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1 DOI: /ncb2153 Figure S1 Ectopic expression of HAUSP up-regulates REST protein. (a) Immunoblotting showed that ectopic expression of HAUSP increased REST protein levels in ENStemA NPCs. (b) Immunofluorescent staining confirmed that ectopic expression of HAUSP up-regulated REST protein levels in nuclei. Differentiated ENStemA cells were transfected to express HA-HAUSP for 48 hours and then fixed for immunofluorescent staining with anti-ha (HA-HAUSP) and anti-rest specific antibodies. The cells expressing HA-HAUSP (green) displayed increased nuclear REST levels (red). The cells expressing HA-HAUSP with increased REST are indicated by arrows. Nuclei are counterstained with DAPI (blue). 1
2 Figure S2 REST knockdown in NPCs phenocopied HAUSP knockdown in neuronal differentiation. (a) Immunoblotting showed that reduced REST protein by shrna to the levels caused by HAUSP knockdown induced similar expression of the neuronal marker TUJ NPCs were infected with lentiviruses expressing shhausp, shrest or the non-targeting (NT) shrna for 72 hours, and harvested for immunoblotting. (b) Immunofluorescent staining confirmed that REST knockdown in NPCs induced neuronal differentiation similar to that induced by HAUSP knockdown NPCs cultured on the coated cover glasses were infected with lentiviruses expressing shhausp, shrest or NT shrna for 5 days, and analyzed by the immunofluorescent staining with specific antibodies against Nestin (an NPC maker, in red) and TUJ1 (a neuronal differentiation marker, in green). (c) Quantified data from b indicated that HAUSP knockdown and REST knockdown in the NPCs induced similar fractions of cells expressing the neuronal marker TUJ1. Data are means ± s.d. [n = 3 (200 cells/ experiment)]. 2
3 Figure S3 HAUSP knockdown reduced self-renewal potential of ENStemA NPCs, which was rescued by REST overexpression. (a) Targeting HAUSP with specific shrna reduced neurosphere formation of NPCs. ENStemA NPCs were infected with lentiviruses expressing shhausp or nontargeting (NT) shrna for 48 hours and allowed to form neurospheres for 9 days. HAUSP knockdown reduced neurosphere size and increased the attachment of neurospheres when cultured in suspension conditions. (b) Reduced HAUSP expression in ENstemA NPCs with two specific shrna (B2 and B5) significantly decreased neurosphere number. NPCs were targeted with shhausp or the control non-targeting (NT) shrna through lentiviral infection for 48 hours and subjected to neurosphere formation assay. The number of neurospheres were counted and compared between cells targeted with shhausp and control non-targeting (NT) shrna. Data are means ± s.d. (n = 4). (c, d) Ectopic expression of REST rescued the reduced neurosphere formation capacity caused by shhausp NPCs were transfected with Flag-REST or vector control through lentiviral infection, infected with lentiviruses expressing shhausp (B5 clone) or control non-targeting (NT) shrna for 96 hours, and allowed to form neurospheres for 9 days. REST overexpression restored neurosphere size reduced by HAUSP knockdown (c) and significantly rescued the reduced neurosphere number caused by HAUSP knockdown (d). Data are means ± s.d. (n = 3). 3
4 Figure S4 HAUSP interacts with REST and stabilizes REST protein through deubiquitination. (a) Interaction of overexpressed HA-REST and Flag- HAUSP in 293T cells. Cell lysates of 293T cells expressing HA-REST and Flag-HAUSP were co-immunoprecipitated with anti-rest specific antibody and then immunoblotted with anti-flag (Flag-HAUSP) and anti- REST antibodies. (b) Reciprocal co-immunoprecipitation with anti-ha (HA-HAUSP). The lysate of 293T cells expressing HA-HAUSP and Flag- REST was immunoprecipitated with anti-ha (HA-HAUSP) antibody and then immunoblotted with anti-flag (Flag-REST) and anti-hausp specific antibodies. Anti-HA (HAUSP) pulled down Flag-REST, indicating interaction of the expressed HAUSP and REST. (c) HAUSP knockdown increased REST ubiquitination in NPCs NPCs were infected with lentiviruses expressing HAUSP-targeting shrna or non-targeting (NT) control shrna for 48 hours, treated with the proteasome inhibitor MG132 for 6 hours, and immunoprecipitated (IP) with an anti-rest specific antibody or the control IgG, and immunobloted with anti-ubiquitin specific antibody. HAUSP knockdown increased REST ubiquitination in NPCs. (d) Ectopic expression of HAUSP suppressed REST ubiquitination NPCs were transfected with Flag-HAUSP or vector control (V) through lentiviral infection for 36 hours and then subjected for analysis of REST ubiquitination. Forced expression of Flag-HAUSP reduced REST ubiquitination and increased REST protein levels. 4
5 Figure S5 Potential conserved binding sequences (P/AXXS) on human, monkey and mouse REST proteins for the HAUSP-mediated deubiquitination are shown (red letters). The real consensus binding site on human REST (310-PYSS-313) required for the HAUSP-mediated REST deubiquitination was identified and indicated by an arrow. 5
6 Figure S6 HAUSP and β-trcp regulate REST ubiquitination. (a) Increased REST ubiquitination results in decreased REST protein levels during neuronal differentiation. ENStemA NPCs were induced by all-trans retinoic acid (RA) for two days. NPCs and differentiated cells were treated with MG132 for 6 hours and harvested for ubiquitination assessment. Cell lysates were immunoprecipitated with an anti-rest antibody (Rabbit) and immunoblotted with anti-ubiquitin and the anti-rest antibodies (mab). (b) HAUSP deubiquitinase counteracts the β-trcp ubiquitin E3 ligase to regulate REST ubiquitination. 293T cells were transfected to express Myc-REST and HA-ubiquitin in combination with HAUSP, Flag-β-TrCP or HAUSP + Flag-β-TrCP as indicated for 48 hours, treated with MG132 for 6 hours, and harvested for ubiquitination assessment. The cell lysates were immunoprecipitated with an anti-ha (HA-Ubiquitin) antibody and immunoblotted with an anti-rest specific antibody. Expression of HAUSP reduced REST polyubiquitination (lanes 1, 2 in top panel). Overexpression of β-trcp increased REST polyubiquitination (lanes 3, 2) but overexpression of HAUSP with β-trcp together abolished the increased REST ubiquitination induced by β-trcp overexpression (lanes 4, 3), indicating that both HAUSPmediated deubiquitination and the β-trcp-mediated ubiquitination play opposite roles to regulate REST protein at post-translational level. 6
7 Figure S7 Lentiviral infection efficiency in neural stem/progenitor cells (NPCs) was determined with GFP-expressing lentiviruses. The infection efficiency in NPCs was examined with the lentiviruses expressing GFP. The majority (>93%) of NPCs were infected by the viruses and expressed GFP, when MOI of 3 was used for the infection. The uninfected cell without GFP expression is indicated by an arrow. 7
8 Figure S8 Uncropped images of immunoblots shown in the main figures (1a, 2a, 2b, 3e, 5a-f, 6c, 6d and 7b-7d). 8
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Supplemental Table S1
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Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna 40 20 0 % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1
SUPPLEMENTARY INFORMATION. Supplementary Figures S1-S9. Supplementary Methods
SUPPLEMENTARY INFORMATION SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane Jian Huang 1,2#, Jie Yan 1,2#, Jian Zhang 3#, Shiguo Zhu 1, Yanli Wang
SUPPLEMENTARY FIGURE LEGENDS
SUPPLEMENTARY FIGURE LEGENDS Supplemental FIG. 1. Localization of myosin Vb in cultured neurons varies with maturation stage. A and B, localization of myosin Vb in cultured hippocampal neurons. A, in DIV
Supplementary Material
Supplementary Material accompanying the manuscript Interleukin 37 is a fundamental inhibitor of innate immunity Marcel F Nold, Claudia A Nold-Petry, Jarod A Zepp, Brent E Palmer, Philip Bufler & Charles
Reviewers' comments: Reviewer #1 (Remarks to the Author):
Reviewers' comments: Reviewer #1 (Remarks to the Author): In this manuscript, Song et al. identified FBXW7 as a new positive regulator for RIG-Itriggered type I IFN signaling pathway. The authors observed
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
SUPPLEMENTAL EXPERIMENTAL PROCEDURES Crystal violet assay Cells were seeded in 24-well plates and cultured in media supplemented with % FBS for 7 days. Media were then removed, plates were briefly washed
Figure S1. HP1α localizes to centromeres in mitosis and interacts with INCENP. (A&B) HeLa
SUPPLEMENTARY FIGURES Figure S1. HP1α localizes to centromeres in mitosis and interacts with INCENP. (A&B) HeLa tet-on cells that stably express HP1α-CFP, HP1β-CFP, or HP1γ-CFP were monitored with livecell
FIG S1 Examination of eif4b expression after virus infection. (A) A549 cells
Supplementary Figure Legends FIG S1 Examination of expression after virus infection. () 549 cells were infected with herpes simplex virus (HSV) (MOI = 1), and harvested at the indicated times, followed
Supplementary Figure 1. Characterization of NMuMG-ErbB2 and NIC breast cancer cells expressing shrnas targeting LPP. NMuMG-ErbB2 cells (a) and NIC
Supplementary Figure 1. Characterization of NMuMG-ErbB2 and NIC breast cancer cells expressing shrnas targeting LPP. NMuMG-ErbB2 cells (a) and NIC cells (b) were engineered to stably express either a LucA-shRNA
SUPPLEMENTARY INFORMATION
DOI: 1.138/ncb3355 a S1A8 + cells/ total.1.8.6.4.2 b S1A8/?-Actin c % T-cell proliferation 3 25 2 15 1 5 T cells Supplementary Figure 1 Inter-tumoral heterogeneity of MDSC accumulation in mammary tumor
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein content relative to GAPDH in two independent experiments.
Supplementary Figure 1: STAT3 suppresses Kras-induced lung tumorigenesis
Supplementary Figure 1: STAT3 suppresses Kras-induced lung tumorigenesis (a) Immunohistochemical (IHC) analysis of tyrosine 705 phosphorylation status of STAT3 (P- STAT3) in tumors and stroma (all-time
Supplementary Figure 1. Characterization of ALDH-positive cell population in MCF-7 cells. (a) Expression level of stem cell markers in MCF-7 cells or
Supplementary Figure 1. Characterization of ALDH-positive cell population in MCF-7 cells. (a) Expression level of stem cell markers in MCF-7 cells or ALDH-positive cell population by qpcr. Data represent
T H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Chairoungdua et al., http://www.jcb.org/cgi/content/full/jcb.201002049/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. Expression of CD9 and CD82 inhibits Wnt/ -catenin
Supplementary Figure 1. Validation of astrocytes. Primary astrocytes were
Supplementary Figure 1. Validation of astrocytes. Primary astrocytes were separated from the glial cultures using a mild trypsinization protocol. Anti-glial fibrillary acidic protein (GFAP) immunofluorescent