Journal: Immunity

Article Title: The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses

doi: 10.1016/j.immuni.2020.11.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BODIPY FL C5-Lactosylceramide complexed to BSA , Thermo Fisher Scientific , Cat# B34402.

Techniques: Recombinant, Derivative Assay, Enzyme-linked Immunosorbent Assay, Sequencing, CRISPR, Biomarker Discovery, Plasmid Preparation, Retroviral, Expressing, Software, Single-cell Analysis, Pore Size, Saline

Journal: Nature Communications

Article Title: Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity

doi: 10.1038/s41467-023-41326-2

Figure Lengend Snippet: a CRISPR-Cas9 genome editing was used to generate PFKFB3 knockout iPS cells. Immunofluorescence confocal imaging of unedited control (CTR) and PFKFB3 knockout (P206, P208) BVO sections showing pericyte coverage (PDGFRβ, magenta) of CD31 + ECs (green). b Percentage of pericyte coverage CTR n = 6 BVOs, P206 n = 7 BVOs, P208 n = 7 BVOs, from three separate preparations. One-two sections per BVO were assessed. c Quantification of vessel density and ( d ) length in CTR n = 6 BVOs, P206 n = 7 BVOs, P208 n = 7 BVOs, from three separate preparations in four different areas per x10 images. One-two sections per BVO were assessed. Values are presented as mean ± SD using two-way ANOVA followed by Tukey’s multiple comparisons tests. ( b : CTR vs. P206 **** p = 0.000000000522; CTR vs. P208 **** p = 0.000000128; c : * p = 0.0374; CTR vs P206 **** p = 0.000027; CTR vs P208 **** p = 0.000000022; d : CTR vs P206 **** p = 0.000088; CTR vs P208 **** p = 0.000005). ns not significant. Bar scales 200 and 50 μm. Effect of PFKFB3 knockout on 13 C label incorporation into metabolites related to ( e ) glycolysis, ( f ) the TCA cycle and ( g ) glycolytic branch pathways after 3 h of incubation with 13 C 6 -glucose. Data represents mean ± SEM, n = 4 independent experiments, statistical significance was assessed by a two-way ANOVA with Holm-Sidak post-hoc test. ( e : LAC ** p = 0.00618; PEP ** p = 0.00531; DHAP * p = 0.02990; G3P **** p = 0.000000798; FBP * p = 0.02749; F6P * p = 0.04479; G6P* p = 0.00473). G6P glucose 6-phosphate, F6P fructose 6-phosphate, FBP fructose 1,6-bisphosphate, G3P glyceraldehyde 3-phosphate, DHAP dihydroxyacetone phosphate, 1,3-BPG 1,3-bisphosphoglycerate, 2-PG 2-phosphoglycerate, PEP phosphoenolpyruvate, LAC lactate, UDP uridine diphosphate, GlcNAc N -acetylglucosamine, Ru5P ribulose 5-phosphate, R5P ribose 5-phosphate, S7P sedoheptulose-7-phosphate, m + x mass isotopologues x. Source data are provided as a Source Data file.

Article Snippet: For BVOs, a 3 h incubation with 5 mM U- 13 C 6 -glucose (Cambridge Isotope Laboratories) in DMEM without glucose and pyruvate (Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin and 1% l -glutamine was performed.

Techniques: CRISPR, Knock-Out, Immunofluorescence, Imaging, Control, Incubation

Journal: Frontiers in Microbiology

Article Title: Akt/AS160 Signaling Pathway Inhibition Impairs Infection by Decreasing Rab14-Controlled Sphingolipids Delivery to Chlamydial Inclusions

doi: 10.3389/fmicb.2019.00666

Figure Lengend Snippet: Sphingolipids transport to the inclusions is interfered by Akt inhibition. (A) Infected cells (MOI 2) were incubated with DMSO or increasing concentrations of iAkt (2, 5, and 10 μM) from 2 to 24 h pi. Before fixation, cells were incubated for 30 min with Ceramide-BODIPY at 4°C in serum-free DMEM. Then, cells were washed with PBS and incubated with DMEM supplemented with FBS for 30 min at 37°C. Finally, bacteria were detected with anti-MOMP coupled to FITC. (B) Intensity profiles obtained by scanning red (SM) and green ( Ct ) fluorescence along a line that crosses inclusions. (C) In experiments conducted as described in (A) , Golgi apparatus was detected with mouse anti-GM130 antibody followed by anti-mouse Cy5-coupled secondary antibody. Bacteria were detected with anti-MOMP coupled to FITC. DAPI stained nuclei (N) and bacterial DNA. Concentration and localization of sphingolipids are depicted in the bottom panels. Fire scale represents the fluorescence intensity associated to sphingolipids. Golgi apparatus is delimited with a white line. (D) Fluorescence intensity corresponding to sphingolipids at the Golgi apparatus was quantified using ImageJ. Data are representative of three independent experiments ( ∗∗∗ p < 0.001, ∗∗ p < 0.01).

Article Snippet: Infected cells grown on 12-mm-diameter glass coverslips in 24-well plates were treated with iAkt for the indicated periods of time; and before its fixation, cells were incubated with 5 μM BODIPY TR ceramide-BSA complex in DMEM or BODIPY FL ceramide-BSA (Molecular Probes, United States) for 30 min at 4°C, then washed 3 times with PBS, and finally, maintained in DMEM without FBS for 30 min at 37°C.

Techniques: Inhibition, Infection, Incubation, Bacteria, Fluorescence, Staining, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Akt/AS160 Signaling Pathway Inhibition Impairs Infection by Decreasing Rab14-Controlled Sphingolipids Delivery to Chlamydial Inclusions

doi: 10.3389/fmicb.2019.00666

Figure Lengend Snippet: Disruption of the Akt/AS160 pathway causes retention of lipids at the Golgi apparatus. (A) Equal amounts of lipids extracted from infected cells incubated with DMSO, 2 μM or 10 μM iAkt were resolved by thin layer chromatography (TLC). A representative TLC from two independent experiments is shown. (B) HeLa cells were transfected with either siRNA-Luc or siRNA-AS160 and, 48 h later, infected with GFP-expressing C. trachomatis (MOI of 1). AS160-silenced and control cells were incubated with DMSO or increasing concentrations of iAkt (2 or 10 μM). After 24 h of infection and before fixation, cells were incubated for 30 min with BODIPY TR ceramide at 4°C in serum-free DMEM. Then, cells were washed with PBS and incubated with DMEM supplemented with FBS for 30 min at 37°C. Golgi apparatus was immunodetected in fixed cells with mouse anti-GM130 monoclonal antibody followed by Cy5 coupled anti-mouse secondary antibody. Bars show the average fluorescence intensity of lipids at the Golgi apparatus. ImageJ was used for this analysis. (C) Representative images of two independent experiments performed as indicated in (B) . In the upper panels, subcellular distribution of sphigolipids appears in red. GFP expressing bacteria are shown in green. Golgi was stained with mouse anti-GM130 followed by anti-mouse Cy5-coupled secondary antibody (white). In the lower panel, fire scale represents the fluorescence intensity associated to sphingolipids. White lines demarcate Golgi structures. Nuclei were stained with DAPI. Asterisks indicate inclusions.

Article Snippet: Infected cells grown on 12-mm-diameter glass coverslips in 24-well plates were treated with iAkt for the indicated periods of time; and before its fixation, cells were incubated with 5 μM BODIPY TR ceramide-BSA complex in DMEM or BODIPY FL ceramide-BSA (Molecular Probes, United States) for 30 min at 4°C, then washed 3 times with PBS, and finally, maintained in DMEM without FBS for 30 min at 37°C.

Techniques: Disruption, Infection, Incubation, Thin Layer Chromatography, Transfection, Expressing, Control, Fluorescence, Bacteria, Staining