Detection of microvesicles and lipid particles in the extracellular environment and analysis of lysosomal exocytosis.

<p>(<b>A</b>) Statistical histogram showing lipid particle counting by NR-FL2 in the extracellular environment for each experimental condition. Each value is expressed as an absolute number ± SD (Results from n ≥ 3 independent experiments); *<i>P</i> < 0.05 <i>vs</i> respective control. Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP starved cells. (<b>B</b>) Statistical histogram showing CD63⁺ events counted in the extracellular environment for each experimental condition. Each value is expressed as an absolute number ± SD (Results from n ≥ 3 independent experiments). (<b>C</b>) Extracellular microparticle/microvesicle detection by FC. (<b>a</b>) Dot plot FSC <i>vs</i> SSC in logarithmic visualization. P1: viable cells; P2: Dako CytoCount counting beads 5.2 μm diameter; P3: beads 1 μm diameter; P4: beads 0.5 μm diameter; (<b>b</b>) Density plot FSC <i>vs</i> FL1 of the negative sample; (<b>c</b>) Density plot FSC <i>vs</i> FL1-CD63 of starved and (<b>d</b>) rapamycin-treated tWT cells. (<b>e</b>) Density plot FSC <i>vs</i> FL1-CD63 of starved and (<b>f</b>) rapamycin-treated tNP cells. (<b>D</b>) Statistical histogram of surface Lamp-1/CD107a detection on fresh cells: MFI values shown are for each experimental condition and from both cell lines. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *<i>P</i> < 0.05 and **<i>P</i> < 0.01 <i>vs</i> respective control. Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP basal condition and a <i>P</i> value < 0.001 (⁺⁺⁺<i>P</i> < 0.001) between tWT and tNP starved cells.</p

Evaluation of intracellular lipid content by Nile Red (NR).

<p>(<b>A</b>) Single confocal optical sections (~0.8 μm thickness) showing overlay of yellow (FL2) and red (FL3) NR fluorescence from all experimental conditions in tWT and tNP cells. Bars: 10 μm. (<b>B</b>) Statistical histogram of FL2 MFI variation of intracellular NR in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); **<i>P</i> < 0.01 <i>vs</i> respective control.</p

Autophagic Vacuole (AV) detection and endocytic compartment evaluation.

<p>(<b>A</b>) Acidic vesicular organelle (AVOs) detection by LysoTracker Green (LTG) in flow cytometry. (<b>i</b>) Statistical histogram of MFI variation of LTG in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments). The difference between cell lines was determined to be significant by two-way ANOVA (***<i>P</i> < 0.001). Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.01 (⁺⁺<i>P</i> < 0.01) between tWT and tNP starved cells (<b>ii</b>) Flow cytometry histogram overlay showing lysotracker green MFI values of tWT and tNP basal condition. (<b>iii</b>) Fold increase related to histogram in (<b>i)</b> starved and rapamycin-treated cells in both cell lines, <i>versus</i> respective control. (<b>B</b>) Autophagic vacuole detection by Monodansylcadaverine (MDC) in fluorescence microscopy and flow cytometry. (<b>i</b>) Microscopy images showing monodansylcadaverine fluorescence from control and starved tWT and tNP cells. White arrows indicate cells rich in AV. Bars: 10μm. (<b>ii</b>) Statistical histogram depicting MFI variation of monodansylcadaverine in tWT and tNP cells for control, starved and rapamycin-treated cells. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments). Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP in basal condition and rapamycin treated and a <i>P</i> value < 0.01 (⁺⁺<i>P</i> < 0.01) between tWT and tNP after starvation treatment (<b>iii</b>) Monodansylcadaverine flow cytometric histograms are overlaid to show the comparison among MFI values of basal condition, starved and rapamycin-treated samples in tWT (upper panel) and tNP (lower panel) lymphocytes. (<b>C</b>) TEM autophagic vacuole detection in control condition of tWT (<b>a</b>, <b>b</b>) and tNP (<b>c-g</b>) cells. Black arrows indicate AVs. Bars: 2 μm for a, b, c, d, f, g; 0.5 μm for e. (<b>D</b>) TEM autophagic vacuole detection in normal (<b>a</b>, <b>e</b>) and NP-B B lymphocytes (<b>b</b>, <b>c</b>, <b>d</b>, <b>f</b>), after nutrient deprivation (<b>e</b>, <b>f</b>) and rapamycin administration (<b>a-d</b>). Black arrows indicate AVs. Black arrowhead indicates a lipid droplet inside AV. Mvb: multivesicular bodies. Bars: 1 μm for a, f; 2 μm for b, c, d, e. (<b>E</b>) Double- and single- membrane autophagic vacuoles from tWT and tNP lymphocytes were counted in control, starvation and rapamycin conditions. Each value is expressed as an absolute number (70 cells counted/experiment). Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.01 between tWT and tNP basal condition and a <i>P</i> value < 0.05 between tWT and tNP rapamycin treated.</p

A proposed model for the pathogenesis of Niemann-Pick disease type B.

<p>Defective ASMase and lysosomal storage could lead to a reduced ability of lysosomes to fuse with autophagosomes. This in turn could result in a partial block of autophagy maturation and defective degradation, accompanied by accumulation of autophagic vacuoles, peroxidized lipid droplets and aberrant mitochondria (autophagic stress). Additional evidence and hypotheses are more fully explained in the text. Dotted lines: proposed mechanism; solid lines: proven mechanism.</p

Evaluation of mitochondrial dysfunctional features by flow cytometry and transmission electron microscopy.

<p>(<b>A</b>) Statistical histogram of MFI variation of NAO in tWT and tNP cells for each experimental condition. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *<i>P</i> < 0.05 <i>vs</i> respective control. The difference between cell lines was significant as shown by two-way ANOVA (***<i>P</i> < 0.001). Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP in basal condition. (<b>B</b>) Flow cytometry histogram overlay depicting NAO MFI values of tWT and tNP basal condition. (<b>C</b>) Statistical histogram of MFI variation of TMRE in tWT and tNP cells for control, starved and rapamycin-treated cells. Mean values were converted to arbitrary units (A.U.) setting control values from wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); *<i>P</i> < 0.05 <i>vs</i> tWT control. The difference between cell lines was significant as shown by two-way ANOVA (*<i>P</i> < 0.05). Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP in basal condition. (<b>D</b>) Electron microscopy analyses of normal (<b>a</b>) and NP-B B lymphocytes (<b>b-g</b>) in control condition (<b>a</b>, <b>b</b>), and after starvation (<b>c</b>), rapamycin (<b>e</b>), nocodazole (<b>f</b>, <b>g</b>) and wortmannin (<b>d</b>) administration. In Niemann-Pick cells, dysfunctional mitochondria are recognizable (arrows): altered morphology is characterized by dilated and distorted cristae (<b>c</b>, <b>d, e</b>) and by stress-damaged features (<b>b</b>, <b>f</b>, <b>g</b>), absent in non-pathological cells (<b>a</b>). Bars: 1 μm for a, b, c, d, f, g; 0.5 μm for e. (<b>E</b>) Mitochondria from non-pathological and pathological cells were counted and classified according to their morphology—mitochondria with standard cristae (<b>i</b>), mitochondria with unusual cristae (<b>ii</b>) and oxidative stress-damaged mitochondria (<b>iii</b>)–in control, starved and rapamycin-treated cells. Each value is expressed as a percentage ± SD (n = 3; 50 cells counted/experiment); *<i>P</i> < 0.05 <i>vs</i> respective control. Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.01 and < 0.05 for standard and unusual cristae numbers respectively (⁺⁺<i>P</i> < 0.01 for standard cristae and ⁺<i>P</i> < 0.05 for unusual cristae) between tWT and tNP in basal condition. (<b>F</b>) ROS detection by CM-H₂DCFDA in flow cytometry. (<b>i</b>) Statistical histogram of MFI expression of CM-H₂DCFDA in tWT and tNP cells. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments); *<i>P</i> < 0.05 and **<i>P</i> < 0.01 <i>vs</i> respective control. Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP in basal condition and after nocodazole treatment; NS: not significant. (<b>ii</b>) CM-H₂DCFDA flow cytometric histograms are overlaid to show the comparison between MFI values of tWT and tNP basal condition. (<b>G</b>) Statistical histogram of MFI expression of MitoSOX in tWT and tNP cells. Mean values were converted to arbitrary units (A.U.) setting as 100 control MFI values of wild-type cells. Each value is expressed as a mean ± SD (Results from n ≥ 3 independent experiments). Two-way ANOVA with Bonferroni post test revealed a <i>P</i> value < 0.05 (⁺<i>P</i> < 0.05) between tWT and tNP in basal condition and after nocodazole treatment, and a <i>P</i> value < 0.01 (⁺⁺<i>P</i> < 0.01) between tWT and tNP after starvation and wortmannin treatments; NS: not significant.</p

Morphological features of MTJ in control and trained rats.

<p>TEM images of MTJs which reveal the different complexity between control (<b>A</b>) and trained groups (RUN-F, <b>B</b>). At high magnification (13500x), tenocytes, located strictly close to MTJ (mtj), display, in comparison to control (<b>C</b>), a growing presence of rough endoplasmic reticulum (►) in trained groups (RUN-F, <b>D</b>). Bars <b>A</b>,<b>B</b>,<b>C</b>,<b>D</b>: 0.5μm.</p

Contractile force levels developed in rat muscles of the three experimental conditions.

<p>Evaluation of muscle strength in response to single twitch (<b>A</b>) and tetanus (<b>B</b>) in CTRL, RUN-S and RUN-F. *p < 0.05; **p < 0.01.</p

mRNA levels of IGF-1 receptor, TbR1, BmpR-1b and Betaglycan in tendon.

<p>Effect of exercise protocols on IGF-1 receptor (<b>A</b>), TbR1 (<b>B</b>), BmpR-1b (<b>C</b>) and Betaglycan (<b>D</b>) mRNA expression in EDL tendon, assessed by real-time PCR. Gene expression is represented as fold-change compared to untrained rats. Data are representative of three independent experiments. *p < 0.05.</p

Morphometric analysis of MTJ complexity.

<p>Morphometric analysis of MTJ base (B) and interface length (IL), and study of muscle-tendon interpenetration (MID) (<b>A</b>). MTJ complexity investigated by means of a modified Sholl analysis (<b>B</b>). m: muscle; t: tendon.</p

mRNA levels of PGC-1α, Vinculin, IGF-1Ea and TGF-β in muscle.

<p>Effects of exercise protocols on PGC-1α (A), Vinculin (B), IGF-1Ea (C) and TGF-β (D) mRNA expression in EDL muscle, assessed by real-time PCR. Gene expression is represented as fold-change compared to untrained rats. Data are representative of three independent experiments. *p < 0.05.</p