Journal: Molecular psychiatry

Article Title: Projection-specific circuits of retrosplenial cortex with differential contributions to spatial cognition.

doi: 10.1038/s41380-024-02819-8

Figure Lengend Snippet: Fig. 2 Monosynaptic rabies viral tracing reveals RSC intrinsic inputs to M2-projecting and AD-projecting RSCg neurons in C57BL/6 mice. A Schematic of monosynaptic retrograde rabies tracing (RV) of synaptic inputs to M2-projecting RSCg cells. The rAAV2-retro-Cre injection in M2 retrogradely drives Cre expression in M2-projecting RSC; then the Cre dependent AAV helper virus (AAV8-hSyn-DIO-TC66T-2A-EGFP-2A- OG) is injected into RSCg, followed by injection of an EnvA-pseudotyped SAD ΔG rabies virus three weeks later. B Immunostaining and microscopy images depict Cre positive neurons in the M2 injection site. rAAV2-retro-Cre labeled neurons are stained by a Cy5-conjugated secondary antibody and visualized as pink in the enlarged inset. C, D Coronal sections of injection in RSCg. Injection sites are shown in white boxes (C) with magnified images in (D). EGFP-labeled AAV-helper virus expressed in M2-projecting Cre+ neurons. Rabies is labeled by DsRed, DAPI is blue. White arrowheads indicate EGFP and DsRed double-labeled starter neurons. E Quantitative mapping of the connection strength of the intrinsic RSC subregion inputs to M2-projetcing RSCg neurons. Connection strength index (CSI) is calculated by the number of presynaptic input neurons divided by the number of starter neurons in RSCg. The individual circle represents one case (n = 5). Data are measured from M2-projecting RSCg neurons (N = 5 cases) and AD-projecting RSCg neurons (N = 5 cases) and are presented as the mean ± s.e.m. F, G Example of input- mapped local RSC subregions along the longitudinal axis. AP number indicates the distance relative to bregma. G represents magnified views corresponding to the panels in (F), indicating that the intrinsic connections vary across different layers along the anterior/posterior axis. H–L follow the same order as (A)–(E) and reveal RSC subregions inputs to AD-projecting RSCg neurons.

Article Snippet: A primary rabbit anti-Cre antibody (NOVUS, 1:500 dilution) was used, followed by a Cy5-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 1:200 dilution).

Techniques: Injection, Expressing, Virus, Immunostaining, Microscopy, Labeling, Staining

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