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: 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