Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes
Fig 1
Evaluation of cell death by Propidium Iodide (PI), Annexin (Anx) and transmission electron microscopy.
(A) Dot plots PI vs SSC from Niemann-Pick (tNP) and wild-type (tWT) B lymphocytes for control (CTRL), starved (STARV), rapamycin (RM), nocodazole (NZ) and wortmannin (WM) treated cells. Rectangular regions show events with low PI uptake (PI+ in R3) and high PI uptake (PI++ in R2), pointing to apoptosis and necrosis, respectively. (B) The statistical histogram shows the total PI uptake percentage (events in R2 and R3) in non-pathological and pathological lymphocytes. Each value is expressed as a percentage ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 and ***P < 0.001 vs respective control. (C) Dot plots Annexin-FITC vs FSC from Niemann-Pick (tNP) and wild-type (tWT) B lymphocytes from control (CTRL) and starved (STARV) cells. Rectangular regions (populations in green) show Anx+ early apoptotic events. (D) The statistical histogram shows Annexin (Anx) positivity percentage in non-pathological and pathological lymphocytes. Each value is expressed as a percentage ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 and ***P < 0.001 vs respective control. (E) TEM images of tWT (a) and tNP (b-e) cells from different experimental conditions: in control condition (a, b), after nutrient deprivation (c), rapamycin (d) and nocodazole (e) administration. In c, d and e autophagic cell death in vacuolated cells is recognizable (arrows: autophagic vacuoles, AVs). Bars: 5 μm for a, b, e; 2 μm for c, d.
