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1 ARTICLE NUMBER: 0 DOI: 0.0/NMICROBIOL.0. The host protein CLUH participates in the subnuclear transport of influenza virus ribonucleoprotein complexes Tomomi Ando,, Seiya Yamayoshi, Yuriko Tomita,, Shinji Watanabe,,a, Tokiko Watanabe,, Yoshihiro Kawaoka,, Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 0-9, Japan Exploratory Research for Advanced Technology Infection-Induced Host Responses Project, Japan Science and Technology Agency, Kawaguchi, Saitama -00, Japan Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA a Current address: Gakuen --, Musashimurayama-shi, Tokyo 0-00, Japan Correspondence should be addressed to S.Y. (yamayo@ims.u-tokyo.ac.jp) and Y.K. (kawaoka@ims.u-tokyo.ac.jp) This PDF file includes Supplementary Figures to NATURE MICROBIOLOGY
2 Supplementary Figures DOI: 0.0/NMICROBIOL.0. a input NFLAG- PB PB PA IP : Normal IgG PB PB PA IP : α-flag PB PB PA b WSN infection ( hpi) input IP IgG α-pb α-cluh α-cluh α-flag α-pb c CLUH/βactin mrna levels (% of sicontrol) sicluh. sicluh. d sicluh. sicluh. α-cluh α-βactin e Cell viability mrna (% (% of of sicontrol) Control) sicluh. sicluh. Virus Titer (log 0 PFU/ml) f MDCK cells sicluh. 0 9 ** ** ** ** P < 0.0 Times (hpi) g sicluh. sicluh. WT sirna resistant c-myc-cluh α-cluh α-c-myc α-βactin h Virus Titer Virus (log titer 0 (log PFU/ml) 0 0 P < 0. ** sicluh. sicluh. WT sirna resistant c-myc-cluh i Virus Titer (log 0 PFU/ml) ** sicluh. siviral NP Virus Titer (log 0 PFU/ml) ** sicluh. siviral NP Virus Titer (log 0 PFU/ml) ** P < 0.0 ** sicluh. siviral NP P HN pdm HN Influenza B Virus Supplementary Fig.. Identification of CLUH as a host protein that contributes to influenza virus replication. (a) Co-immunoprecipitation of endogenous CLUH with NFLAG-PB. NATURE MICROBIOLOGY
3 DOI: 0.0/NMICROBIOL NFLAG-tagged PB (NFLAG-PB), PB (NFLAG-PB), and PA (NFLAG-PA)-expressing plasmids were transfected individually into HEK9 cells and immunoprecipitated by using an anti-flag antibody. (b) Interaction of PB with CLUH in infected cells as assessed by use of co-immunoprecipitation. HEK9 cells were infected with WSN at an MOI of 0, and immunoprecipitated by using an anti-pb antibody. (c, d) Down-regulation of mrna and protein expression of CLUH by treatment with an sirna against CLUH. Cells were transfected with non-targeting control sirna (sicontrol) or sirnas against CLUH (sicluh. or sicluh.). The mrnas encoding CLUH and β-actin were measured by RT-qPCR (c), and CLUH was detected by use of western blotting (d). (e) Cell viability of CLUH-depleted cells. Cells were transfected with sicontrol, sicluh., or sicluh., and then subjected to a cell viability assay. (f) Virus growth in CLUH-depleted MDCK cells, infected with WSN at an MOI of **P < 0.0 (two-way ANOVA with repeated measures followed by Tukey s tests). (g) Protein expression in CLUH-expressing cells. c-myc-cluh(wt)- or c-myc-cluh(sirna-resistant)-stably-expressing cells were transfected with sicontrol or sicluh.. Protein expression was analyzed by use of western blotting. (h) Virus growth in cells expressing sirna-resistant CLUH and treated with sirna against CLUH. c-myc-cluh(wt) or c-myc-cluh(sirna-resistant)-stably-expressing cells were transfected with sicontrol or sicluh., and infected with WSN at an MOI of Virus titers were determined at hpi. **P < 0.0 (one-way ANOVA followed by Tukey s tests). (i) Growth of clinical isolates in CLUH-depleted cells. Cells were transfected with sicontrol, sicluh., or siviral NP, and infected with A/California/0/009 (HNpdm, left), A/Yokohama/UT-KA/0 (HN, middle), and B/Yokohama/UT-KA/0 (Influenza B Virus, right) at an MOI of 0.0. Virus titers were determined at hpi. Since the control siviral NP was designed for influenza A virus, the virus titer in the siviral NP-treated cells infected with NATURE MICROBIOLOGY
4 DOI: 0.0/NMICROBIOL.0. B/Yokohama/UT-KA/0 was comparable to that in the sicontrol-transfected cells. **P < 0.0 (one-way ANOVA followed by Tukey s tests). (c, e, f, h, i) The values represent the means of triplicate experiments ± SD. (a i) The results shown are representative of three independent experiments. NATURE MICROBIOLOGY
5 i i i DOI: 0.0/NMICROBIOL.0. a α-cluh α-crm α-pb α-np α-ha α-m α-ns α-βactin sicluh hpi CRM (% of sicontrol) hpi PB (% of sicontrol) hpi NP (% of sicontrol) hpi HA (% of sicontrol) hpi M (% of sicontrol) hpi NS (% of sicontrol) hpi b vrna copies (x0 ) sicluh. 0 0 Time (hpi) Supplementary Fig.. The effect of CLUH depletion on the early steps of virus propagation. (a) The expression of viral proteins in CLUH-depleted cells. Cells were transfected with sicontrol or sicluh., and were infected with WSN at an MOI of 0. The cells were then harvested at the indicating times after infection. Proteins were detected by western blotting and then quantitated (normalized to the expression of β-actin). The results shown are representative of three independent experiments. Quantification of the expression level of each protein is indicated at the bottom of the graphs. Error bars represent means ± SD from three independent NATURE MICROBIOLOGY
6 DOI: 0.0/NMICROBIOL.0. experiments. (b) The numbers of vrna copies in CLUH-depleted cells. Cells were transfected with sicontrol or sicluh., and infected with WSN at an MOI of 0. The number of vrna copies was determined by strand-specific real-time RT-qPCR (see Supplementary Methods). The results shown are representative of three independent experiments. The values represent the means of triplicate experiments and are expressed as the means ± SD. NATURE MICROBIOLOGY
7 DOI: 0.0/NMICROBIOL.0. vrnp WSN infection ( hpi) NS Hoechst sicluh. M CLUH Hoechst sicluh. sicluh. HA CLUH Hoechst Supplementary Fig.. The localization of NS, M, and HA in CLUH-depleted cells. Cells were transfected with sicontrol or sicluh., and infected with WSN at an MOI of 0. At hpi, the cells were fixed with % paraformaldehyde, permeabilized with 0.% Triton X-00, and subjected to immunofluorescence analysis. All images were obtained by using confocal microscopy. The results shown are representative of three independent experiments. NATURE MICROBIOLOGY
8 DOI: 0.0/NMICROBIOL.0. MitoTracker CLUH Hoechst 9 hpi Mock Supplementary Fig.. The localization of CLUH and mitochondria in uninfected or infected cells. Cells were infected with WSN at an MOI of 0 or left uninfected. At 9 hpi, the cells were stained by using Mitotracker, fixed with % paraformaldehyde, permeabilized with 0.% Triton X-00, and subjected to immunofluorescence analysis. Bar: 0 µm. All images were obtained by using confocal microscopy. The results shown are representative of three independent experiments. 9 0 NATURE MICROBIOLOGY
9 DOI: 0.0/NMICROBIOL.0. hole cells nuclear matrix CLUH Hoechst CLUH Hoechst LMB + LMB Supplementary Fig.. Intracellular localization of CLUH in uninfected cells. Cells were treated without (top) or with (bottom) Leptomycin B (LMB), an inhibitor of the CRM-dependent nuclear export pathway, for h, and were then fixed with % paraformaldehyde before (whole cells) or after treatment with DNase I and high-salt buffer (nuclear matrix). White lines in the Hoechst channel indicate the nuclei. Bar: 0 µm. All images were obtained by using confocal microscopy. The results shown are representative of three independent experiments. 9 0 NATURE MICROBIOLOGY 9
10 DOI: 0.0/NMICROBIOL.0. a whole cells PB transfection PB CLUH Hoechst PB transfection Cytoplasm Nucleoplasm/Cytoplasm Nucleoplasm n=0 PB / M co-transfection PB / M co-transfection n= CLUH localization in transfected cells (Cell population (%) ) b nuclear matrix M transfection M CLUH Hoechst M transfection fluorescense ( ) fluorescense (+) n=0 PB / M co-transfection PB / M co-transfection CLUH signal in nuclear matrix of transfected cells (Cell population (%) ) n= 9 0 Supplementary Fig.. Intracellular localization of CLUH in PB and/or M over-expressing cells. (a) Intracellular localization of CLUH in PB, or PB and M, over-expressing cells was analyzed by using an anti-pb antibody and an anti-cluh antibody. Percentages of cells in which CLUH was detected in the cytoplasm only, the nucleoplasm only, or both are shown for cells transfected with a PB-expression plasmid alone or with plasmids expressing PB and M. (b) CLUH localization in the nuclear matrix of M, or M and PB, over-expressing cells was analyzed by using an anti-m antibody and an anti-cluh antibody. The numbers of cells positive for CLUH were determined and are expressed as a percentage of the total number of cells. (a, b) The total number of cells counted is indicated to the right of the graph. All images were obtained by using confocal microscopy. Bar: 0 µm. (a, b) The results shown are representative of three independent experiments. 0 NATURE MICROBIOLOGY
11 DOI: 0.0/NMICROBIOL.0. FLAG whole cells CLUH Hoechst FLAG nuclear matrix CLUH Hoechst NFLAG-NS transfection NFLAG-NS transfection NFLAG-M transfection NFLAG-NA transfection NFLAG-HA transfection NFLAG-NP transfection NFLAG-PA transfection NFLAG-PB transfection Supplementary Fig.. The intracellular localization of CLUH in viral protein-overexpressing cells. Cells were transfected with the indicated plasmid for the NATURE MICROBIOLOGY
12 DOI: 0.0/NMICROBIOL.0. expression of N-terminally FLAG-tagged (NFLAG-) viral protein, and then fixed with % paraformaldehyde before (whole cells) or after treatment with DNase I and high-salt buffer (nuclear matrix), and subjected to immunofluorescence analysis by using an anti-flag antibody and an anti-cluh antibody. White lines in the Hoechst channel indicate nuclei. Bar: 0 µm. All images were obtained by using confocal microscopy. The results shown are representative of three independent experiments. NATURE MICROBIOLOGY
13 DOI: 0.0/NMICROBIOL.0. M WSN infection ( hpi) nuclear matrix PML Hoechst HPα CLUH Hoechst HKme CLUH Hoechst 9 Supplementary Fig.. The localization of CLUH and marker proteins of the nuclear compartments in virus-infected cells. Cells were infected with WSN at an MOI of 0. At hpi, the cells were fixed after treatment with DNase I and high-salt buffer (nuclear matrix), and subjected to immunofluorescence analysis by using an anti-cluh or an anti-m antibody and an anti-hpα antibody, an anti-trimethyl Histone H (Lys) (HKme) antibody, or an anti-pml antibody. White lines in the Hoechst channel indicate the nuclei. Bar: 0 µm. All images were obtained by using confocal microscopy. The results shown are representative of three independent experiments. 0 NATURE MICROBIOLOGY
14 DOI: 0.0/NMICROBIOL.0. PB WSN infection ( hpi) nuclear matrix CLUH Hoechst x 9 Supplementary Fig. 9. The co-localization of CLUH and PB in the nuclear matrix of infected cells. Cells were infected with WSN at an MOI of 0. At hpi, the cells were fixed after treatment with DNase I and high-salt buffer (nuclear matrix), and subjected to immunofluorescence analysis by using an anti-cluh and an anti-pb antibody. White lines in the Hoechst channel indicate the nuclei. Bar: 0 µm. The image was obtained by using confocal microscopy. This figure is excerpted from Figure c. The right image is an enlargement of the cell enclosed in the white box. Some of the co-localized PB and CLUH are indicated by the arrowheads. 0 NATURE MICROBIOLOGY
15 DOI: 0.0/NMICROBIOL.0. input M transfection IgG IP α-cluh α-cluh α-m Supplementary Fig. 0. Co-immunoprecipitation of endogenous CLUH with M. M-expressing plasmid was transfected into HEK9 cells and the interaction of M with CLUH was analyzed by co-immunoprecipitation using an anti-cluh antibody. The results shown are representative of three independent experiments. NATURE MICROBIOLOGY
16 DOI: 0.0/NMICROBIOL.0. Mean Intensity of CLUH at the nuclear periphery ** P < 0.0 LMB ( ) " LMB (+) 9 0 Supplementary Fig.. CLUH is enriched at the nuclear periphery upon LMB treatment. Localization of vrnp and CLUH at the nuclear matrix of cells infected with WSN at an MOI of 0 in the presence or absence of LMB was analyzed by using an anti-vrnp antibody and an anti-cluh antibody. Experiments were performed in parallel with those described in the legend to Fig. d. The mean fluorescence intensity at the nuclear periphery was analyzed from the signal in a specific confocal section. The values are expressed as means ± SD (n = 0 cells); **P < 0.0 (one-sided Mann-Whitney test). The results shown are representative of three independent experiments. NATURE MICROBIOLOGY
17 DOI: 0.0/NMICROBIOL.0. Cyt Nuc ch ch0 LMB * Cyt Nuc ch ch0 AS si AS si AS si AS si * AS : sicontrol si : sicluh. α-cluh α-mek/ α-hmgb α-tfiib α-histone H α-pb α-np α-m 9 0 Supplementary Fig.. Confirmation of subcellular fractionation using host marker proteins. Cells were infected with WSN at an MOI of, and treated with LMB from hpi (left), or were transfected with sicontrol or sicluh., and then infected with WSN at an MOI of (right). At hpi, cells were fractionated into cytoplasmic ( Cyt ), nucleoplasmic ( Nuc ), 0 mm NaCl-extractable chromatin ( ch0 ), and 00 mm NaCl-extractable chromatin ( ch00 ) fractions. Fractionation was confirmed by western blotting using the following antibodies against host marker proteins: MEK/ (cytoplasm), HMGB (cytoplasm and nucleoplasm), TFIIB (0 mm NaCl-extractable chromatin), and Histone H (00 mm NaCl-extractable chromatin). The amounts of the viral proteins, including components of the vrnp nuclear export complex such as PB, NP, and M, were not affected by CLUH depletion or LMB treatment, because excess viral proteins are present in infected cells and not all of them form vrnps. *Non-specific bands. Experiments were performed in parallel with those described in the legend to Fig. e,f. The results shown are representative of three independent experiments. NATURE MICROBIOLOGY
18 DOI: 0.0/NMICROBIOL.0. Supplementary Fig.. Full blot images of all western blots. NATURE MICROBIOLOGY
19 DOI: 0.0/NMICROBIOL.0. NATURE MICROBIOLOGY 9
20 DOI: 0.0/NMICROBIOL.0. 0 NATURE MICROBIOLOGY
21 DOI: 0.0/NMICROBIOL.0. NATURE MICROBIOLOGY
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Supplementary Figure 1 sirna and shrna mediated depletion of ATP7A results in loss of melanosomal ATP7A staining. a-h, sirna mediated ATP7A depletion. Immunofluorescence microscopy (IFM) analysis of ATP7A
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES Luciferase Assays Cells were seeded on 24well plates and grown to 7% confluency. Cells were then transfected with ng of reporter constructs and 1 ng of the renilla
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Supplementary Figure 1 EPRS expression in multiple cell lines upon viral infection. (a,b) EPRS is slightly induced upon viral induction. qpcr of Eprs mrna (a) and immunoblot analysis of corresponding endogenous
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Title of file for HTML: Supplementary Information Description: Supplementary Figures and Supplementary Tables Title of file for HTML: Supplementary Movie 1 Description: Nuclear morphology and dynamics
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Supplementary Figure Legends Figure S1 gene targeting strategy for disruption of chicken gene, related to Figure 1 (f)-(i). (a) The locus and the targeting constructs showing HpaI restriction sites. The
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doi:10.1038/nature09732 Supplementary Figure 1: Depletion of Fbw7 results in elevated Mcl-1 abundance. a, Total thymocytes from 8-wk-old Lck-Cre/Fbw7 +/fl (Control) or Lck-Cre/Fbw7 fl/fl (Fbw7 KO) mice
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Supplementary Figure 1 Schematic and results of screening the combinatorial antibody library for Sox2 replacement activity. A single batch of MEFs were plated and transduced with doxycycline inducible
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DOI: 10.1038/ncb2386 Figure 1 Src-containing puncta are not focal adhesions, podosomes or endosomes. (a) FAK-/- were stained with anti-py416 Src (green) and either (in red) the focal adhesion protein paxillin,
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DOI:.38/ncb327 a b Sequence coverage (%) 4 3 2 IP: -GFP isoform IP: GFP IP: -GFP IP: GFP Sequence coverage (%) 4 3 2 IP: -GFP IP: GFP 33 52 58 isoform 2 33 49 47 IP: Control IP: Peptide Sequence Start
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