Journal: iScience

Article Title: Yip1 interacting factor homolog B mediates the unconventional secretion of ORF8 during SARS-CoV-2 infection

doi: 10.1016/j.isci.2024.111551

Figure Lengend Snippet:

Article Snippet: anti-IL17RA , Thermo Fisher Scientific , PA5-47199.

Techniques: Virus, Recombinant, Modification, One Step RT-PCR, Software

Journal: PLOS Pathogens

Article Title: Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1011128

Figure Lengend Snippet: (A) Schematic diagram of SARS-CoV-2 infectious cDNA clone generated by a reverse genetic system. The cDNA fragments F1-F7 were synthesized and assembled into full-length SARS-CoV-2 cDNA, and RNA transcription, electroporation, and virus production were carried out in Vero E6 cells. (B) Schematic diagram of SARS-CoV-2 infection model. Calu-3 epithelial cells were infected with SARS-CoV-2 at a MOI of 0.01. After 12 hours, cell culture supernatant was centrifuged and divided into two parts for western blotting and ELISA, respectively. (C) The secretion of ORF8 in (B) was detected by western blotting and ELISA. (D) Jurkat cells or Calu-3 epithelial cells were infected with HIV-1 or SARS-CoV-2. After 12 hours, the secretion of ORF8, Nef and 3CL pro was detected by western blotting. (E) Schematic diagram of time-dependent ORF8 secretion upon SARS-CoV-2 infection. Twelve hours after SARS-CoV-2 infection, cell culture medium was replaced, and the amount of ORF8 protein in the supernatant was detected by ELISA every 2 hours. (F-H) Schematic diagram of ORF8-deleted SARS-CoV-2 variant (F). ORF8 coding sequence was deleted from the cDNA of F7 fragment. SARS-CoV-2 ΔORF8 variant was used to infect Calu-3 cells, THP-1 DM cells, and the co-culture system. The secretion of cytokines and chemokines related to cytokine storm was detected by ELISA (G, H). (I) Calu-3 Ace2 +/+ , Calu-3 Ace2 -/- , THP-1 DM Il17ra +/+ and THP-1 DM Il17ra -/- cells were used to form four kinds of culture systems. The secretion of cytokines and chemokines in different cell culture systems was detected by ELISA. Representative images from n = 3 biological replicates are shown (C, D). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (C, E, G-I). Unpaired two-tailed Student t test (C) and one-way ANOVA followed by Bonferroni post hoc test (G, I) were used for data analysis. *, p < 0.05, **, p < 0.01.

Article Snippet: Blots were probed with the indicated antibodies: anti-ORF8 (NBP3-05720), anti-GAPDH (NBP2-27103) (Novus Biologicals), anti-3CL pro (GTX135470) (GeneTex), anti-NEF (MA1-71507), anti-IL17RA (PA5-47199) (Thermo Fisher Scientific), anti-GFP (ab6556), anti-V5 (ab9137) (Abcam), anti-Flag (AF0036), anti-His (AF5060), anti-GST (AF5063), anti-HA (AF0039) (Beyotime Biotechnology, Shanghai, China).

Techniques: Generated, Synthesized, Electroporation, Infection, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Variant Assay, Sequencing, Co-Culture Assay, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1011128

Figure Lengend Snippet: (A) Schematic diagram of glycosylation identification based on HLPC-MS/MS. DMSO (control), Brefeldin A (3 μg/mL) or Monensin (2 μM) was used to pretreat Calu-3 cells for 2 hours. The supernatant was collected for HLPC-MS/MS analysis. (B) SARS-CoV-2 ORF8 secreted through conventional pattern has N78 glycosylation. An increase of 2.9890 Da of Asn residue was used to determine N-linked glycosylation. (C-G) Calu-3 cells were infected with SARS-CoV-2 ORF8-N78Q variant (C, D, F), or transfected with ORF8 N78Q plasmid (C, E, G). After 12 hours, the supernatant was collected and divided into three parts. One part was used to purify ORF8 protein, followed by PNGase F digestion and western blotting (C); the second part was used to stimulate THP-1 DM cells (D, E); the third part was used to purified ORF8 protein and then stimulate THP-1 DM cells at a final concentration of 10 ng/mL (F, G). After 12 hours, the release of cytokines and chemokines was detected by ELISA (D-G). (H) Calu-3 cells were infected with SARS-CoV-2 ORF8-N78Q variant, or transfected with ORF8-N78Q plasmid. Twelve hours later, the supernatant was used to stimulate THP-1 DM cells for another 12 hours. The interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. (I, J) Calu-3 cells were infected with SARS-CoV-2 ORF8-N78Q variant (I), or transfected with ORF8-N78Q plasmid (J). After 12 hours, the supernatant was collected to purify ORF8 protein. After PNGase F digestion, the ORF8 protein was used to stimulate THP-1 DM cells for 12 hours, and the interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. (K, L) Brefeldin A or Monensin was used to pretreat Calu-3 cells for 2 hours, followed by infection with SARS-CoV-2 ORF8-N78Q variant (K), or transfection with ORF8-N78Q plasmid (L). Twelve hours later, the supernatant was used to stimulate THP-1 DM cells for another 12 hours. The interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. Representative images from n = 3 biological replicates are shown (C, H-L). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (D-G). One-way ANOVA followed by Bonferroni post hoc test (D-G) was used for data analysis. *, p < 0.05, **, p < 0.01.

Article Snippet: Blots were probed with the indicated antibodies: anti-ORF8 (NBP3-05720), anti-GAPDH (NBP2-27103) (Novus Biologicals), anti-3CL pro (GTX135470) (GeneTex), anti-NEF (MA1-71507), anti-IL17RA (PA5-47199) (Thermo Fisher Scientific), anti-GFP (ab6556), anti-V5 (ab9137) (Abcam), anti-Flag (AF0036), anti-His (AF5060), anti-GST (AF5063), anti-HA (AF0039) (Beyotime Biotechnology, Shanghai, China).

Techniques: Tandem Mass Spectroscopy, Infection, Variant Assay, Transfection, Plasmid Preparation, Western Blot, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: PLOS Pathogens

Article Title: Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1011128

Figure Lengend Snippet: (A) Unglycosylated ORF8 (ORF8) or synthetic N-linked-glycosylated ORF8 (ORF8-N-Glyc) (1 μg/mL) was added into the culture medium of THP-1 DM cells to stimulate IL-17 pathway. The interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. (B) ORF8 or ORF8-N-Glyc protein was added into the culture medium of THP-1 DM cells at a concentration of 10 ng/mL. The release of cytokines and chemokines was detected by ELISA. (C) Schematic diagram of ORF8 protein treatment model. Mice were treated with aerosols of PBS (n = 13), unglycosylated ORF8 (n = 18) or synthetic N-linked-glycosylated ORF8 proteins (n = 17) (200 μg/mouse) every other day. For IL17RA antibody injections (n = 12), each mouse received 200 μg per injection every three days as indicated. (D-G) Survival curves were calculated using the Kaplan-Meier method (D). Lung lesions were detected by H&E staining at day 7 post infection (dpi) (E). The release of cytokines and chemokines in lungs (F) and livers (G) were detected by ELISA at 7 dpi. (H-J) Hamsters were intranasally infected with 4×10 5 PFU SARS-CoV-2 (n = 18) or ORF8-deleted SARS-CoV-2 variant (n = 18). Weight loss data were documented daily (H). Lung lesions were detected by H&E staining at 7 dpi (I). The relative expression of cytokines and chemokines in lungs were detected by qRT-PCR at 2, 4 and 7 dpi (J). Representative images from n = 3 biological replicates are shown (A, E, I). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (B, F, G, J). Log-rank (Mantel-Cox) test (D) and one-way ANOVA followed by Bonferroni post hoc test (B, F, G, J) were used for data analysis. Scale bar = 500 μm. *, p < 0.05, **, p < 0.01. Abbreviations: n.s., not significant.

Article Snippet: Blots were probed with the indicated antibodies: anti-ORF8 (NBP3-05720), anti-GAPDH (NBP2-27103) (Novus Biologicals), anti-3CL pro (GTX135470) (GeneTex), anti-NEF (MA1-71507), anti-IL17RA (PA5-47199) (Thermo Fisher Scientific), anti-GFP (ab6556), anti-V5 (ab9137) (Abcam), anti-Flag (AF0036), anti-His (AF5060), anti-GST (AF5063), anti-HA (AF0039) (Beyotime Biotechnology, Shanghai, China).

Techniques: Immunoprecipitation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Injection, Staining, Infection, Variant Assay, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry

doi: 10.1038/srep04376

Figure Lengend Snippet: Novel interactors of AMPK associated with regulation of actin cytoskeleton

Article Snippet: The resultant proteins were separated on an 8% to 15% SDS-PAGE gel and immunoblotted with antibodies against AMPK-α1 and AMPK-β1 (Cell Signal, Boston, MA, USA), MYH9 (sc-47199), IQGAP1 (sc-10792), gelsolin (sc-48769) and RhoA (sc-179) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Rac1 (05-389) (Millipore, Billerica, MA 01821, USA).

Techniques: Molecular Weight

Journal: Scientific Reports

Article Title: Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry

doi: 10.1038/srep04376

Figure Lengend Snippet: (A) Network analysis of AMPK interactome, showing that the AMPK complex interacts directly with multiple proteins associated with the regulation of the actin cytoskeleton. Proteins and protein-protein interactions associated with regulation of the actin cytoskeleton were extracted from the KEGG pathway and STRING 9.1 databases. All input proteins associated with actin regulation are depicted as small gray spheres. Interacting proteins in our dataset are depicted as green spheres, and interacting proteins validated by Co-IP are shown as yellow spheres. Bait proteins are shown as red hexagons. Protein-protein interactions extracted from the STRING database are shown as grey lines. Interactions identified in this study are shown as green lines. Co-IP-validated interactions are shown as red dotted lines. (B) Validation of protein-protein interactions by pulldown approach. A novel interacting protein (Iqgap1) was validated by co-IP. Well-known proteins (Gsn and Vim) were also validated. Vim was used as a positive control to confirm homologous interactions. (C) Validation of protein-protein interactions using direct IP. AMPK-α1, AMPK-β1, Myh9, Iqgap1, Rhoa, and Rac1 were validated by co-IP. The co-IP results for novel interactors (Myh9, Iqgap1, Rhoa, and Rac1) that were associated with regulation of the actin cytoskeleton corresponded well with the AP-MS results.

Article Snippet: The resultant proteins were separated on an 8% to 15% SDS-PAGE gel and immunoblotted with antibodies against AMPK-α1 and AMPK-β1 (Cell Signal, Boston, MA, USA), MYH9 (sc-47199), IQGAP1 (sc-10792), gelsolin (sc-48769) and RhoA (sc-179) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Rac1 (05-389) (Millipore, Billerica, MA 01821, USA).

Techniques: Co-Immunoprecipitation Assay, Positive Control