Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Differentially expressed genes (DEGs, P-FDR < 0.05) were identified by RNA-seq analysis in IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1 KO HepG2 cells after 72 hrs of induction by dox, comparing to controls (dox-conditions). Cutoff threshold (fold change > +/-1.5) is indicated by dotted lines. (A) Analysis of all DEGs (n=3251) detected for IFN-λ4-GFP or IFN-λ4-GFP-IFNLR1 KO cells. In blue - DEGs (n=2,735) specific to IFN-λ4-GFP and considered IFNLR1-dependent. In black - DEGs (n=145) shared between both groups and considered IFNLR1-independent. In orange - DEGs (n=371) specific to IFN-λ4-GFP-IFNLR1 KO . (B) DEGs of IFN-λ4-GFP analyzed in IFN-λ3-GFP transcriptome. In black - DEGs (n=1,506) shared in IFN-λ4-GFP and IFN-λ3-GFP and in blue - IFN-λ4-signature DEGs (n=1,229) detected in IFN-λ4-GFP but not in IFN-λ3-GFP producing cells. Additional details are provided in Fig. S3 and Table S6. (C , D) Cell cycle analysis of cells synchronized by 24 hrs of serum starvation, treated with or without dox (0.5 µg/ml) for 72 hrs and analyzed by flow cytometry after PI staining. The plot shows a representative picture and the percentage of cells in each phase of the cell cycle. All data are shown as mean± SEM from triplicate experiments. *, P < 0.05. (E , F) Bromodeoxyuridine (BRDU, %) incorporation indicating cell proliferation in HepG2 cells expressing IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1KO. Cells were cocultured with HepG2 cells labeled with Far Red proliferation dye, dox-induced for 72 hrs and treated with BRDU for 3 hrs before analysis. Gates show HepG2 cells exposed to IFN-λs (IFN-λ treated cells) and HepG2 expressing IFN-λs. P-values compare corresponding dox+ vs. dox-HepG2 cells, ** p<0.01, Student’s T-test. Graphs represent one of three independent experiments, each in biological triplicates.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: RNA Sequencing, Cell Cycle Assay, Flow Cytometry, Staining, BrdU Incorporation Assay, Expressing, Labeling

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: (A) Unsupervised clustering of activities of 54 regulons that were significantly and directionally enriched with HepG2-DEGs in the set of 885 of TCGA-LIHC regulons. The heatmap shows differences in activity scores (dES) for the IFN-λ4-enriched regulons organized by GSEA-2T results for the IFN-λ4-GFP, IFN-λ3-GFP and IFN-λ4-GFP-IFNLR1 KO DEG signatures. Cluster I: regulons with dES > 0 in IFN-λ4-GFP; cluster II: regulons with dES < 0 in IFN-λ4-GFP. Table S7 provides the regulon activity scores presented in Figure 2A. (B-G) GSEA-2T plots for IRF1 and IRF2, respectively, in DEG signatures for (B-C) IFN-λ3-GFP, ( D-E ) IFN-λ4-GFP, and ( F-G ) IFN-λ4-GFP-IFNLR1 KO . (H) Inhibition of proliferation in IFN-λ4-GFP HepG2 cells, for one of three independent experiments. Cells were treated with IRF1 siRNA for 24 hrs, labeled with Far Red proliferation dye and dox-induced at indicated concentrations for 72 hrs. Proliferation was assessed by flow cytometry with a graph representing the geometric mean expression of Far Red proliferation dye with higher values indicating reduced cell proliferation. P-values compare dox-treated control siRNA with dox-treated IRF1 siRNA. ** p<0.01, Student’s T-test. Below: Western blots showing IRF1 protein levels following siRNA knockdown.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Activity Assay, Inhibition, Labeling, Flow Cytometry, Expressing, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Representative confocal images of HepG2 cells transduced with a mammalian baculovirus delivery system (BacMam) of GFP-tagged proteins targeting specific organelles - lysosomes, Golgi, early and late endosomes. After transduction for 6 hrs, cells were transiently transfected with Halo-tagged constructs for IFN-λ4 or control for indicated times, stained with cell-permeant Halo-tag ligand TMR (red), and imaged. (A) Confocal images showing IFN-λ4 accumulation in lysosomes but not in early endosomes. (B) Late endosomal trafficking of IFN-λ4, with the inset showing larger magnification. (C) Unfolded protein response (UPR) is represented by lysosomal enlargement after protein accumulation. (D) Live images of IFN-λ4-expressing HepG2 cells undergoing apoptosis, characterized by membrane blebbing and cell death. Images were scanned every minute for 12 hrs. Scale bars – 10 um. (E) Apoptosis detection with ApoTox-Glo assays in corresponding untreated and dox-induced cells for indicated time points. RLU, relative luminescence units. (F) Graph showing counts from colony formation assay for HepG2 cells expressing IFN-λ4 or IFNLR1 KO grown in 6-well plates with or without dox for 13 days. Cell colonies were stained with crystal violet and manually counted. The graph represents the number of colonies as a percentage of initial plated counts. (G,H) mRNA (G) and protein levels ( H ) of DDIT3 after siRNA knockdown tested by qRT-PCR and Western blot assays, respectively. (I-J) Apoptosis (I) and cell viability (J) assays were performed after siRNA knockdown of DDIT3 in dox-induced IFN-λ4-GFP cells. * p<0.05, ** p< 0.01, *** p<0.001.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Transduction, Transfection, Construct, Control, Staining, Expressing, Membrane, Colony Assay, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Viral RNA in the supernatant of Calu-3 cells infected with indicated SARS-CoV-2 variants was quantified by qRT–PCR at 48 h postinfection (MOI 0.05). Cells were treated preinfection with increasing concentrations of indicated IFNs (α2 at 5 and 500 IU/ml, β, and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml). Dots represent the mean of n = 3 + SEM. (B) Percentage of viral RNA in the supernatant compared with the no IFN control (set to 100%) at 500 IU/ml IFN (100 ng/ml for IFN-λ1) as indicated. Bars represent the mean of n = 3 ± SEM. (C) Infectious SARS-CoV-2 particles determined by the TCID50 assay in the supernatant of Calu-3 cells infected with indicated SARS-CoV-2 variants (MOI 0.05, 48 h postinfection). Cells were left untreated or were pretreated with 500 IU/ml IFN-β or IFN-γ. Bars represent the mean of n = 3 ± SEM. d.l., detection limit. (B, C, D) Correlation of infectious titer of viral particles ((C), TCID50) with viral RNA in the supernatant ((B), qRT–PCR), r, Pearson’s correlation. Statistical significance was calculated using t tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR, Control, TCID50 Assay

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Metabolic activity of Calu-3 cells as assessed by MTT assay after treatment with IFNs (α2, β, and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml) as in . Dots represent the mean of n = 3 ± SEM. (B) Mean of viral RNA in the supernatant of Calu-3 cells infected with indicated SARS-CoV-2 variants quantified by qRT–PCR at 6 h (wash CTRL) and 48 h postinfection (MOI 0.05). Bars represent the mean of n = 3 ± SEM (undetermined values in the 6 h sample were excluded from the calculation). (C) Representative immunofluorescence images of Calu-3 cells infected with Omicron BA.1 (MOI 0.05) or uninfected. Cells were stained for SARS-CoV-2 nucleocapsid (red), SARS-CoV-2 spike (green), and nuclei (blue) at 24 or 48 h postinfection. Scale bar = 50 μm. (D) Metabolic activity of iAT2 cells as assessed by MTT assay after treatment with IFNs (α2, β, and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml) as in . Dots represent the mean of n = 3 ± SEM.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Activity Assay, MTT Assay, Infection, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Viral RNA in the supernatant of iAT2 cells infected with indicated SARS-CoV-2 variants was quantified by qRT–PCR at 48 h postinfection (MOI 0.5). The cells were treated 24 h preinfection with increasing concentrations of indicated IFNs (α2 at 5 and 500 IU/ml, β and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml). Dots represent the mean of n = 3–4 + SEM. (B) Percentage of viral RNA in the supernatant compared with the no IFN control (set to 100%) at 500 IU/ml IFN (100 ng/ml for IFN-λ1) as indicated. Bars represent the mean of n = 3–4 ± SEM. Statistical significance was calculated using t tests. * P < 0.05.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR, Control

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Viral RNA levels harvested from the apical surfaces of ALI cultures infected with indicated SARS-CoV-2 variants 1 h post-treatment with IFNs (α2, β, and γ at 0.5, 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml) quantified by qRT–PCR at 5 d postinfection (MOI 0.5). Individual donors are indicated. (B) Viral RNA levels harvested from the apical surfaces of ALI cultures infected with SARS-CoV-2 omicron BA.1 (MOI 0.5) quantified by qRT–PCR at 2 h (wash CTRL), and 3- and 5-d postinfection. Individual donors are indicated.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1

doi: 10.26508/lsa.202201745

Figure Lengend Snippet: (A) Normalized viral RNA of ALI cultures from three donors (Donors A, B, and C) infected with indicated SARS-CoV-2 variants (MOI 0.5) quantified by qRT–PCR at 5 d postinfection. RNA was harvested from the apical surfaces (top panel) or intracellularly (bottom panel). ALI cultures were treated 1 h preinfection with increasing concentrations of indicated IFNs (α2, β and γ at 5, 50, and 500 IU/ml or λ1 at 1, 10, and 100 ng/ml). All values are normalized to the no IFN control (set to 100%) and dots represent the mean of n = 3 (δ and ο BA.1) or n = 2 (NL-02-2020) individual donors + SEM. (B) Percentage of viral RNA from the apical surfaces of ALI cultures compared with the no IFN control (set to 100%) at 500 IU/ml IFN (100 ng/ml for IFN-λ1) as indicated. Bars represent the mean of n = 3 (δ and ο BA.1) or n = 2 (NL-02-2020), individual donors are represented by different symbols and connected with lines. Statistical significance was calculated using ratio paired t tests compared with NL-02-2020. * P < 0.05; ** P < 0.01.

Article Snippet: 24 and 96 h post-seeding, the cells were stimulated with increasing amounts of IFNs (α2 [Cat#11101-2; R&D Systems], β [Cat#8499-IF-010/CF; R&D Systems], and γ [Cat#285-IF-100/CF; R&D Systems] using 5, 50, and 500 IU/ml or IFN-λ1 [Cat#1598-IL-025/CF; R&D Systems] using 1, 10, and 100 ng/ml) in 0.5 ml of medium.

Techniques: Infection, Quantitative RT-PCR, Control

Journal: Clinical Cancer Research

Article Title: Overexpression of PD-L1 Significantly Associates with Tumor Aggressiveness and Postoperative Recurrence in Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.ccr-08-1608

Figure Lengend Snippet: Fig. 1. PD-Ls expression in HCC cell lines and tissue samples.Western blots detected constitutive PD-L1and PD-L2 expressions on 7 HCC cell lines (A). Total protein (50 Ag) was loaded per lane. Consecutive sections were used for immunohistochemical study on expression of PD-L1and PD-L2 on HCC tumor tissues. Representative cases of concurrent high or low (B) as well as either high (C) expression of PD-L1and PD-L2 on HCC tumor tissues were shown. Positive cells were stained brown. Magnification, 200.

Article Snippet: The cells lysing, protein extraction, concentration detection, and Western analysis were done as we described previously (31, 34) with the following primary antibodies: goat antihuman PD-L1 (1:1,000) and PD-L2 (1:1,000) monoclonal antibodies (R&D Systems) with h-actin (1:500; Chemicon) as an internal loading control.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Clinical Cancer Research

Article Title: Overexpression of PD-L1 Significantly Associates with Tumor Aggressiveness and Postoperative Recurrence in Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.ccr-08-1608

Figure Lengend Snippet: Fig. 2. Kaplan-Meier curves of survival differences among HCC patients. DFS and OS for expression of PD-L1 (A), PD-L2 (B), and their combination (C) were found to be statistically significant. Significant differences in DFS and OS were validated in an independent cohort based on PD-L1 expression (D). P values were determined by the log-rank test.

Article Snippet: The cells lysing, protein extraction, concentration detection, and Western analysis were done as we described previously (31, 34) with the following primary antibodies: goat antihuman PD-L1 (1:1,000) and PD-L2 (1:1,000) monoclonal antibodies (R&D Systems) with h-actin (1:500; Chemicon) as an internal loading control.

Techniques: Expressing