Journal: Frontiers in Plant Science

Article Title: DspA/E Contributes to Apoplastic Accumulation of ROS in Non-host A. thaliana

doi: 10.3389/fpls.2016.00545

Figure Lengend Snippet: Erwinia amylovora induces accumulation of H 2 O 2 . (A–C) Leaves of 5-weeks-old plants were infiltrated on the upper half of the leaf blade with water (mock), E. amylovora ( Ea ) wild-type strain, T3SS-deficient mutant ( tts ) or dspA/E mutant ( dspA/E ). (A) Leaves stained with DCFH-DA show intracellular H 2 O 2 accumulation in green. (B) Brown stains indicate accumulation of H 2 O 2 in leaves stained with DAB, the graph to the right shows DAB staining intensity. The stars indicate a significant difference according to Mann and Whitney’s statistical test ( p < 0.05). (C) Localization of H 2 O 2 deposits in DAB-stained leaves observed under a photon microscope. Bar: 50 μm; pl: plastid; pe: staining at the periphery of the cell. Similar results were obtained for three independent experiments. Top panel: palissadic parenchyma; bottom panel: spongy parenchyma.

Article Snippet: Cut samples were collected on electron microscope grids (200 mesh) for further observation on a Zeiss EM912 OMEGA electron microscope at a magnification of 10000 to 50000 X.

Techniques: Mutagenesis, Staining, Microscopy

Journal: Human Molecular Genetics

Article Title: Autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of Fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type 2I

doi: 10.1093/hmg/ddx252

Figure Lengend Snippet: Exosomes characterization. Nanosight Tracking Analysis (NTA) of exosomes derived from MOI 20 infected MDC1C CD133+ blood-derived cells ( A ) and FKRP L276I KI satellite cells ( B ). Plots represent the average value of three recordings performed for each sample. Size and particle distribution (concentration) are reported. FACS immunophenotyping of MOI 20 transduced MDC1C CD133+ stem cells- ( C ) and MOI 20 transduced FKRP L276I KI satellite cells- released exosomes ( D ). Each study included at least 100,000 events. 92,2% of CD133+-derived exosomes were CD63+ and 82,5% were CD81+; 88% of SCs-derived exosomes were CD63+ and 89% CD81+. Magnified view of Transmission Electron Microscopy (TEM) micrograph of exosomes released from transduced MOI 20 MDC1C CD133+ blood-derived stem cells (mean value 92,7 nm ± 39,5 nm SD) ( E ) and FKRP L276I KI satellite cells (mean value 109 nm ± 46,3 nm SD) ( F ). Exosome diameters were measured using NIH ImageJ software. Scale bar, 200 nm. Western blot analysis of exosomal TSG-101, AGO2 and FKRP proteins extracted from MOI 20 transduced MDC1C CD133+ stem cells- (MOI 20 INF MDC1C CD133+ EXOs, lane 2) and MOI 20 FKRP L276I KI satellite cells- (MOI 20 INF L276I KI FKRP EXOs, lane 4) derived exosomes. MOI 20 infected CD133+ blood-derived cell lysate (MOI 20 INF MDC1C CD133+, lane 1) and MOI 20 infected satellite cells lysate (MOI 20 INF L276I KI FKRP, lane 3) were loaded as positive controls.

Article Snippet: Anti-TSG 101 (1:200) (Abcam; Cambridge, UK), anti-Ago2 (1:200) (Proteintech, Europe; Manchester, UK), and anti FKRP-STEM (1:600) antibodies were incubated overnight at 4 °C in the blocking solution or, alternatively, exploiting the Signal-boost Immunoreaction Enhancer Kit (Calbiochem; Inalco, Italy), when the enhancement of the signal was required.

Techniques: Derivative Assay, Infection, Concentration Assay, Transmission Assay, Electron Microscopy, Software, Western Blot

Journal: Human Molecular Genetics

Article Title: Autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of Fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type 2I

doi: 10.1093/hmg/ddx252

Figure Lengend Snippet: Western blot analysis of plasma- derived exosomes. Exosomes were isolated from the plasma of 12 months- old FKRP L276I KI mice injected with 35 × 10 3 (lane 35K), 75 × 10 3 (lane 75K), 100 × 10 3 (lane 100K) FKRP L276I KI SCs transduced cells. Plasma was collected immediately before the animal sacrifice. Exosomes were evaluated for the expression of FKRP protein and TSG-101 exosomal marker expression. Plasma-derived exosomes isolated from not injected mice were exploited as control (lane NI) ( A ). TEM image representative of plasma exosomes morphology (mean size 90 nm ± 36,6 nm SD). Scale bar: 200nm ( B ). NTA graph representative of plasma-derived exosomes concentration and dimensions ( C ). Immunoblotting analysis of FKRP L276I KI engineered SCs-injected mice hearts. FKRP expression ( D ) and α-DG glycosylation ( E ) were evaluated in N = 4 mice transplanted with 35 × 10 3 (lanes #1, #2, #3, #4), N = 5 with 75 × 10 3 (lanes #5, #6, #7, #8, #9), and N = 4 with 100 × 10 3 (lanes #10, #11, #12, #13) engineered SCs. Not injected FKRP L276I KI heart muscle was used as negative control (lane NI). C57BL heart muscle was exploited as positive control (lane CTR+) (D, E).

Article Snippet: Anti-TSG 101 (1:200) (Abcam; Cambridge, UK), anti-Ago2 (1:200) (Proteintech, Europe; Manchester, UK), and anti FKRP-STEM (1:600) antibodies were incubated overnight at 4 °C in the blocking solution or, alternatively, exploiting the Signal-boost Immunoreaction Enhancer Kit (Calbiochem; Inalco, Italy), when the enhancement of the signal was required.

Techniques: Western Blot, Derivative Assay, Isolation, Injection, Expressing, Marker, Concentration Assay, Negative Control, Positive Control