxSH-1 or TaxSH-2). The cells were then treated with 0.5 mM of sodium arsenite for 80 minutes, washed, further cultured in fresh medium for one hour, then stained with anti-Tax (red) antibodies and Hoechst33258 (blue). The arrows indicate apoptotic cells with condensed nuclei. The bar indicates 20 m.

Supplemental Figure 2. USP10 in an HTLV-1-transformed T-cell line controls the SG-forming activity and sensitivity to arsenite-induced apoptosis. (A) An HTLV-1-transformed T-cell line MT-4 was infected with lentiviruses encoding human USP10 shRNA (sh-USP10-1 or sh-USP10-3) or control nontargeting shRNA (sh-NT), and the cells were then cultured in the presence of puromycin. Cell lysates prepared from the selected cells were characterized using a Western blot analysis with anti-USP10, anti-Tax and anti-α-tubulin antibodies. (B) USP10-knockdown MT-4 cells and control cells were stained with CM-H2DCFDA, and the ROS levels (DCFDA-F) in the cells were quantitatively measured using a cell imaging software program. (C) USP10-knockdown MT-4 cells and control cells were treated with 0.25 mM of sodium arsenite for 0, 20 and 30 minutes, then stained with anti-G3BP1 antibodies and Hoechst33258, similar to that described in Figure 5A, to measure SG formation. The SG (%) is presented. (D) USP10-knockdown MT-4 cells and control cells were treated with 5 μM of sodium arsenite for 48 hours, then stained with propidium iodide (PI). The proportion of the sub-G1 fraction was measured using flow cytometry. In all experiments, the values denote the mean ± s.d.; *P < 0.05; ***P < 0.001.

Supplemental Figure 3. The sensitivity to apoptosis induced by arsenic trioxide

(As2O3) in HTLV-1-infected T-cells correlates with the SG-forming activity. (A) Jurkat and SLB-1 cells were treated with 0.1, 0.5 and 1.0 mM of As2O3 for 30 minutes, then stained with anti-USP10 antibodies (green), anti-G3BP1 antibodies (red) and Hoechst33258 (blue). The bar indicates 10 m. (B) The SG (%) is presented. (C) Jurkat and SLB-1 cells were incubated with or without 10 mM of NAC, then further treated with 5 μM of As2O3 for 48 hours and stained with PI. The proportion of the sub-G1 population (apoptotic cells) was measured using flow cytometry. In all experiments, the values denote the mean ± s.d.; ***P < 0.001.

Takahashi et al. Supplemental Figure 1

Tax

Control

Tax

62-353

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arsenite Merge

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Takahashi et al. Supplemental Figure 2

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Takahashi et al. Supplemental Figure 3

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