Journal: Cellular signalling

Article Title: SARS-CoV-2 NSP16 promotes IL-6 production by regulating the stabilization of HIF-1α.

doi: 10.1016/j.cellsig.2024.111387

Figure Lengend Snippet: Fig. 4. SARS-CoV-2 NSP16 interacts with HIF-1α. (A) HEK293T cells (1.0 × 106) were co-transfected with Flag- HIF-1α (2.0 μg) and HA-SARS-CoV-2 NSP16 (2.0 μg) plasmids. At 48 h post transfection, equal amount of supernatant from whole-cell lysates were used for Co-IP with an IgG or anti-HA-antibody and then detected by IB with anti-Flag and anti-HA antibodies. (B) and (C) Colocalization of SARS-CoV-2 NSP16 with HIF-1α. HEK293T cells (3.0 × 105) were transfected with Flag- HIF-1α (2.0 μg) and HA-SARS-CoV-2 NSP16 (2.0 μg) plasmids, and at 24 h post transfection the cells treated with CoCl2 (100 uM) or left untreated. At 6 h after CoCl2 treatment, the cells were immunoblotted with mouse anti-Flag antibody or rabbit anti-HA antibody and CoraLite488-conjugated affiniPure goat anti-mouse IgG antibody or CoraLite594-conjugated goat anti-rabbit IgG antibody. Immunofluorescence data quantified at specific cell diameters were showed in (B.i.) the merge of Flag-HIF-1α, (B.ii.) the merge of HA-NSP16, (B.iii.) the merge of Flag-HIF-1α and HA-NSP16 in normal condition, and (C.i.) the merge of Flag-HIF-1α, (C.ii.) the merge of HA-NSP16, and (C.iii.) the merge of Flag-HIF-1α and HA-NSP16 in hypoxia condition. The nuclei were stained with DAPI. Confocal microscopy (Leica-LCS- SP8-STED) was used for image analysis. Scale bars: 10 μm.

Article Snippet: The cells were then washed three times with PBS, and incubated with a CoraLite488-conjugated affiniPure goat anti-mouse IgG antibody (1:200, Proteintech) and a CoraLite594-conjugated goat anti-rabbit IgG antibody (1:200, Proteintech) for another 2 h at room temperature.

Techniques: Transfection, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Confocal Microscopy

Journal: Cellular signalling

Article Title: SARS-CoV-2 NSP16 promotes IL-6 production by regulating the stabilization of HIF-1α.

doi: 10.1016/j.cellsig.2024.111387

Figure Lengend Snippet: Fig. 5. Domain mapping of SARS-CoV-2 NSP16 interaction sites in HIF-1α and HIF-2α. (A and B) Schematic diagrams of HIF-1α (A) and HIF-2α (B) domain structures. Mapping of SARS-CoV-2 NSP16 interacting domains of HIF-1α (C) and HIF-2α (D). HEK293T cells (1.0 × 106) were transfected with StrepII-SARS-CoV-2 NSP16 (2.0 μg) along with various Flag-tagged HIF-1α or HIF-2α wild type or truncated mutants (2.0 μg) as indicated. At 48 h post transfection, the cell lysates were harvested and immunoprecipitated with an anti-Flag antibody and then the precipitates were analyzed by IB with anti-StrepII or anti-Flag antibodies. For (C) and (D), the experiments were repeated at least three times with similar results. (E and F) Co-localization of SARS-CoV-2 NSP16 and HIF-1α truncated mutants. HEK23T cells (3.0 × 105) were transfected with indicated Flag-tagged HIF-1α mutants (2.0 μg). At 24 h post transfection, the cells were probed with mouse anti-Flag antibody or rabbit anti-HA antibody and CoraLite488-conjugated affiniPure goat anti-mouse IgG antibody or CoraLite594-conjugated goat anti-rabbit IgG antibody. The nuclei were stained with DAPI. Confocal microscopy (Leica-LCS-SP8-STED) was used for image analysis. Scale bars, 10.0 μm.

Article Snippet: The cells were then washed three times with PBS, and incubated with a CoraLite488-conjugated affiniPure goat anti-mouse IgG antibody (1:200, Proteintech) and a CoraLite594-conjugated goat anti-rabbit IgG antibody (1:200, Proteintech) for another 2 h at room temperature.

Techniques: Transfection, Immunoprecipitation, Staining, Confocal Microscopy