Journal: Journal of Lipid Research

Article Title: ACAT1/SOAT1 maintains adipogenic ability in preadipocytes by regulating cholesterol homeostasis

doi: 10.1016/j.jlr.2024.100680

Figure Lengend Snippet: ACAT1 deficiency in preadipocytes impairs adipogenesis. A: Schematic of knockout (KO) of ACAT1 in WAT-SVF with adenovirus (AV) and the subsequent adipogenesis. Ing-WAT derived SVF cells from Acat1 flox/flox mice were infected with CTRL-AV or CRE-AV (adenovirus expressing Cre recombinase) to generate either CTRL cells or ACAT1-KO cells, respectively. The ACAT1 protein levels were determined by Western blot (B). After differentiation, the adipocytes were stained with Oil Red O (C), and mRNA level of genes involved in adipogenic transcription program and TG synthesis were quantified by qPCR (D). ACAT1 was knocked down with lentivirus-mediated shRNA in human SGBS preadipocytes, which were further differentiated to mature adipocytes, and lipid droplets (LDs) were captured under a light microscope (E), and mRNA levels were quantified with qPCR (F). In 3T3-L1 cell line, shCTRL and shSOAT1 preadipocytes were collected to quantify for the mRNA levels of indicated genes with qPCR (G), and after differentiation, the LDs were stained with Nile red (H). Data is presented as Mean ± SEM (n = 3–5) and analyzed by student t test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Adenoviruses expressing human ACAT1 (pADM-CMV-ACAT1-mCherry), catalytical dead ACAT1 (H460A, refer to the previous study ( )) or control sequence (pADM-CMV-mCherry) were purchased from VigeneBio.

Techniques: Knock-Out, Derivative Assay, Infection, Expressing, Western Blot, Staining, shRNA, Light Microscopy

Journal: mBio

Article Title: Host-Directed Antimicrobial Drugs with Broad-Spectrum Efficacy against Intracellular Bacterial Pathogens

doi: 10.1128/mBio.01534-14

Figure Lengend Snippet: Identification of antibacterial compounds that are nontoxic to host cells. (A) Six hundred forty FDA-approved drugs were first screened for their inhibition of the intracellular growth of bacteria in THP-1 cells. Then, they were screened to eliminate those toxic to THP-1 cells. Finally, host-acting compounds were identified by removing known antimicrobials. (B) Dual-fluorescence images of THP-1 cells treated with representative compounds. (Top) Agents with no effect on the intracellular C. burnetii growth or host cells; (middle) cytotoxic agents; (bottom) agents inhibiting bacterial growth but not cytotoxic. Each image represents the overlay of two fluorescence channels: 590/650 nm to detect mCherry-expressing C. burnetii and 340/380 nm to detect filipin-stained cell membranes. The pseudogreen color was used to visually improve the detection of C. burnetii . Scale bar, 100 µm. (C) Agents causing different degrees of inhibition of C. burnetii intracellular growth and host cell cytotoxicity. Circles, relative prevalence of Coxiella -containing vacuoles (CCVs); triangles, relative mCherry fluorescence; gray bars, relative viability at day 5; RFU, relative fluorescence units. (D) Effect of selected compounds on CCV size (area of individual CCV in each image). (E) Growth of C. burnetii in THP-1 cells treated with selected compounds at 33 µM. Intracellular bacterial abundance was followed over 5 days by the mCherry fluorescence of the bacteria.

Article Snippet: The intracellular growth of C. burnetii was assessed by the fluorescence of the constitutively expressed mCherry protein using a Tecan Infinite M200 PRO fluorescence plate reader with 580-nm excitation and 620-nm emission ( ).

Techniques: Inhibition, Fluorescence, Expressing, Staining

Journal: MedComm

Article Title: Intermedin promotes vessel fusion by inducing VE‐cadherin accumulation at potential fusion sites and to achieve a dynamic balance between VE‐cadherin‐complex dissociation/reconstitution

doi: 10.1002/mco2.9

Figure Lengend Snippet: The active form of Rab4 or Rab11 is required for VEC transportation. A‐D, HUVECs were co‐transfected with lentivirus expressing VEC/Rab4 WT or VEC/Rab11 WT , treated without IMD (A and B) or with IMD (C and D), and observed under continuous microphotography with a 15‐s interval. Arrows indicate the long‐range moving VEC + /Rab4 WT+ or VEC + /Rab11 WT+ vesicles. E‐G, HUVECs were transfected with shRNA to knockdown Rab4 (shR‐47596 targets ORF; shR‐47597 targets 3’‐UTR) or Rab11 (shR‐377861 targets ORF; shR‐411101 targets 3’‐UTR), and rescued by transfection of Lentiviral Rab4 WT or Rab11 WT , and incubated with or without IMD. The number of long‐range moving VEC + vesicles and their moving distance and speed were quantified. H and I, HUVECs were co‐transfected with Lentiviral VEC (red) and Rab4 WT /Rab4 Act /Rab4 Neg (green) or Rab11 WT /Rab11 Act /Rab11 Neg (green), respectively. J‐O, The number of long‐range moving VEC + /Rab + vesicles and their moving distance and speed were quantified. All quantifications use 10 randomly chosen fields from two independent experiments. Data were presented as scatter plots with mean ± SEM. Significance was assessed by one‐way ANOVA (Kruskal‐Wallis test) followed by nonparametric Dunn's post hoc analysis. Act: active; IMD −/− : intermedin knockout; KD: knockdown; Neg: negative; shRNA: small hairpin RNA; VEC: vascular endothelial cadherin; WT: wildtype

Article Snippet: Lentiviral vectors expression mCherry‐VEC, eGFP‐Rab4 WT , eGFP‐Rab4 Act ([Q67L] mutation), eGFP‐Rab4 Neg ([S27N] mutation), eGFP‐Rab11 WT , eGFP‐Rab11 Act ([Q70L] mutation), and eGFP‐Rab11 Neg ([S25N] mutation) were customized and provided by Genechem.

Techniques: Transfection, Expressing, shRNA, Knockdown, Incubation, Knock-Out

Journal: Viruses

Article Title: Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner

doi: 10.3390/v16040502

Figure Lengend Snippet: Effect of SLFN14 on viral replication. ( A ) Role of the RNA Polymerase III-RIG-I-IFN signaling pathway in the SLFN14 activity. HEK293T cells co-transfected with HIVluc expression plasmid and an empty plasmid (control cells) or a plasmid expressing human SLFN14 were treated or not with inhibitors indicated. Luciferase activity measured in these cells was expressed as % of control cells. Data correspond to a triplicate experiment and are representative of three independent experiments. Although not indicated, statistically significant differences ( p ≤ 0.0001) were found between the control and each of the other groups, as calculated with two-way ANOVA and Dunnett post-hoc tests. ( B ) Effect of SLFN14 on HIV-1 replication. ( I ) CD4 and CXCR4 expression. HEK293T cells were co-transfected with plasmids expressing CD4 and a bicistronic plasmid encoding CXCR4 and mCherry, and either, an empty plasmid (control cells) or a human SLFN14 expression plasmid. CD4 was detected by immunostaining and MFI values of CD4 and mCherry (CXCR4) were quantified by flow cytometry. ( II ) These cells were infected with HIV-1 wild-type and viral replication was followed by measuring HIV-1 p24 in the cell supernatant. Data pertain to a triplicate experiment and they are representative of two independent experiments. Statistically significant differences were calculated with one-way ANOVA and Bonferroni post-hoc tests **** p ≤ 0.0001. Figure is created by Valenzuela et al.

Article Snippet: The CXCR4 expression plasmid contains an independent mCherry expression cassette (bicistronic plasmid). pCI Luc contains firefly luciferase cDNA cloned MluI/Xba I in pCI (Promega, Madison, WI, USA). pNLENG1-ES-IRES (a gift of D.N.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Immunostaining, Flow Cytometry, Infection

Journal: PLoS Genetics

Article Title: Curly Encodes Dual Oxidase, Which Acts with Heme Peroxidase Curly Su to Shape the Adult Drosophila Wing

doi: 10.1371/journal.pgen.1005625

Figure Lengend Snippet: (A) Knockdown of cysu , but not the other seven Drosophila heme peroxidases, suppresses the Curly wing phenotype. (B) An endogenously tagged mCherry::Cysu is expressed in the late pupal wing. (C) Knockdown of Cysu in the dorsal wing compartment causes downward cupping and bending of the wing resembling Duox knockdown wings. (D) Knockdown of Cysu with apGal4 in a Cy K background causes defects in scutellum and notum formation. (E) Cysu::mCherry is expressed in the thorax of control flies, but not those expressing cysu RNAi with apGal4.

Article Snippet: mCherry-tagged Cysu expressing flies were made by BestGene by injecting mimic construct #1315 into Mi{MIC}CG5873 MI11428 (BDSC 56608) [ ].

Techniques: Knockdown, Control, Expressing

Journal: PLoS Genetics

Article Title: Curly Encodes Dual Oxidase, Which Acts with Heme Peroxidase Curly Su to Shape the Adult Drosophila Wing

doi: 10.1371/journal.pgen.1005625

Figure Lengend Snippet: Drosophila strains.

Article Snippet: mCherry-tagged Cysu expressing flies were made by BestGene by injecting mimic construct #1315 into Mi{MIC}CG5873 MI11428 (BDSC 56608) [ ].

Techniques: