Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: The association of p62 with cytosolic Salmonella increases shortly after initiation of intracellular replication. (A) GFP-p62- or GFP-expressing HeLa cells were infected with wild-type (WT) or ∆ sifA Salmonella . Cells at 1 hpi and 5 hpi were stained with LAMP-1 (red) and DAPI (cyan). “Zoom” represents a magnified picture of the boxed area. Scale bar, 10 µm. (B to D) Quantification of the percentage of p62 + WT or p62 + ∆ sifA bacteria (B), the percentage of p62 + WT or p62 + ∆ sifA bacteria that are not associated with LAMP-1 (C), and the percentage of LAMP-1 − WT or LAMP-1 − ∆ sifA bacteria (D) in GFP-p62-expressing cells at 1, 5, and 8 hpi (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Staining, Bacteria

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: Dynamic association of p62 and/or LC3 correlates with Salmonella replication in the cytosol of HeLa cells. (A) GFP-LC3-expressing cells were infected with mKO-expressing Salmonella . After infection for 4.5 h (0 min), cells were imaged at 30-s intervals with a spinning-disc confocal microscope. A series of live-cell imaging data is shown, and the elapsed time (minutes) is shown in the corner of each picture. Arrowheads 1, 2, 3, and 4 indicate replicating bacteria, whereas arrowheads 5 and 6 indicate nonreplicating bacteria. Scale bar, 10 µm. (B) GFP-p62-expressing cells were infected with mKO-expressing Salmonella . After infection for 4.5 h (0 h), the cells were imaged at 20-s intervals with a spinning-disc confocal microscope. Boxed areas indicate where p62 associates with cytosolic Salmonella . Arrows 1 to 5 indicate bright bacteria that are not replicating; arrowheads 6 and 7 in column 1 (0 h) indicate light-red bacteria that start to replicate. Scale bar, 5 µm.

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Microscopy, Live Cell Imaging, Bacteria

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: p62 and autophagy favors cytosolic replication of Salmonella . (A) Western blot analyses of HeLa cells treated with control siRNA (si-NT) or siRNA targeting p62 (si-p62), LC3 (si-LC3), Atg5 (si-Atg5), and p62 and LC3 (si-p62+LC3). These cells were infected with WT Salmonella . Calnexin was used as a loading control. (B) Microscopic analyses of infected cells containing 1 to 5, 6 to 10, 11 to 20, or greater than 20 bacteria per cell at 2 hpi (top) and 6 hpi (bottom). The number of bacteria in at least 50 infected cells was enumerated, and the percentage of cells containing the indicated number of bacteria was presented (mean ± SD, n = 3). (C) Replication of Salmonella was compared in HeLa cells used in panel A. The fold replication was calculated as fold increases of the number of bacteria at late time points compared to the number of bacteria at 2 hpi (mean ± SD, n = 3). (D) HeLa cells were treated with control siRNA (si-NT) or siRNA targeting TBK1 (TANK-binding kinase-1) (si-TBK1), TBK1 and LC3 (si-TBK1+LC3), and TBK1 and ATG5 (si-TBK1+ATG5) and infected with Salmonella for 2 h (top) or 6 h (bottom). The percentage of cells containing the indicated number of bacteria was quantified as in panel B (mean ± SD; n = 3). (E) GFP-LC3-expressing HeLa cells were treated with si-NT, si-TBK1, si-TBK1+LC3, or si-TBK1+Atg5. The percentage of LC3 + bacteria or LAMP-1 − bacteria at 5 hpi was quantified (mean ± SD, n = 3). (F) Western blot analyses of HeLa cells used in panel D.

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Western Blot, Control, Infection, Bacteria, Binding Assay, Expressing

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: The effector SopB is required for Salmonella to associate with autophagosomes. (A) GFP-LC3-expressing cells were infected with the indicated strains. The ∆ sopB /pACDE mutant expresses the wild-type SopB in a ∆ sopB background. Unlike other strains that are individually used to infect cells, ∆ invA and ∆ sipB bacteria were used in a coinfection model. The percentage of LC3 + bacteria at 5 hpi was quantified for each strain (mean ± SD, n = 3). (B) Western blot analysis of HeLa cells used in panel A. (C) HeLa cells treated with si-NT or si-LC3 were infected with the indicated strains. The fold replication of these strains at 6 hpi was determined (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Expressing, Infection, Mutagenesis, Bacteria, Western Blot

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: Cells containing hyperreplicating bacteria and showing decreased autophagy are positive for caspase-1 and caspase-3/7 activation and undergo cell death. (A) GFP-p62-expressing HeLa cells were infected with Salmonella for 8 h and stained with DAPI (blue). A cell containing hyperreplicating bacteria (more than 50 bacteria per cell, i.e., HRBC cells) and showing abnormal nuclear morphology is shown. Scale bar, 10 µm. (B) At least 100 HRBC cells were divided into two groups. The first group exhibited a low level of p62 signals colocalized with Salmonella (<1% of intracellular bacteria), and the second group exhibited a relatively high level of p62 signals (>2% of intracellular bacteria). The number of cells showing abnormal nuclear morphology (such as chromatin condensation and DNA fragmentation) was further quantified and expressed as the percentage of cells with abnormal nuclei in each group of cells (mean ± SD, n = 3). (C) A GFP-p62-expressing HRBC cell shows a relatively enhanced autophagy and normal nuclear morphology. Scale bar, 10 µm. (D) Cells stably expressing monomeric red fluorescent protein (mRFP)-LC3 were infected with WT Salmonella for 7 h and stained with DAPI (blue) and active caspase-1 probe FAM-YVAD-FMK (green) or caspase-3/7 probe FAM-DEVD-FMK (similar to caspase-1 staining [data not shown]). (E) Quantification of the percentage of casapase-1 (left)- or caspase-3/7 (right)-positive cells in uninfected cells, HRBC cells showing decreased autophagy, and HRBC cells showing normal autophagy at 8 hpi (mean ± SD, n = 3).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Bacteria, Activation Assay, Expressing, Infection, Staining, Stable Transfection

Journal: mBio

Article Title: Autophagy Facilitates Salmonella Replication in HeLa Cells

doi: 10.1128/mBio.00865-14

Figure Lengend Snippet: A proposed model for p62-dependent autophagy favoring Salmonella replication in the cytosol of HeLa cells. In this proposed model, Salmonella likely acquires nutrients supplied by autophagy for its replication. At least three populations of bacteria are present in infected HeLa cells. The first population of bacteria remains in intact Salmonella -containing vacuoles (SCVs). These bacteria replicate slowly, as they do not associate with autophagosomes and are difficult to acquire nutrients from the cytosol. The second population of bacteria is partially exposed to the cytosol through the gaps between damaged SCVs . These bacteria are ineffectively coated with ubiquitin and associated with autophagosomes to a low level. They acquire a limited amount of nutrients for replication and replicate faster than the first population of bacteria. The third population of bacteria is completely exposed to the cytosol. These bacteria are effectively coated with ubiquitin and associated with autophagosomes to a high level. They acquire sufficient nutrients from the cytosol and replicate the fastest. Notably, some cells containing the third population of bacteria will undergo cell death and detach from the epithelial layer, resulting in the release of hyperreplicating bacteria that are capable of initiating secondary infections in neighboring cells. The whereabouts of the first and second populations of bacteria at 8 hpi were not determined (ND).

Article Snippet: HeLa human epithelial cells (ATCC CCL-2) and 293T/17 human kidney epithelial cells (ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), and 1% GlutaMax (Gibco).

Techniques: Bacteria, Infection, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 2. Visual Characterization of GP-Vi- rosomes (A) PMA-differentiated THP-I cells were trans- duced with parental lentiviral vectors (mock) or lentiviral vectors expressing either GP fused to GFP or GP only. Cells and supernatants were harvested 72 h later and analyzed using western blot for the presence of GP, CD81, and actin. Shown is one representative of three experiments. (B) Three-dimensional (3D) SIM live-cell imaging of GP-GFP transduced THP-I (left) or HeLa cells (right) at 3 days post-infection (dpi). Shown is the start of the imaging series at a membrane-prox- imal region. Recorded stacks are presented at maximum intensity projections (MIPs). Scale bar, 2 mm. (C) Time lapse of the magnified areas indicated in (B). 3D SIM live-cell imaging in 20 s intervals. Re- corded stacks are presented at MIP. Top: THP-I; bottom: HeLa cells. Scale bar, 1 mm. See also Videos S1, S2, S3, and S4. (D and E) Heterogeneity and relative vesicle size distribution of GP-virosomes in (D) THP-I (n = 86) and (E) HeLa cells (n = 112). For examples of size measurements, see Figure S1 (THP-I) and Figure S2 (HeLa). (F–R) Transmission electron microscopy of GP- expressing HeLa cells (F–H) and (I–L) vesicles from mock cells or (M–P) GP-expressing cells as well as negative staining and immuno-gold electron mi- croscopy (EM) against GP for vesicles isolated from (Q) mock or (R) GP-expressing cells. Scale bars, 500 nm (F–H) and 100 nm (I–R). See also Figure S3 for representative images of non-GP-expressing control HeLa cells and Fig- ure S4 for immuno-gold EM of vesicles isolated from mock or GP-expressing cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Expressing, Western Blot, Live Cell Imaging, Infection, Imaging, Membrane, Transmission Assay, Electron Microscopy, Negative Staining, Isolation, Control

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 3. Tetherin Suppresses the Release of EBOV-GP (A) 293T cells were transfected to express EBOV-GP or an empty control plasmid (mock) and increasing amounts of tetherin. 24 hours post-trans- fection, GP levels in the media and in the cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (B) HeLa cells were transfected to express EBOV-GP and tetherin or an empty control plasmid. 24 hours post-transfection, cell culture media as well as cell lysates were analyzed as described in (A). (C) HeLa cells were transfected with a tetherin-specific or a non-targeting siRNA as control. 24 hours later, the cells were additionally transfected to express GP or a non-coding control plasmid. After another 24 h, GP and tetherin steady-state levels in cell culture media and in cell lysates were analyzed using western blot. The results were verified in at least three addi- tional experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Cell Culture

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 5. The EBOV-GP RBD and TMD Are Determinants of GP Release (A) 293T cells were transfected to express EBOV-GP or the indicated GP mutants together with tetherin or a non-coding control plasmid. 24 hours post- transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in cell lysates were analyzed using western blot. (B) HeLa cells were transfected to express EBOV-GP or the indicated GP mutants together with a tetherin-specific siRNA or a non-targeting siRNA as negative control. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in cell lysates were analyzed using western blot. (C) HeLa cells were transfected to express EBOV-GP or the ELE variant together with GFP from a bicistronic plasmid or the respective control plasmid only expressing GFP (mock). 24 hours post-transfection, a proximity ligation assay was performed with antibodies against EBOV-GP1 and tetherin, respectively.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot, Negative Control, Variant Assay, Expressing, Proximity Ligation Assay

Journal: Cell reports

Article Title: Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

doi: 10.1016/j.celrep.2019.01.065

Figure Lengend Snippet: Figure 6. Ebolavirus GP Release Is Associ- ated with Increased Viral Pathogenicity (A) 293T cells were transfected to express EBOV- GP, SUDV-GP, TAFV-GP, or RESTV-GP together with tetherin or a non-coding control plasmid. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (B) HeLa cells were transfected to express EBOV- GP, SUDV-GP, TAFV-GP, or RESTV-GP, and GP levels in cell culture media and cell lysates were quantified using western blot. (C) 293T cells were transfected to express EBOV- GP or the GP-A82V variant derived from the 2014 Ebola outbreak in West Africa together with teth- erin or a non-coding control plasmid. 24 hours post-transfection, GP levels in cell culture media and cell lysates as well as tetherin levels in the cell lysates were determined using western blot. (D) HeLa cells were transfected to express EBOV- GP or the GP-A82V variant and GP levels in cell culture media and cell lysates were quantified us- ing western blot. All results were verified in at least three independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit-anti-GP1 Wrensch et al., 2015 PMID: 26034199 Rabbit-anti-GP2 Icosagen Cat#A2-100-100 KZ52 EBOV neutralizing antibody Parren et al., 2002; provided by S. Becker PMID: 12021376 Mouse-anti-Tetherin Abnova Cat#H00000684-B02P; RRID:AB_1204015 Rabbit-anti-Tetherin NIH AIDS Reagent Progam Cat#11721 Mouse-anti-Flag (F-tag-01) Abcam Cat#ab18230; RRID:AB_444336 Rabbit-anti-V5 Abcam Cat#ab9116; RRID:AB_307024 Rabbit-anti-alpha-tubulin Thermo Cat#PA5-22060; RRID:AB_11154084 Rabbit-anti-HA Thermo Cat#71-5500; RRID:AB_2533988 Mouse-anti-Actin Sigma Cat#A3853; RRID:AB_262137 Mouse-anti-CD81 (clone B11) SantaCruz Cat#sc-166029; RRID:AB_2275892 Mouse-anti-TSG101 (clone C2) SantaCruz Cat#sc-7964; RRID:AB_671392 Mouse-anti-Calnexin (clone AF18) SantaCruz Cat#sc-23954; RRID:AB_626783 HRP-conjugated goat anti-mouse Dianova Cat#A21010 RRID:AB_2728771 HRP-conjugated goat anti-rabbit Dianova Cat#A21020 Goat-anti-Rabbit 800CW Li-Cor Cat#926-32211; RRID:AB_621843 Goat-anti-Mouse 680RD Li-Cor Cat#926-68070; RRID:AB_10956588 Rabbit polyclonal V5 (CoIP) Diagenode #C15410270 Bacterial and Virus Strains NEB stable competent E.coli NEB Cat#C3040H Chemicals, Peptides, and Recombinant Proteins Puromycin Thermo Cat#A11138-03 Lipofectamin2000 Thermo Cat#11668019 Saponin Applichem Cat#A4518,0100 Protease inhibitor cocktail (Complete Mini) Roche Cat#04693124001 Critical Commercial Assays Proteome Profiler Human Cytokine Array Kit Panel A R&D Systems Cat#ARY005B Duolink In Situ Detection Reagents Red Sigma-Aldrich Cat#DUO92008 Duolink In Situ PLA Probe Anti-Rabbit PLUS Sigma-Aldrich Cat#DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS Sigma-Aldrich Cat#DUO92004 Cell Culture Lysis Reagent Promega Cat#E1531 Beetle-Juice BIG KIT PJK Cat#102511 Renilla luciferase: coelenterazine Roth Cat#4094.4 Experimental Models: Cell Lines 293T cells (embryonal kidney) DSMZ Cat#ACC635 HeLa cells (cervix carcinoma) DSMZ Cat#ACC57 THP-1-shSamHD1 Gramberg et al., 2013 PMID: 23497255 Oligonucleotides Fwd_EBOV_GP_XbaI: 50-CGTCTAGAATATGGGCGTTAGAGGAAT ATTGC-30 This paper N/A Rev_EBOV_GP_MluI: 50-TACGCGTTTCTAAAAGACAAATTTGCAT ATACAG-30 This paper N/A (Continued on next page) e1 Cell Reports 26, 1841–1853.e1–e6, February 12, 2019

Techniques: Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot, Variant Assay, Derivative Assay