Supplementary Information
TRC8 suppresses tumorigenesis through targeting heme oxygenase-1 for ubiquitination and degradation
Pu-Hua Lin, Wan-Min Lan and Lee-Young Chau
Supplementary materials and methods
Real-time quantitative PCR.HEK293T cells (2.5x105) were transfected with plasmid DNA encoding HO-1 (0.4 μg) and increasing amounts of TRC8 plasmid DNA (0.1~ 0.8 μg) as indicated. After 30 h, the total RNAs were isolated using the Trizol Reagent (Invitrogen). Reverse transcription was performed using 2 µg DNase I treated total RNA, 100 ng random hexamers, 10 nmole dNTPs and 200 units of Superscript III reverse transcriptase (Invitrogen). Real-time quantitative PCR for human HO-1 and GAPDH was then performed using LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Applied Science). The primers used for HO-1 were: 5’- TTCTCCGATGGGTCCT-3’ and 5’-TGTGCTTTTCGTTGGG-3’. The primers used for GAPDH were :5’- ACTTCAACAGCGACAC-3’ and 5’-GTGAGGGTCTCTCTCTT-3’.
Immunofluorescence. HEK293T cells were seeded on cover glasses coated with 0.1% gelatin. Cells were then transfected withHA-tagged TRC8 cDNA in combination with DsRed2-ER (BD Biosciences Clontech) or Flag-tagged HO-1 cDNA as indicated. After 30 h, cells were washed twice with PBS, fixed in 4% paraformaldehyde for 15 min, and followed by 3 washes with phosphate-buffered saline (PBS). Fixed cells were then permeabilized by 0.1% Triton X-100 in PBS for 30 min, followed by blocking with 3% BSA for 1 h. Cells were then incubated in 200 μl of PBS containing 0.1 % Triton X100, mouse anti-HA antibody (1:100 dilution) and rabbit anti-HO-1 antibody (1: 300 dilution). Negative control was performed by incubating fixed cells with normal mouse IgG (1: 100 dilution ; Santa Cruz) and normal rabbit IgG (1: 300 dilution; Santa Cruz). After incubation at 4oC for 18 h, cells were washed 3 times with PBS and then incubated with FITC-AffiniPure Goat anti-mouse IgG (1: 1000 dilution ; Jackson ImmunoResearch Inc.) and Alexa-Fluor 568-conjugated goat anti-rabbit IgG (1: 1000 dilution ; Molecular Probes) for 1 h at room temperature. After 3 washes with PBS, cells was counterstained with DAPI. The cover glasses were then mounted with 50% glycerol on glass slides. The fluorescence immunostains were examined by UltraView confocal microscope (Perkin Elmer, Wellesley, MA, USA). The laser excitation beam 405 nm (DAPI), 488 nm (FITC), 568 nm (cy-3) passed through a 45o mirror into the microscope and then onto the samples. Photomicrographs were captured using a 100x oil immersion objective lens with a numerical aperture (NA) of 1.4 (at room temperature). Representative images were processed with Velocity Software (Mannheim, Germany). Scale bar, 5 μm.
In vitro ubiquitination. A glutathione S-transferase (GST)-fused HO-1 construct was
prepared by subcloning a cDNA fragment encoding human HO-1 protein (amino acids
7-247) into the pGEX-4T-1 vector. Both GST and GST-fused HO-1 proteins were then
induced in E. coli BL-21 and purified by glutathione sepharose chromatography.
HEK293T cells plated in three 10-cm dishes were transfected with HA-tagged TRC8 cDNA
for 30 hr. Cells were then lysed in buffer containing 50 mMTris-HCl, pH 7.5,150 mM
NaCl, 0.5% NP-40, and a protease inhibitor mixture. After centrifugation at 13,000 xg for
15 min at 4oC, the supernatant was removed and the HA-tagged TRC8 protein waspurified
withanti-HA affinity agarose gel(Sigma). After the final wash with buffer, the anti-HA
affinity agarose bound withHA-tagged TRC8 was resuspended in 30 μl of buffer containing
50 mM Tris-HCl, pH 7.5,4mM MgCl2, and 0.2 % NP-40, and used immediately for
ubiquitination assay. An in vitro ubiquitination assaywas performed by incubating 1μg
of GST or GST-HO-1with or without 250 ngE1(Calbiochem), 500 ng GST-UbcH5b (E2)
(Calbiochem), and 10μlof HA-taggedTRC8-bound agarose in 30 μl of ubiquitination buffer
containing 50 mMTris-HCl, pH 7.5, 4mM MgCl2, 1mM DTT, 2.5μg ubiquitin
(BostonBiochem), 1 mM ATP, 10 mM creatine phosphate,and 16IU
phosphocreatine kinase at 30 °C for 2 hr with shaking. The reaction mixture was then
centrifuged at5,000 xg for 2 min at room temperature. Both supernatantand pellet
(HA-tagged TRC8 bound agarose )fractions wereboiled in SDS sample buffer for 5
min and then subjected to SDS-PAGE and Western blot analysis. The primaryantibodies
usedwere anti-TRC8 (Abcam; 1:3000), anti-GST (Santa Cruz;1:3000), andanti-ubiquitin
(Santa Cruz; 1:1000).
Supplementary Figure Legends
Supplementary Figure S1. TRC8 has no effect on HO-1 mRNA expression. HEK293T cells were transfected with cDNAs encoding HO-1 (0.4 μg) and increasing amount of TRC8 (0.1~0.8 μg) as indicated for 30 h. Total RNAs were then isolated, and the mRNA levels of HO-1 and GAPDH were quantified by real-time RT-PCR. Data shown are the mean ± S.E. of 3 independent experiments.
Supplementary Figure S2.Colocalization of full length HO-1 and TRC8 in ER. HEK293T cells were transfected with HA-tagged TRC8 cDNA together with DsRed2-ERcDNA (A),Flag-tagged full length HO-1cDNA (B), or Flag-tagged truncated HO-1 (t-HO-1) cDNA(C) as indicated. After 30 hr, cells were fixed andsubjected to immunofluorescence staining and confocal microscopic examination. Bar, 5μm.
Supplementary Figure S3. In vitro ubiquitination of HO-1 by TRC8. GST or GST-fused HO-1 protein was incubated with or without purified HA-tagged TRC8 bound to anti-HA affinity agarose, E1 and E2 as indicated in a reaction buffer containing ubiquitin, ATP and ATP regeneration system at 30oC for 2 hr with shaking. The production of ubiquitinated high molecular weight protein species was then examined by Western blot analysis using specific anti-ubiquitin antibody.
Supplementary Figure S4. Colocalization of HO-1 with HA-T270 and HA-T316 ∆211-270 in ER. HEK293T cells were transfected with full length HO-1 cDNA together with HA-T270 (A) or HA-T316 ∆211-270 (B) construct as indicated. After 30 hr, cells were fixed and subjected to immunofluorescence staining and confocal microscopic examination. Bar, 5μm.
Supplementary Figure S5. SiRNA-mediated HO-1 knockdown in TRC8-depleted U2OS cells. Control and TRC-8 knockdown U2OS cell lines were transfected with control scrambled siRNA (si-NC) or specific siRNA targeting HO-1 (si-HO-1) for 48 h. The expression level of HO-1 and β-actin (internal control) were examined by Western blot analysis and quantified by densitometry. The level in control cells transfected with scrambled siRNA was set to 100. Data shown are the mean ±S.E. for 3 independent experiments.