IL-28R alpha/IFN-lambda R1 Antibody NBP1-84381 from Novus Biologicals, a Bio-Techne Brand

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Frontiers in immunology
January 01 2021

Fig 1: IFN-?4 is a misfolded protein that induces ER stress. 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-?4 or control for indicated times, stained with cell-permeant Halo-tag ligand TMR (red), and imaged. (A) Confocal images showing IFN-?4 accumulation in lysosomes but not in early endosomes. (B) Late endosomal trafficking of IFN-?4, with the inset showing larger magnification. (C) Unfolded protein response (UPR) is represented by lysosomal enlargement after protein accumulation. (D) Live images of IFN-?4-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-?3, IFN-?4 or IFNLR1 KO grown in 6-well plates with or without dox for 13 days. Cell colonies were stained with crystal violet and counted with ImageJ software. The graph represents the number of colonies as a percentage of initial plated counts. n.s., not significant, ***p < 0.001, ****p < 0.0001, Student’s T-test.
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Frontiers in immunology
January 01 2021

Fig 2: IFN-?4 inhibits the proliferation of HepG2 cells. RNA-sequencing analysis was used to identify differentially expressed genes (DEGs, P-FDR < 0.05) in IFN-?3-GFP, IFN-?4-GFP and IFN-?4-GFP-IFNLR1KO HepG2 cells after 72 hrs of induction by dox, compared to controls (dox- conditions). The cutoff threshold (fold change > +/-1.5) is indicated by dotted lines. (A) Analysis of all DEGs (n = 3251) detected for IFN-?4-GFP or IFN-?4-GFP-IFNLR1KO cells. In blue - DEGs (n = 2,735) specific to IFN-?4-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-?4-GFP-IFNLR1KO. (B) DEGs of IFN-?4-GFP analyzed in IFN-?3-GFP transcriptome. In black - DEGs (n = 1,506) shared in IFN-?4-GFP and IFN-?3-GFP and in blue - IFN-?4-signature DEGs (n = 1,229) detected in IFN-?4-GFP but not in IFN-?3-GFP producing cells. Additional details are provided in Figure S2 and Table S1. (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, Student’s T-test comparing no dox to dox+ for each cell cycle stage. (E, F) Bromodeoxyuridine (BrdU, %) incorporation indicating cell proliferation in HepG2 cells expressing IFN-?3-GFP, IFN-?4-GFP and IFN-?4-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-?s (IFN-? treated cells) and HepG2 expressing IFN-?s. 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.