Journal: Nature Communications

Article Title: PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

doi: 10.1038/ncomms7692

Figure Lengend Snippet: ( a ) CPT1A mRNA was analysed by real-time quantitative PCR and relative expression of mRNA of each time point and culture condition over the levels expressed in unstimulated cells (defined as 1) is shown (* P <0.05, T CC+PD1 versus T CC , Student’s t -test; ♦ P <0.05, T CC+PD1 versus UT and T CC versus UT, analysis of variance (ANOVA); n =3 experiments). ( b ) CD4 + primary human T cells were cultured under the indicated conditions. Cell lysates were prepared at the indicated time points and expression of CPT1A and β-actin were assessed by SDS–PAGE and immunoblot. Results are representative of three experiments. ( c ) Fatty acid β-oxidation rate after culture under the indicated conditions for various time intervals was examined. Values of T CC and T CC+PD1 cells were compared with unstimulated (UT) cells ( ♦ P <0.05, ANOVA; n =3) and values of T CC+PD1 were compared with T CC cells (* P <0.05, Student’s t -test; n =3).

Article Snippet: The anti-FASN (#3189) was from Cell Signaling Technologies, Danvers, MA, and the anti-CPT1A (#TA303144) antibody was from OriGene Technologies Inc., Rockville, MD.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, SDS Page, Western Blot

Journal: Nature Communications

Article Title: PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

doi: 10.1038/ncomms7692

Figure Lengend Snippet: ( a ) CD4 + primary human T cells were cultured with tosyl-activated magnetic beads conjugated with aCD3/aCD28/IgG (T CC ) in the presence of either LY294002 (LY, 10 μM), UO126 (UO, 10 μM) or their combination. Cell lysates were prepared at the indicated time points and expression of CPT1A and β-actin was assessed by SDS–PAGE and immunoblot. Results are representative of three experiments. ( b – d ) In parallel experiments, rate of fatty acid β-oxidation was examined. Values of T CC +inhibitor cultures were compared with T CC (* P <0.05, Student’s t -test; n =3) and values of T CC and T CC +inhibitor cultures were compared with unstimulated (UT) ( ♦ P <0.05, analysis of variance (ANOVA); n =3). ( e ) At 72 h of culture under the indicated conditions, spare respiratory capacity (SRC) was determined. Values of T CC and T CC +inhibitor cells were compared with unstimulated (UT) cells ( ♦ P <0.05, ANOVA; n =3) and values in T CC +inhibitor cells were compared with T CC (* P <0.05, Student’s t -test; n =3). ( f ) T cells were cultured with tosyl-activated magnetic beads conjugated with aCD3/aCD28/PD-L1-Ig or with tosylatcivated magnetic beads conjugated with aCD3/aCD28/IgG in the presence of either LY294002 (LY, 10 μM), UO126 (UO, 10 μM) or their combination. Cell lysates were prepared at the indicated time points and expression of ATGL and β-actin was assessed by SDS–PAGE and immunoblot.

Article Snippet: The anti-FASN (#3189) was from Cell Signaling Technologies, Danvers, MA, and the anti-CPT1A (#TA303144) antibody was from OriGene Technologies Inc., Rockville, MD.

Techniques: Cell Culture, Magnetic Beads, Expressing, SDS Page, Western Blot

Journal: Nature Communications

Article Title: PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

doi: 10.1038/ncomms7692

Figure Lengend Snippet: CD4 + primary human T cells were either left unstimulated (UT) or were incubated with magnetic beads conjugated with αCD3/αCD28/IgG (T cells co-stimulated (T CC )) or magnetic beads conjugated with αCD3/αCD28/αCTLA-4 mAbs (T cells co-stimulated+ CTLA-4 (T CC+CTLA4 )). ( a ) Expression of Glut1 after culture under the indicated conditions and time intervals was examined by flow cytometry. Results are representative of three experiments. ( b – f ) mRNA for HK2, SNAT1, SNAT2, CPT1A and ATGL was analysed by real-time quantitative PCR and relative expression of mRNA of each time point and culture condition over the levels expressed in unstimulated cells (defined as 1) is shown. ( g ) Fatty acid β-oxidation rate after culture under the indicated conditions for various time intervals was examined. ( h , i ) Analysis of lactate ( h ) and 3-hyroxybutyrate ( i ), end metabolites of glycolysis and fatty acid β-oxidation, respectively, was performed in culture supernatants. (For the studies shown in all panels: * P <0.05, T CC+CTLA4 versus T CC , Student’s t -test; ♦ P <0.05, T CC versus UT and T CC+CTLA4 versus UT, analysis of variance; n =3 experiments).

Article Snippet: The anti-FASN (#3189) was from Cell Signaling Technologies, Danvers, MA, and the anti-CPT1A (#TA303144) antibody was from OriGene Technologies Inc., Rockville, MD.

Techniques: Incubation, Magnetic Beads, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

doi: 10.1038/ncomms7692

Figure Lengend Snippet: CD4 + human T cells were pre-activated with anti-CD3-and-anti-CD28 mAbs for 4 h or for 24 h and subsequently were collected, were left to rest for 3 h and re-cultured with tosyl-activated magnetic beads conjugated with αCD3/αCD28/PD-L1-Ig (Tpr4hr→PD-1 and Tpr24hr→PD-1). In the same experiment CD4 + T cells without pre-activation were stimulated with tosyl-activated magnetic beads conjugated with αCD3/αCD28/IgG (T CC ) and used as positive control for optimal stimulation without PD-1. ( a ) Expression of Glut1 after culture under the indicated conditions and time intervals was examined by flow cytometry. ( b , c ) mRNA for HK2 and CPT1A was analysed by real-time quantitative PCR and relative expression of mRNA of each time point and culture condition over the levels expressed in unstimulated cells (defined as 1) is shown. ( d ) Fatty acid β-oxidation rate after culture under the indicated conditions for various time intervals was examined. ( e , f ) Analysis of lactate ( e ) and 3-hydroxybutyrate ( f ), end metabolites of glycolysis and fatty acid β-oxidation, respectively, was performed in culture supernatants (for the studies shown in all panels: * P <0.05, Tpr4hr→PD-1 versus T CC or Tpr4hr→PD-1 versus T CC , Student’s t -test; ♦ P <0.05, T CC versus UT, Tpr4hr→PD-1 versus UT and Tpr4hr→PD-1 versus UT, analysis of variance; n =3 experiments).

Article Snippet: The anti-FASN (#3189) was from Cell Signaling Technologies, Danvers, MA, and the anti-CPT1A (#TA303144) antibody was from OriGene Technologies Inc., Rockville, MD.

Techniques: Cell Culture, Magnetic Beads, Activation Assay, Positive Control, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Medicine

Article Title: Mesoporous Silica-Based Nanoparticles as Non-Viral Gene Delivery Platform for Treating Retinitis Pigmentosa

doi: 10.3390/jcm11082170

Figure Lengend Snippet: Transfection efficacy of PRPF31 gene using the nanoparticle delivery system PRPF31 -GFP/N-MSiNPs in a human cell line. Panels ( a – g ), immunofluorescence images of HEK-293 cells transfected with N-MSiNPs loaded with the PRPF31 -GFP plasmid. GFP fluorescent signal ( a , d ), in green; anti-PRPF31 fluorescent signal ( e ), in red. Cell nuclei were stained with DAPI ( b , f ), in blue. Merged images of the different channels are shown in ( c , g ). ( h ) WB of HEK-293 transfected cells showing the expression levels of the PRPF31 transgene fused to GFP. Protein extracts of HEK-293 treated with only Lipofectamine (negative control), in lane 1; from cells transfected with PRPF31 -GFP using Lipofectamine, in lane 2; from cells incubated with empty N-MSiNPs, in lane 3; and from cells transfected with N-MSiNPs loaded with PRPF31 -GFP, in lane 4. WBs were performed with anti-GFP (upper panel) and anti-PRPF31 (central panel) antibodies. GAPDH was used as a loading control (bottom panel). The (*) in the upper and central panels marks the expected molecular weight (80 kDa) of the PRPF31-GFP fused protein visualized by anti-GFP and ant-PRPF31 antibodies. The (#) in the central panel marks the expected molecular weight (55 kDa) of the endogenous PRPF31 protein. Scale bars represent 25 and 50 µm.

Article Snippet: Incubation with primary antibodies anti-PRPF31 (1:100; OriGene Technologies Inc., TA302582) and anti-GFP (1:200; Cell signaling 2956) was performed for 1 h at room temperature.

Techniques: Transfection, Immunofluorescence, Plasmid Preparation, Staining, Expressing, Negative Control, Incubation, Molecular Weight

Journal: Journal of Clinical Medicine

Article Title: Mesoporous Silica-Based Nanoparticles as Non-Viral Gene Delivery Platform for Treating Retinitis Pigmentosa

doi: 10.3390/jcm11082170

Figure Lengend Snippet: Fundus images and the retinal section of PRPF31 -GFP/N-MSiNP subretinally injected mice. ( a ) Fundus image. ( b ) Fluorescence fundus. ( c ) Detail of the injection site showing GFP fluorescent dots. Immunostaining of retinal sections for GFP ( d ), in green; PRPF31 ( e ), in red. Nuclei stained with DAPI ( f ), in blue. ( g ) Merged images of the different channels. GFP and PRPF31 signal co-localized mainly in RPE cells. RPE = retinal pigment epithelium, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Scale bar represents 50 µm.

Article Snippet: Incubation with primary antibodies anti-PRPF31 (1:100; OriGene Technologies Inc., TA302582) and anti-GFP (1:200; Cell signaling 2956) was performed for 1 h at room temperature.

Techniques: Injection, Fluorescence, Immunostaining, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae

doi: 10.3389/fcimb.2022.921410

Figure Lengend Snippet: Dot blot using anti-GP63 (upper row) confirmed the presence of this GPI-anchored extracellular vesicle (EV) marker in EVs of both species and dot blot using anti-GFP (lower row) confirmed the presence of this cytosolic EV marker within EVs of both species. (A, F) L. tarentolae GFP+ extract as a positive control for tEV (35 µg protein in total). (B, G) L. tarentolae GFP+ EVs or tEV (1 µg in total). (C, H) L. major GFP+ extract as a positive control for mEV (35 µg in total). (D, I) L. major GFP+ EVs or mEV (1 µg in total). (E, J) Negative control (phosphate-buffered saline). The middle row depicts a separate dot blot using normal mouse sera as the primary antibody, which serves as a negative control. (a) L. tarentolae GFP+ extract (same as A, F ). (b) L. tarentolae GFP+ EVs or tEV (similar to B, G ). (c) L. major GFP+ extract (same as C, H ). (d) L. major GFP+ EVs or mEV (similar to D, I ). PC, positive control; tEV, L. tarentolae EV; mEV, L. major EV; NC, negative control.

Article Snippet: The GP63 antibody was provided by the Pasteur Institute of Iran, the anti-GFP-HRP goat polyclonal antibody was obtained from Acris antibodies GmbH (Germany), and the beta actin monoclonal antibody was purchased from ProteinTech Group Inc. (Chicago, IL, USA).

Techniques: Dot Blot, Marker, Positive Control, Negative Control

Journal: Molecular Psychiatry

Article Title: Inhibition of colony stimulating factor 1 receptor corrects maternal inflammation-induced microglial and synaptic dysfunction and behavioral abnormalities

doi: 10.1038/s41380-020-0671-2

Figure Lengend Snippet: a Gene ontology (GO) biological processes pathway analysis shows that MIA microglia increase synaptogenic functions while repopulated microglia recover homeostatic functions. Left (red): Top significantly enriched GO biological process terms increased by MIA and decreased by repopulation. Right (purple): Top significantly enriched GO biological process terms decreased by MIA and increased by repopulation. These GO findings were verified using GORILLA. b IPA of genes with differential expression in microglia between MIA versus Saline (RNA-seq data). Pathway analysis reveals MIA-induced upregulation of neuritogenic gene expression, specifically in developmental stages, based on activation z -score. Red denotes pathway activated in E17 MIA microglia. c Genes in “neuritogenesis/formation of cellular protrusions” function. Hierarchal clustering of gene sets based on relative expression values; red: high relative expression, blue: low relative expression. Cluster 1 represents genes increased in adult MIA microglia but reduced in MIA + MG-REP including Ctnnd2, Ncam2, and Ntrk2 . Cluster 2 represents genes increased in immature MIA microglia including Ncam2, Ntn, Ptn and Wnt5a . Cluster3 represents genes decreased in immature MIA microglia including Plau. In situ hybridization (ISH) and immunofluorescence of E17 Saline or MIA offspring in the cortical plate region. d mRNA of cellular protrusion/ neuritogenic genes ( Ctnnd2, Ncam2, Ntn, Ptn, and Wnt5a ) were detected by florescent-labeled antisense cRNA probes (red) but not by scramble cRNA probe (not detected: N.D.), and the sections were immunostained for IBA1 (green) and DAPI (blue). e The number of IBA1 + cells expressing the cellular protrusion/neuritogenic genes were quantified in the cortical plate region. n = (4–5/2) male mice/ litters per molecule for Saline and MIA, n = 3 for scramble control probe. * p < 0.05, ** p < 0.01, ns denotes no significance, by unpaired Student t test. Graphs indicate mean ± s.e.m. ELISA verification of selected RNA-seq molecules: CTNND2 ( f ), NCAM2 ( g ), NTRK2 ( h ), NTN ( i) , PTN ( j ) and WNT5A ( k ) in acutely isolated microglia. MIA increases protein expression of cellular protrusion/neuriotgenic molecules in microglia that were normalized via repopulation. n = (6/4, 6/3, 5/3, 6/3) female mice/litters for P60 Saline + CTRL, MIA + CTRL, Saline + MG-REP and MIA + MG-REP. CTNND2: Prenatal treatment effect, F (1,19) = 157.1, p < 0.0001, Drug effect, F (1,19) = 262.7, p < 0.0001, Interaction effect, F (1,19) = 201, p < 0.0001, NCAM2: Prenatal treatment effect, F (1,19) = 23.76, p = 0.0001, Drug effect, F (1,19) = 26.29, p < 0.0001, Interaction effect, F (1,19) = 17.63, p = 0.0005, NTRK2: Prenatal treatment effect, F (1,18) = 13.99, p = 0.0015, Drug effect, F (1,18) = 12.45, p = 0.0024, Interaction effect, F (1,18) = 0.06203, p = 0.8061, NTN: Prenatal treatment effect, F (1,19) = 0.01669, p = 0.8986, Drug effect, F (1,19) = 0.8, p = 0.3823, Interaction effect, F (1,19) = 6.121, p = 0.0230, PTN: Prenatal treatment effect, F (1,19) = 10.31, p = 0.0046, Drug effect, F (1,19) = 52.02, p < 0.0001, Interaction effect, F (1,19) = 0.002927 p = 0.9574, WNT5A: Prenatal treatment effect, F (1,19) = 5.581, p = 0.0290, Drug effect, F (1,19) = 1.550, p = 0.2282, Interaction effect, F (1,19) = 0.0834 p = 0.7799, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as determined by 2-way ANOVA (alpha = 0.05) with Tukey’s post-hoc. # p < 0.05 for main effect of MIA. Graphs indicate mean ± s.e.m.

Article Snippet: For the detection of NCAM2, CTNND2 and WNT5A, custom ELISA kits were developed according to the manufacturer’s instruction using anti-NCAM2 goat polyclonal antibody (0.3 μg/well Acris Antibodies GmbH, AP32136PU-N), biotinylated anti-NCAM2 goat polyclonal antibody using Antibody Biotinylation Kit (0.3 μg/ml, Pierce/Thermo Scientific, 90407), anti-CTNND2 mouse monoclonal antibody (0.3 μg/well, Santa Cruz Biotechnology, SC-81793, clone 40.1), biotinylated anti-CTNND2 rabbit antibody (1 μg/ml, Abcam, EPR17628), anti-WNT5A goat polyclonal antibody (0.3 μg/well, R&D Systems, AF645), and biotinylated anti-WNT5A antibody using Antibody Biotinylation Kit (1 μg/ml, Pierce).

Techniques: Expressing, RNA Sequencing Assay, Activation Assay, In Situ Hybridization, Immunofluorescence, Labeling, Enzyme-linked Immunosorbent Assay, Isolation