pik fyve inhibitor  (MedChemExpress)

Journal: Human Molecular Genetics

Article Title: Mitochondria–lysosome membrane contacts are defective in GDAP1-related Charcot–Marie–Tooth disease

doi: 10.1093/hmg/ddaa243

Figure Lengend Snippet: GDAP1 regulates membrane biogenesis in MAMs and the activity of PYK fyve . ( A ) Representative images of GFP-FYVE positive vesicles in transfected SH-SY5Y and G4 cells. Box-plot shows vesicle diameter (μm). Scale bar: 10 μm, 3D scale bar:1 μm. ( B ) PLA of GDAP1 and PIK fyve in untreated SH-SY5Y cells and after autophagy induction (EBSS). Quantification of the number of dots per cell is shown in the right panel ( n = 261 untreated SH-SY5Y cells and n = 229 SH-SY5Y EBSS cells; three independent experiments). ( C ) Co-IP assay of endogenous GDAP1 and PIK fyve in SH-SY5Y cells. Quantification is shown in the right panel (three independent experiments). ( D – F ) Representative images of lysosomes stained with LysoTracker Red (D) or α-LAMP-1 (E) in SH-SY5Y and G4 cells and with α-LAMP-1 in eMNs (F). ( G ) Representative images of α-LAMP-1 staining in untreated neuroblastoma cells and after Apilimod treatment (left panel) and lysosomal area distribution (right panel) ( n = 2339 SH-SY5Y lysosomes, n = 3128 G4 lysosomes, n = 4487 SH-SY5Y Apilimod lysosomes and n = 2338 G4 Apilimod lysosomes; two independent experiments). Scale bar: 10 μm. ( H and I ) Representative images of TFEB staining and quantification of nuclear TFEB intensity in neuroblastoma cells (H) ( n = 459 SH-SY5Y cells, n = 399 G4 cells; three independent experiments) and eMNs (I) ( n = 95 Wild-type cells, n = 66 Gdap1 −/− cells; three independent primary cultures). Scale bars: 10 μm. ( J ) Colocalization of GFP-LC3 vesicles with LAMP-1 staining in SH-SY5Y and G4 cells. Analysis of Mander’s Overlapping Coefficient (MOC) of the vesicles is shown in the graphic below ( n = 278 SH-SY5Y cells, n = 260 G4 cells; three independent experiments). Scale bars: 10 μm. Data information: In (A, B), the box plot lines correspond from the bottom of the box to top: 25th percentile, median percentile, 75th percentile. The whiskers extend to the minimum and maximum values. Outliers are represented as dots. In (C, G, H, I and J) data represent mean ± SD and individual values are displayed as dots. Mann–Whitney U test (A, B), Student’s t -test (C, H–J) and Kolmogorov–Smirnov test (G). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The PIK fyve kinase inhibitor Apilimod (Selleckchem) was used at 20 n m for 60 min at 37°C.

Techniques: Membrane, Activity Assay, Transfection, Co-Immunoprecipitation Assay, Staining, MANN-WHITNEY

Journal: Human Molecular Genetics

Article Title: Mitochondria–lysosome membrane contacts are defective in GDAP1-related Charcot–Marie–Tooth disease

doi: 10.1093/hmg/ddaa243

Figure Lengend Snippet: GDAP1 function in mitochondrial MCSs and proposed pathophysiological mechanisms in deficient cells. ( A ) GDAP1 is located in both the outer mitochondrial membrane and the mitochondria-associated membranes (MAMs), the membrane contact sites (MCSs) between mitochondria and ER. GDAP1 participates in membrane biogenesis of early autophagic vesicles, interacting with STX17 and LC3-I/II, allowing proper basal autophagic flux. GDAP1 through its interaction with the lysosomal protein LAMP-1, constitutes a new tether of mitochondria-lysosome MCSs, regulating mitochondrial and lysosomal dynamics. GDAP1 also interacts with the kinase PIK fyve, involved in lysosomal identity, maturation, and transport. The figure also includes the known participation of GDAP1 in both retrograde and anterograde movements mediated by RAB6 and caytaxin, respectively, which locates mitochondria at Ca 2+ microdomains. ( B ) Cells lacking GDAP1 present an oxidative microenvironment due to a decrease in GSH levels. The depletion of GDAP1 causes defects in vesicle biogenesis from MAMs, leading to autophagosome accumulation and slowing down the autophagic flux. The lack of GDAP1 also affects mitochondria-lysosome MCSs inducing (1) enlarged lysosomes and the activation of TFEB, which is recruited to the nucleus activating the expression of autophagic genes and (2) mitochondrial network abnormalities. In Ca 2+ microdomains, mitochondrial localization is also altered affecting SOCE activity. Illustration created with BioRender ( https://biorender.com/ ).

Article Snippet: The PIK fyve kinase inhibitor Apilimod (Selleckchem) was used at 20 n m for 60 min at 37°C.

Techniques: Membrane, Activation Assay, Expressing, Activity Assay