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

Journal: Cell reports

Article Title: Housing mice near vs. below thermoneutrality affects drug-induced weight loss but does not improve prediction of efficacy in humans

doi: 10.1016/j.celrep.2024.114501

Figure Lengend Snippet: Primer information

Article Snippet: Sema3a , Mm00436469_m1.

Techniques:

Journal: Nature

Article Title: Rewiring the Taste System

doi: 10.1038/nature23299

Figure Lengend Snippet: a , Confocal microscopy images of circumvallate papilla (CV) taste buds from a T1R3::Sema3A mouse demonstrating transgene expression (marked by Flag tag; green) in T1R3-expressing TRCs (red). b, The Sema7A transgene of T2R::Sema7A animals (green) is expressed in a subset of TRCs not overlapping with T1R3 (red). c, Sema 7A (green) in PKD2L1::Sema7A animals is expressed in sour TRCs (marked by CAR4, red).

Article Snippet: Taqman probes were as follows: t1r3 (Mm0167446_g1), t2r5 (Mm0098502_s1), pkd2l1 (Mm00619572_m1), sema3A (Mm01230889_m1), bdnf (Mm04230607_s1), slit2 (Mm01216521_m1), dscaml1 (Mm01174253_m1), pcdhgb7 (Mm04209449_m1), tspan18 (Mm00806028_m1), nrxn3 (Mm04279482_m1), sema 7 A (Mm00441361_m1) and actb (Mm01205647_g1).

Techniques: Confocal Microscopy, Expressing, FLAG-tag

Journal: Nature

Article Title: Rewiring the Taste System

doi: 10.1038/nature23299

Figure Lengend Snippet: The table shows the distribution of geniculate ganglion neurons according to their responses to each of the five basic taste qualities. All responsive neurons are included in the table. Shown are numbers of cells responding to the various stimuli in Sema3A mutant animals, and their littermate controls; all the data are included in Fig. 2 . Note that since bitter-sour tuned neurons reflect the activation of T2Rs (see ref. 21 ), they were grouped in the table and in Fig. 2 .

Article Snippet: Taqman probes were as follows: t1r3 (Mm0167446_g1), t2r5 (Mm0098502_s1), pkd2l1 (Mm00619572_m1), sema3A (Mm01230889_m1), bdnf (Mm04230607_s1), slit2 (Mm01216521_m1), dscaml1 (Mm01174253_m1), pcdhgb7 (Mm04209449_m1), tspan18 (Mm00806028_m1), nrxn3 (Mm04279482_m1), sema 7 A (Mm00441361_m1) and actb (Mm01205647_g1).

Techniques: Mutagenesis, Activation Assay, Control

Journal: Nature

Article Title: Rewiring the Taste System

doi: 10.1038/nature23299

Figure Lengend Snippet: The table shows the distribution of geniculate ganglion neurons according to their responses to each of the five basic taste qualities in T1R3::Sema3A and control animals; shown are numbers of cells responding to the various stimuli. All the data are included in Fig. 3 . Note that as bitter-sour neurons reflect responses of T2Rs (see ref. 21 ) they were grouped.

Article Snippet: Taqman probes were as follows: t1r3 (Mm0167446_g1), t2r5 (Mm0098502_s1), pkd2l1 (Mm00619572_m1), sema3A (Mm01230889_m1), bdnf (Mm04230607_s1), slit2 (Mm01216521_m1), dscaml1 (Mm01174253_m1), pcdhgb7 (Mm04209449_m1), tspan18 (Mm00806028_m1), nrxn3 (Mm04279482_m1), sema 7 A (Mm00441361_m1) and actb (Mm01205647_g1).

Techniques: Control

Journal: PLoS Biology

Article Title: Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

doi: 10.1371/journal.pbio.1001808

Figure Lengend Snippet: (A) Schematic diagram illustrating the anatomy of the hypothalamic-pituitary-gonadal axis in a sagittal view. In rodents, GnRH cell bodies (green circles) are diffusely distributed in the preoptic region and send neuroendocrine axons (green fibers) towards the ME of the hypothalamus, where they release the neurohormone into pituitary portal blood vessels (red arrow) for delivery to the anterior pituitary. At the adenohypophysis, GnRH elicits the secretion of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which stimulate gametogenesis and gonadal-steroid secretion and thus support reproductive function. cc, corpus calosum; ac, anterior comissure; oc, optic chiasma; 3V, third ventricle. (B) Representative dark-field photomicrographs of a coronal section of an adult female rat ME, showing Sema3A mRNA localized using a radioactive probe (bright dots indicating silver grains, top panel). Note the presence of Sema3A mRNA in the capillary zone of the ME (white arrow) and in intrainfundibular capillary loops (arrowhead) containing PV1-immunoreactive fenestrated endothelial cells (right panel, green immunofluorescence), and its relative paucity in the parenchyma. Sema3A mRNA expression is also seen in various nuclei of the mediobasal hypothalamus (MBH) that lie adjacent to the ME but do not contain PV1-immunoreactive blood vessels. V3, third ventricle. Scale bar, 100 µm. (C) PV1-positive cell (PV1-pos) isolation by FACS (schematic diagram and dot plot, top) and real-time PCR analysis of PV1, Sema3A, estrogen receptor alpha (ERα), and ERβ transcripts. (D) Representative immunofluorescence images showing the localization of Sema3A immunoreactivity (green) in coronal sections of the ME of adult female mice. Fenestrated vascular endothelial cells are labeled by the monoclonal antibody MECA32, which binds to mouse PV1 (red). Note that Sema3A immunoreactivity is localized in portal blood capillaries of the external zone of the ME (inset) as well as some intrainfundibular capillary loops present in the nervous parenchyma (arrowhead); Sema3A immunolabelling is of very high intensity at the level of the capillary zone, but is also seen in the adjacent nervous parenchyma, progressively vanishing at deeper levels of the tissue. Nuclei are counterstained in blue using Hoechst. (Insets) High-magnification images of the areas indicated by dashed lines. Black arrowheads at the periphery of the pictures indicate the planes of the individual images that make up the photomontage; each panel is composed of an assembly of four images captured sequentially for each fluorophore using the MosaiX module of the AxioVision 4.6 system (Zeiss, Germany) and a Zeiss 20× objective (N.A. 0.8). Scale bar, 100 µm (30 µm in inset). (E) Detection by RT-PCR of Sema3A mRNA in total RNA extracts from ME explants microdissected from adult female rats and immunopurified ME endothelial cells (ECs). ML, 100 bp molecular ladder; H 2 O, PCR negative control without cDNA; +/−, Sema3A amplicon (395 bp) with (+) or without (−) RT. (F) Western blot analysis of Sema3A protein levels in the adult ME and 48 h ME EC-conditioned medium (EC-CM). Each lane was loaded with 35 µg of protein. While all Sema3A isoforms (65, 95, and 125 kDa) are detected in protein extracts from the adult female ME, only the 65 kDa Sema3A isoform is present in the EC-CM. (G) Western blot (top) and quantitative analysis (bottom, relative to actin) of Sema3A protein levels showing a difference in 65 kDa Sema3A expression between the afternoon of diestrus (Di16h) and proestrus (Pro16h), whereas the protein levels of 125 and 95 kDa Sema3A remain unchanged. Band intensity was quantified using Scion software. ** t (8) = 4.709, p = 0.0015 ( n = 5 independent experiments). (H) Western blot analysis for Sema3A (top; upper image) and actin (top; lower image) in the ME of control ovariectomized (OVX) female rats and those treated with 17β-estradiol 3-benzoate (E2), progesterone (P), or E2+P ( n = 5 independent experiments per treatment). E2 induces a significant increase in 65 kDa Sema3A expression in the ME of OVX rats when compared with the other treatment groups (one-way ANOVA, F (8,11) = 27.779, p <0.001; Tukey's test, *** p <0.001), whereas progesterone inhibits this increase (Tukey's test, p = 0.003). Bar graph, mean ratio (±SEM) of Sema3A expression to that of actin ( p <0.05, one-way ANOVA). Data are represented as means ± SEM.

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): PV1 (PV1_Rn00571706_m1), Sema3A (Sema3A_Rn00436469_m1), ERα (Esr1_Rn01640372_m1), ERβ (Esr2_Rn00562610_m1), and housekeeping ribosomal RNA (Rn45_Rn03928990_g1).

Techniques: Immunofluorescence, Expressing, Isolation, Real-time Polymerase Chain Reaction, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Amplification, Western Blot, Software, Control

Journal: PLoS Biology

Article Title: Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

doi: 10.1371/journal.pbio.1001808

Figure Lengend Snippet: (A) Representative electron micrographs of GnRH-immunoreactive axon terminals (green) from diestrous female rat hypothalamic explants containing the ME, incubated for 30 min in the presence (right panel) or absence (left panel) of Sema3A. Under basal unstimulated conditions (left panel), GnRH nerve endings (n, arrowhead, green) are distant from the pericapillary space (p.s., pink). Sema3A treatment (right panel) causes GnRH axon terminals to advance towards the pericapillary space (p.s., pink), from which they remain separated by only a few nanometers (arrows). Cap, pituitary portal blood capillaries. Scale bar, 1 µm. (B) Quantitative analysis of the percentage of GnRH nerve terminals located less than 1 µm from the pericapillary space in the external zone of the ME, in explants from diestrous (left panel) and proestrous (right panel) rats treated with Sema3A, a Nrp1-neutralizing antibody (Nrp1-Ab), a Sema3A-neutralizing antibody (Sema3A-Ab), both Nrp1-Ab and Sema3A, and in controls. (Left panel) One-way ANOVA, F (2,11) = 54.875, p <0.001. (Right panel) One-way ANOVA, F (3,12) = 37.093, p <0.001. Tukey's test, *** p <0.001, ** p <0.01 for pairs of groups as indicated; n = 3–4 animals per group. (C) Genetic strategy to invalidate Nrp1 expression specifically in GnRH-expressing cells in mice. (D) Immunofluorescence analysis of coronal brain sections from adult female Nrp1 loxP/loxP (left) and GnRH:: Cre; Nrp1 loxP/loxP (right) littermates using antibodies to GnRH (green) and Nrp1 (red). Note the markedly reduced Nrp1 immunoreactivity in the dorsolateral part of the ME, where most GnRH axon fibers occur, in GnRH:: Cre; Nrp1 loxP/loxP mice (asterisk) when compared to Nrp1 loxP/loxP animals (arrow), confirming the efficient ablation of Nrp1 in GnRH neurons of the former. Scale bar, 50 µm. (E) Quantitative analysis of the percentage of GnRH nerve terminals located less than 1 µm from the pericapillary space in the external zone of the ME in explants from control Nrp1 loxP/loxP and GnRH:: Cre; Nrp1 loxP/loxP mice and those treated with Sema3A. One-way ANOVA, F (3,15) = 9.894, p = 0.0015. Tukey's test, * p <0.05; n = 3–4 animals per group. Data are represented as means ± SEM.

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): PV1 (PV1_Rn00571706_m1), Sema3A (Sema3A_Rn00436469_m1), ERα (Esr1_Rn01640372_m1), ERβ (Esr2_Rn00562610_m1), and housekeeping ribosomal RNA (Rn45_Rn03928990_g1).

Techniques: Incubation, Expressing, Immunofluorescence, Control

Journal: PLoS Biology

Article Title: Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

doi: 10.1371/journal.pbio.1001808

Figure Lengend Snippet: (A) Schematic representation of a sagittal view of a mouse embryo at E12.5, showing the distribution of GnRH neurons (green dots) within the head. Primary cultures were performed from microdissected nasal compartment (NC) explants, which contain most GnRH neurons at this embryonic stage. FB, forebrain; OE, olfactory epithelium; VNO, vomeronasal organ. (B) Representative images showing the binding of the Sema3A-neutralizing antibody (red) to cultured cells surrounding GFP-expressing GnRH neurons (green). (C) Representative images showing the morphology of cultured GnRH neurons under control conditions and after treatment with the Sema3A-neutralizing antibody and bar graph quantifying the mean length of their axon-like processes (control, N = 3 independent experiments, n = 146 cells; Sema3A-Ab, N = 3 independent experiments, n = 143 cells). Total number of cultures, 24 from 4 litters. Data are represented as means ± SEM. Unpaired Student's t test, t (285) = 4.823, p <0.0001.

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): PV1 (PV1_Rn00571706_m1), Sema3A (Sema3A_Rn00436469_m1), ERα (Esr1_Rn01640372_m1), ERβ (Esr2_Rn00562610_m1), and housekeeping ribosomal RNA (Rn45_Rn03928990_g1).

Techniques: Binding Assay, Cell Culture, Expressing, Control

Journal: PLoS Biology

Article Title: Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

doi: 10.1371/journal.pbio.1001808

Figure Lengend Snippet: (A) Three-dimensional matrix assays using co-cultures of ME explants dissected from adult female rats (control, n = 3; Nrp1-Ab, n = 4) and cell aggregates of immortalized GnV-3 cells, in the absence (top panels) or presence (bottom panels) of an Nrp1-neutralizing antibody (Nrp1-Ab). Co-cultures were fixed and stained with Alexa 588–X phalloidin. GnV-3 cell aggregates show neurite extension under control conditions, whereas neurite sprouting is strongly inhibited by Nrp1-Ab. White arrowheads in the left panels indicate the merging point of the two individual images composing each picture. (B) Quantitative analysis of the area covered by phalloidin staining surrounding the aggregates (top panel; n = 3 in controls, n = 4 in Nrp1-Ab-treated aggregates; unpaired Student's t test, t (5) = 7.424, p <0.001) and GnV-3 neurite length (bottom panel; n = 3 in controls, n = 4 in Nrp1-Ab-treated aggregates; unpaired Student's t test, t (5) = 5.610, p <0.005), respectively. (C) Co-cultures of GnV-3 cell aggregates placed around aggregates of COS-7 cells transfected with full-length (95 kDa) Sema3A (Sema3A-FL), 65 kDa Sema3A (p65-Sema3A), or the control vector ( n = 7), in the presence (Sema3A-FL+Nrp1-Ab, n = 4; p65-Sema3A+Nrp1-Ab, n = 6) or absence of Nrp1-Ab (Sema3A-FL, n = 4; p65-Sema3A, n = 4), as shown in the schematic drawing. Sema3A-FL and p65-Sema3A are equally effective at inducing GnV-3 neurite growth when compared to control conditions (middle panels), while neurite growth is prevented by the Nrp1-neutralizing antibody (right panels). (D) Quantitative analysis of GnV-3 neurite length (one-way ANOVA with Tukey's post hoc test, F (4,24) = 38.058, p <0.0001). Data are represented as means ± SEM. Scale bars, 100 µm in (A), 50 µm in (C).

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): PV1 (PV1_Rn00571706_m1), Sema3A (Sema3A_Rn00436469_m1), ERα (Esr1_Rn01640372_m1), ERβ (Esr2_Rn00562610_m1), and housekeeping ribosomal RNA (Rn45_Rn03928990_g1).

Techniques: Control, Staining, Transfection, Plasmid Preparation

Journal: PLoS Biology

Article Title: Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

doi: 10.1371/journal.pbio.1001808

Figure Lengend Snippet: (A, Upper panel) Schematic diagram representing the stereotaxic implantation of a 28 gauge infusion cannula connected to a subcutaneously implanted mini-osmotic pump in the ME of cycling female rats, for the delivery of Nrp1- or Sema3A-neutralizing antibodies (0.2 µg/µl, 0.5 µl/h). (Lower panel) Representative estrous cycle profiles showing the disruption of estrous cyclicity by the infusion of Nrp1-Ab but not of PBS into the ME. Infusion was started on day 0 (downward arrow) and ended 7 d later (upward arrow), when the pump contents were exhausted. Di, diestrus; Pro, proestrus; Es, estrus. (B) Quantitative analysis of alterations in ovarian cyclicity (number of days in each phase) caused by Nrp1-Ab or Sema3A-Ab infusion ( n = 6 animals in the PBS and Nrp1 groups; n = 5 animals in the Sema3A group). Diestrus, two-way repeated-measures ANOVA, F (14,33) = 19.073, p <0.001, Tukey's test, ** p = 0.003 and p = 0.005 between before and after infusion within Nrp-1- and Sema3A-treated groups, respectively. Proestrus, two-way repeated measures ANOVA, F (14,33) = 31.119, p <0.001, Tukey's test, *** p <0.001 and ** p = 0.002 between before and after infusion within the Nrp-1- and Sema3A-treated groups, respectively. Estrus, two-way repeated measures ANOVA, F (14,33) = 0.084, p = 0.776. Data represented as means ± SEM.

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): PV1 (PV1_Rn00571706_m1), Sema3A (Sema3A_Rn00436469_m1), ERα (Esr1_Rn01640372_m1), ERβ (Esr2_Rn00562610_m1), and housekeeping ribosomal RNA (Rn45_Rn03928990_g1).

Techniques: Disruption

Journal: PLoS Biology

Article Title: Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

doi: 10.1371/journal.pbio.1001808

Figure Lengend Snippet: (A) Representative image showing Tomato expression (red) in the capillary zone (asterisk) onto which GnRH axon terminals abut (green) in the ME of tdTomato loxP/+ mice into which the TAT-Cre recombinant protein was injected intravenously (i.v.). Note that Tomato is also expressed in tanycytes (arrowhead), whose cell bodies line the floor of the third ventricle (3V) but that do not express Sema3A mRNA (see ). (B) Quantitative RT-PCR analysis of Sema3A mRNA expression in the ME, t (13) = 3.372, ** p = 0.005, and in the adjacent mediobasal hypothalamus (MBH), t (10) = −0.287, p = 0.780, in Sema3a loxP/loxP mice treated i.v. with vehicle ( n = 7) or TAT-Cre ( n = 8 and 5, respectively). (C) Preovulatory LH levels in TAT-Cre ( n = 8) or vehicle-injected ( n = 7) Sema3a loxP/loxP mice, t (13) = 2.188, * p = 0.048. Data are represented as means ± SEM.

Article Snippet: Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystems): PV1 (PV1_Rn00571706_m1), Sema3A (Sema3A_Rn00436469_m1), ERα (Esr1_Rn01640372_m1), ERβ (Esr2_Rn00562610_m1), and housekeeping ribosomal RNA (Rn45_Rn03928990_g1).

Techniques: Expressing, Recombinant, Injection, Quantitative RT-PCR