Journal: Nature communications

Article Title: Regulation of local GTP availability controls RAC1 activity and cell invasion.

doi: 10.1038/s41467-021-26324-6

Figure Lengend Snippet: Fig. 3 Localization of regions with high RAC1 activity and high local GTP concertation. a Schematic representation of the RAC1 FRET (Förster resonance energy transfer) biosensor. CRIB Cdc42/Rac interactive binding motif. b MDA-MB-231 cells co-expressed the RAC1 biosensor and GEVAL30 or GEVALNull. The signal of each individual biosensor was imaged and rendered as described in Methods. c MDA-MB-231 cells co-expressed the RAC1 biosensor and GEVAL30 or GEVALNull as in (b) and assayed for the correlation between biosensor activities. For each cell, biosensor data were collected in a series of images taken at 1-min intervals over the course of 30 min and analyzed as described in Methods. Pixel-wide Pearson correlation between RAC1 activity (measured as FRET (Förster resonance energy transfer) index for the RAC1 biosensor) and GTP index (measured as the activity of the indicated GEVAL variant) was calculated for each image. The correlation values for the image series corresponding to each individual cell were summarized as “bar-and-whiskers” plots, with “whiskers” indicating the first and the fourth quartiles, the horizontal line (median) splitting the bars into the second and the third quartiles, and “X” indicating quartiles, as well as the mean correlation coefficient (r) for each series. The mean correlation coefficients were compared by Mann–Whitney test. d Cells expressing the indicated constructs were probed in RAC1 activity assay as described in Methods. Note that the difference in migration of RAC1T17N compared to other proteins is likely due to the fact that RAC1T17N is fused to one Myc-tag, whereas other RAC1 proteins—to two Myc tags. Shown are representative images of at least two independent experiments. e Quantification of (d); n = 2 biologically independent samples (left panel); n = 3 biologically independent samples (right panel) The data represents average ± SEM.

Article Snippet: Expression vectors for RAC1WT, RAC1P29S, RAC1Q61L, and RAC1T17N were purchased from Addgene (#128580, #128581, #128582, and #12984, respectively).

Techniques: Activity Assay, Förster Resonance Energy Transfer, Binding Assay, Variant Assay, MANN-WHITNEY, Expressing, Construct, Migration

Journal: EBioMedicine

Article Title: Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro.

doi: 10.1016/j.ebiom.2021.103712

Figure Lengend Snippet: Figure 3. SARS-CoV-2 induces N-glycosylation through NF-kB/STT3A axis. (a) Locus compare plot for the comparison between the STT3A eQTLs and the GWAS result, the COVID-19 A2 analysis for patients with severe respiratory symptoms via COVID-19 Host Genetics Initiative (COVID-19-HGI) project. The points are colored based on linkage disequilibrium (LD) bins. rs201008304, the indel variant in strong LD with the lead STT3A eQTL, is the top and most significant SNP in COVID-19 A2 analysis. (b) The eQTL plot for the association between STT3A variant types [A/A (n = 437), A/AT (n = 46), and AT/AT (n = 0)] expression and rs201008304 in cells-cultured fibroblasts was queried from the GTEx. (c) Forest plot shows the risk of rs201008304 in different COVID-19 phenotypes, originally from the summarized statistic results. (d) Representative data of immunohistochemistry staining of STT3A (red cells) and N protein in lung tissues of AAV-Ctrl and AAV-hACE2 mice infected by SARS-CoV2 at 5 dpi. Images were captured at 40x objective lens magnification. Scale bar, 100 mm. Three randomly selected STT3A/nuclei expression fields in lung tissues were exported from CaseViewer software (3DHistech) and quantified by the HistoQuant plugin in QuantCenter (3DHistech). Statistical method: t test, *p < 0.05, (n = 3). (e) Western blotting of S protein in Vero E6 cells after knockdown of STT3A or STT3B. Circle, g-spike; arrow, ng-spike. (f) ChIP analysis of enrichment of NF-kB in STT3A promoters after SARS-CoV-2 infection for 1 d. The enrichment levels were analyzed by RT-qPCR and shown as the per- centage of input. Statistical method: two-way ANOVA, Tukey post hoc tests, ***p < 0.001 (n = 3 with mean § SD shown).

Article Snippet: The deleted NF-kB binding motif construct had a deletion at bases 363CGTAGTTTCC 353 in the STT3A promoter. pBabe-PuroIkBalpha-mut (RRID:Addgene_15291; Addgene plasmid #15291, Watertown, MA, USA) [21] is a dominant-negative mutant NF-kB inhibitor (IkBaSR) and inhibits NF-kB activity.

Techniques: Glycoproteomics, Comparison, Variant Assay, Expressing, Cell Culture, Immunohistochemistry, Staining, Infection, Software, Western Blot, Knockdown, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro.

doi: 10.1016/j.ebiom.2021.103712

Figure Lengend Snippet: Figure 4. Impairment of STT3A compromises SARS-CoV-2 infectivity. (a) Luciferase assay was used to monitor the infection rate of pseudovirions generated from shSTT3A or shSTT3B HEK293T cells in HEK293T-ACE2 cells. Expression of spike in pseudovirus produced from shSTT3A and shSTT3B. Statistical method: one-way ANOVA, Tukey post hoc tests. **p < 0.01 (n = 3 with mean § SD shown). (b) Western blot analysis of N protein expression in A549-ACE2 cells infected with SARS-CoV-2 generated from control, shSTT3A or shSTT3B Vero E6 cells (left panel). N protein intensity was quantified by ImageJ (right panel). (c) Western blot analysis of spike and N protein expression in DMSO and NGI-1 treated SARS-CoV-2 infected Vero E6 cells. (d) Representative immunofluorescence images of N protein expression in DMSO and NGI-1 treated Vero E6 cell after SARS-CoV-2 infection. Scale bar, 100 mm. (e) SARS-CoV-2 neutralization IC50 of NGI-1 in Vero E6. Vero E6 cells was pretreated with NGI-1 1 h ahead at the indicated doses prior to SARS-CoV-2 infection (red). Cell viability of Vero E6 cells following NGI-1 treatment determined by MTT assay (black). Data shown are means § SD from representative triplicates. (f) Luciferase activity was measured at 48 h to determine SARS-CoV-2, Alpha, and Beta variants pseudoviral infectivity in DMSO and NGI-1 treated HEK293T-ACE2; Data shown are means § SD from represen- tative triplicates.

Article Snippet: The deleted NF-kB binding motif construct had a deletion at bases 363CGTAGTTTCC 353 in the STT3A promoter. pBabe-PuroIkBalpha-mut (RRID:Addgene_15291; Addgene plasmid #15291, Watertown, MA, USA) [21] is a dominant-negative mutant NF-kB inhibitor (IkBaSR) and inhibits NF-kB activity.

Techniques: Infection, Luciferase, Generated, Expressing, Produced, Western Blot, Control, Neutralization, MTT Assay, Activity Assay

Journal: Journal of lipid research

Article Title: Disruption of nucleotide biosynthesis reprograms mitochondrial metabolism to inhibit adipogenesis.

doi: 10.1016/j.jlr.2024.100641

Figure Lengend Snippet: Fig. 4. PPARγ overexpression rescues mitochondrial function induced by the loss of de novo nucleotide biosynthesis. A: OXPHOS protein levels were measured in primary SVF cells that were differentiated and treated with PPARγ inhibitor SR 16832 (2 μM or 10 μM) for 6 days (B) 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM 5FU or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. C: 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM MIZ or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. D: Live cell imaging with MTG and TMRE staining was used to assess changes in mito- chondrial membrane potential relative to mitochondrial mass in the presence or absence of 25 μM MIZ. E: ImageJ was used to measure the ratio of MTG and TMRE. All experiments were repeated two or three times with two or three biological replicates. Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ****P < 0.0001.

Article Snippet: PPARγ2 plasmid (Addgene #8859) or pBabe control was transfected into PLAT-E cells.

Techniques: Over Expression, Stable Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Live Cell Imaging, Staining, Membrane