Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Anakinra restores cellular proteostasis by coupling mitochondrial redox balance to autophagy

doi: 10.1172/JCI144983

Figure Lengend Snippet: ( A ) H1L1 cells were treated with different doses of kynurenine and anakinra for 6 hours and assessed for luciferase assay. ( B ) Representative immunoblots of IL1RA, HSP90, AIP, and AhR and ( C ) ARNT and AhR in cell lysates in which AhR was immunoprecipitated from Il1r1 –/– MEF cells treated with 10 μg/mL anakinra or 10 μM FICZ. In B and C , data are representative of 1 out of 2 independent experiments and the relative densitometric analysis are reported. ( D and E ) Il1r1 –/– MEF cells were treated with 10 μg/mL anakinra for 2 and 8 hours and analyzed for gene expression by a custom QuantiGene plex gene expression assay. Fold changes are reported as heatmap for 2 and 8 hours ( D ) and histograms for 8 hours ( E ) (data are representative of 1 out of 2 independent experiments). ( F ) Il1r1 –/– MEF cells were treated with 10 μg/mL anakinra and analyzed for mRNA expression of selected genes by RT-PCR ( n = 3 independent samples) and protein expression of IDO1 and SOCS3 by immunoblotting (representative experiment). The relative densitometric analysis is reported. ( G ) Il1r1 –/– MEF cells were treated with either 10 μg/mL anakinra for different times or 10 ng/mL IFN-γ as positive control for 48 hours and assessed for kynurenine production by ELISA. ( H ) Il1r1 –/– MEF cells were treated with 10 μg/mL anakinra in the presence or absence of 10 μM epacadostat and assessed for kynurenine production by ELISA and Cyp1a1 gene expression by RT-PCR ( n = 3 independent samples). * P < 0.05, ** P < 0.01, *** P < 0.001, treated versus untreated (None or 0 h) cells. One-way ANOVA, Bonferroni post hoc test.

Article Snippet: The proteins were separated by electrophoresis on SDS-PAGE and detected using specific antibodies against LC3b I and II (Cell Signaling Technology, 2775S), p62 (Cell Signaling Technology, 5414S), IDO1 (cv152, as described in ref. ), AhR (Invitrogen, MA1-514), IL-1Ra (Invitrogen, PA5-21776), ARNT (Cell Signaling Technology, 5537), HSP90 (Proteintech, 13171-1-AP), AIP (Proteintech, 18176-1-AP), and SOCS3 (Cell Signaling Technology, 2923S).

Techniques: Luciferase, Western Blot, Immunoprecipitation, Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Oncology

Article Title: Germacrone Regulates HBXIP-Mediated Cell Cycle, Apoptosis and Promotes the Formation of Autophagosomes to Inhibit the Proliferation of Gastric Cancer Cells

doi: 10.3389/fonc.2020.537322

Figure Lengend Snippet: Label-free proteomic and bioinformatic analysis of proteins associated with the cell cycle, apoptosis, and autophagy. (A–C) The DAVID database was used for analysis of molecular function, cellular component, and biological process. (D) A heat map was constructed based on the abundance of 111 proteins. D1, D2, and D3 represent the DMSO 1, DMSO 2, and DMSO 3 groups; G1, G2, and G3 represent the germacrone 1, germacrone 2, and germacrone 3 groups. (E) A protein interaction network was constructed in Cytoscape based on the information provided by the STRING database. Red indicates increased expression of the protein in the germacrone group; green indicates reduced expression of the protein in the germacrone group. (F, G) . The expression of HBXIP, HSP70, and ANXA1 was detected to verify the accuracy of the proteomics results.

Article Snippet: The membranes were incubated overnight at 4°C with antibodies against GAPDH (Cat. No. ab128915, Abcam, Massachusetts, USA), KI67 (Cat. No. ab197234, Abcam), cyclin-D1 (Cat. No. ab134175, Abcam), CDK4 (Cat. No. ab108357), CDK2 (Cat. No. ab32147, Abcam), cyclin-E1 (Cat No. ab33911, Abcam), cleaved caspase-3 (Cat. No. 11648-2-AP, Proteintech, Wuhan, China), p62 (Cat. No. ab109012, Abcam), LC3 I/II (Cat. No. D3U4C, Cell Signaling Technology, Boston, USA), ANXA 1 (Cat. No. BA3701, Boster Biological Technology, Wuhan, China), HSP70 (Cat. No. BA0928, Boster Biological Technology), and HBXIP (Cat. No. 14492-1-AP, Proteintech).

Techniques: Construct, Expressing

Journal: Cell reports. Medicine

Article Title: Hepatitis Delta Virus Acts as an Immunogenic Adjuvant in Hepatitis B Virus-Infected Hepatocytes.

doi: 10.1016/j.xcrm.2020.100060

Figure Lengend Snippet: Figure 1. Establishment of an In Vitro HBV/HDV Infection System in HepG2-hNTCP Cells and PHHs (A) Schematic of the experimental procedure. HepG2-hNTCP cells or PHHs were seeded and treated with 2% DMSO for 4 h. Cells were then inoculated with HBV at a MOI of 3,000 genome equivalents (GE) per cell for 24 h and subsequently with HDV at a MOI of 500 GE/cell for another 24 h. Infection status of the cells was analyzed 7 days post-infection. (B) HBV and HDV mRNA expression in infected target cells (HepG2-hNTCP and PHH) analyzed using customized NanoString probes. The relative positions of each NanoString probe targeting the HBV and HDV genome are annotated as probes 1 to 3. Bar graphs show the average normalized counts of probes 1 and 2 expressed on a log10 scale and probe 3 expressed on a linear scale (n = 2 for each cell type). (C) Expression of HDV RNA was quantified by the PrimeFlow RNA assay. A representative dot plot is shown (left), and bars on the right show the average fre- quency of HDV RNA+ cells in infected PHH (n = 6; p = 0.0073). (D) Quantification of HBsAg and HBcAg expression in infected HepG2-hNTCP cells (n = 5) and PHHs (n = 3) by flow cytometry. Bars indicate the average fre- quency of HBsAg+ and HBcAg+ cells in the respective infection, and each dot represents a single experiment. *p = 0.01–0.05 and **p = 0.001–0.01. Non-significant p values are indicated as N.S. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Pacific Orange live/dead stain Invitrogen L34959 Anti-human HBcAg Abcam #7841 Anti-human HBsAg Raybiotech #MD050186 Goat-anti-rabbit CF555 Sigma Aldrich SAB46000068 Goat-anti-mouse APC Life Technologies AB_2536211 Goat-anti-mouse PE Life Technologies AB_2539848 HBs183-191 TCR-like antibody Sastry et al., 2011 N/A HBc18-27 TCR-like antibody Sastry et al., 2011 N/A Anti-human CD107a FITC BD Biosciences 560949 Anti-human CD8 V500 BD Horizon 560774 Anti-human IFN-g PeCy7 eBiosciences AB_469682 Anti-human TNF-a PE BD Bioscience 559321 HBs183-191 PE pentamer ProImmune WB3290-PE HBs370 PE pentamer ProImmune WB4073-PE EBV PE pentamer ProImmune WB2144-PE Biological Samples Human pathological liver tissue Asian American Liver Centre, Gleneagles Hospital https://www.aamg.co/liver/ Human HBV/HDV patient blood Fonsazione IRCCS Ca’Granda Ospedale Maggiore Policlinico https://www.policlinico.mi.it/ Human healthy blood Health Sciences Authority https://www.hsa.gov.sg/ Chemicals, Peptides, and Recombinant Proteins Recombinant Human IL-2 R&D systems 202-1L-050 Anti-CD3 eBioscience AB_468854 Ficoll-Paque PLUS GE Healthcare 17-1440-02 BD Cytofix/Cytoperm Fixation BD Bioscience 51-2090 KZ Recombinant Human IFN-a2a PBL 11100-1 Recombinant Human IFN-b Peprotech 300-02BC Recombinant Human IL-29/IFN-l1 R&D systems 1598-IL-025 Critical Commercial Assays Primeflow assay with customized probeset Invitrogen EB 16488 Nanostring human immunology version 2 and customized probes assay Nanostring technologies XT-CSO-HIM2-12 FASER PE amplification kit Miltenyi 130-091-764 One Shot Top10 E. coli kit Invitrogen C404003 QIAamp minELute virus spin kit QIAGEN 57704w Rneasy Mini kit QIAGEN 74004 mMESSAGE mMACHINE T7 Ultra kit Ambion AM1345 Nucelofactor kit Lonza V4XP-3024 Human serum albumin ELISA quantification kit Bethyl Laboratories E88-129 RNAScope Fluorescent Multiplex kit Advanced Cell Diagnostics 320850 HBV Artus kit QIAGEN 4506265 Quantitative HBsAg kit Abbott Architect Platform 6C36 (Continued on next page) e1 Cell Reports Medicine 1, 100060, July 21, 2020

Techniques: In Vitro, Infection, Expressing, Cytometry

Journal: Cell reports. Medicine

Article Title: Hepatitis Delta Virus Acts as an Immunogenic Adjuvant in Hepatitis B Virus-Infected Hepatocytes.

doi: 10.1016/j.xcrm.2020.100060

Figure Lengend Snippet: Figure 6. HDV/HBV Infection Increases the Antiviral Efficacy of HBs183-Specific TCR-Redirected T Cells In Vivo (A) Schematic representation of the experimental design: human liver chimeric mice were stably infected with HBV and 10 weeks after with HDV. After 8 weeks of double infection, mice were treated with the indicated number of TCR T cells or left untreated. (B) Median HBV viremia changes relative to baseline levels in HBV/HDV-co-infected mice at days 4, 8, and 12 after the first injection of HBs183-specific TCR T cells (n = 3), EBV-specific TCR T cells (n = 3), or untreated animals (n = 6). (C) Bars represent the fold changes of HBV DNA viremia in the HBV-mono-infected or HBV/HDV-infected mice observed at day 12 (4 days after the last TCR-T cell infusion). (D) Changes in ALT levels in HBV/HDV-co-infected mice after treatment with indicated TCR T cells in relation to day 0 levels (BL, baseline) (n = 3 mice per group). (E) Fold changes of cccDNA, HBV pgRNA, total RNA, and HBsAg in the HBV/HDV-infected mice (n = 3 per group) observed 4 days after the last infusion of the indicated TCR-T cells. (F) Median HDV viremia changes relative to baseline levels in HBV/HDV-co-infected mice after 3 injections of the indicated TCR T cells (n = 6 for untreated, n = 3 for both EBV- and HBs183-specific T cells). (G) RNA in situ hybridization in liver tissues of humanized mice chronically infected with HBV (left) or HBV/HDV (right). HBV pgRNA (left) and antigenomic HDV RNA (right) are depicted in red and hCXCL10 is shown in green. Nuclei are stained with DAPI (blue).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Pacific Orange live/dead stain Invitrogen L34959 Anti-human HBcAg Abcam #7841 Anti-human HBsAg Raybiotech #MD050186 Goat-anti-rabbit CF555 Sigma Aldrich SAB46000068 Goat-anti-mouse APC Life Technologies AB_2536211 Goat-anti-mouse PE Life Technologies AB_2539848 HBs183-191 TCR-like antibody Sastry et al., 2011 N/A HBc18-27 TCR-like antibody Sastry et al., 2011 N/A Anti-human CD107a FITC BD Biosciences 560949 Anti-human CD8 V500 BD Horizon 560774 Anti-human IFN-g PeCy7 eBiosciences AB_469682 Anti-human TNF-a PE BD Bioscience 559321 HBs183-191 PE pentamer ProImmune WB3290-PE HBs370 PE pentamer ProImmune WB4073-PE EBV PE pentamer ProImmune WB2144-PE Biological Samples Human pathological liver tissue Asian American Liver Centre, Gleneagles Hospital https://www.aamg.co/liver/ Human HBV/HDV patient blood Fonsazione IRCCS Ca’Granda Ospedale Maggiore Policlinico https://www.policlinico.mi.it/ Human healthy blood Health Sciences Authority https://www.hsa.gov.sg/ Chemicals, Peptides, and Recombinant Proteins Recombinant Human IL-2 R&D systems 202-1L-050 Anti-CD3 eBioscience AB_468854 Ficoll-Paque PLUS GE Healthcare 17-1440-02 BD Cytofix/Cytoperm Fixation BD Bioscience 51-2090 KZ Recombinant Human IFN-a2a PBL 11100-1 Recombinant Human IFN-b Peprotech 300-02BC Recombinant Human IL-29/IFN-l1 R&D systems 1598-IL-025 Critical Commercial Assays Primeflow assay with customized probeset Invitrogen EB 16488 Nanostring human immunology version 2 and customized probes assay Nanostring technologies XT-CSO-HIM2-12 FASER PE amplification kit Miltenyi 130-091-764 One Shot Top10 E. coli kit Invitrogen C404003 QIAamp minELute virus spin kit QIAGEN 57704w Rneasy Mini kit QIAGEN 74004 mMESSAGE mMACHINE T7 Ultra kit Ambion AM1345 Nucelofactor kit Lonza V4XP-3024 Human serum albumin ELISA quantification kit Bethyl Laboratories E88-129 RNAScope Fluorescent Multiplex kit Advanced Cell Diagnostics 320850 HBV Artus kit QIAGEN 4506265 Quantitative HBsAg kit Abbott Architect Platform 6C36 (Continued on next page) e1 Cell Reports Medicine 1, 100060, July 21, 2020

Techniques: Infection, In Vivo, Stable Transfection, Injection, RNA In Situ Hybridization, Staining

Journal: Biomicrofluidics

Article Title: Fabrication of anti-protein-fouling poly(ethylene glycol) microfluidic chip electrophoresis by sandwich photolithography

doi: 10.1063/1.4959239

Figure Lengend Snippet: Separation of three proteins under different voltages using PEG MCE. Conditions: buffer, 40 mM phosphate (pH = 6.0); sample, 0.5 mg·ml−1 for each protein; microchannel, 14 cm × 50 ...

Article Snippet: C. Protein separation performance of PEG MCE Fig. shows the separation of a mixture of three proteins (BSA, Lys, and Cyt-c) using the PEG MCE equipped with electrochemical detector at different voltages.

Techniques:

Journal: Biomicrofluidics

Article Title: Fabrication of anti-protein-fouling poly(ethylene glycol) microfluidic chip electrophoresis by sandwich photolithography

doi: 10.1063/1.4959239

Figure Lengend Snippet: Comparison of the anti-protein-fouling PEG MCE with traditional PDMS MCE in protein separation performance. Separation conditions: buffer, 40 mM phosphate (pH = 6.0); applied voltage, +200 V; sample, 0.5 mg·ml ...

Article Snippet: C. Protein separation performance of PEG MCE Fig. shows the separation of a mixture of three proteins (BSA, Lys, and Cyt-c) using the PEG MCE equipped with electrochemical detector at different voltages.

Techniques: Comparison