Figure 3:

PPT1 inhibition leads to lysosomal inhibition. (A) A375P cells expressing the mCherry-eGFP-LC3B reporter were treated with HCQ (3 μM, 1 hr), Lys05, (3 μM, 1 hr), or DC661 (3 μM, 1 hr) in the presence or absence of N-tert-Butylhydroxylamine (NTBHA, 2 mM), and subsequently analyzed by microscopy. Shown are quantitation. (B) A375P WT PPT1 or KO PPT1 cells were treated with HDSF (60 μM), HCQ (3 μM), Lys05 (3 μM) or DC661 (3 μM) for 6 hours before being stained with Lysosensor. To the right is quantitation of Lysosensor intensity. (C) A375P WT PPT1 or KO PPT1 cells were treated with HCQ, Lys05, or DC661 (3 μM, 0 – 6 hr). Lysate was immunoblotted (D) A375P WT PPT1 or KO PPT1 cells were treated with HCQ (30 μM), Lys05 (3 μM), or DC661 (1 μM, 72 hrs) and cell numbers were quantified as shown. (E) B16 WT Ppt1 or KO Ppt1 cells were treated with HCQ, Lys05, or DC661 (3 μM, 1 hr). Lysate was immunoblotted. (F) Proximity ligation assay for the p18 (Ragulator) – V1A (vATPase subunit) interaction in WT PPT1 and KO PPT1 cells. Shown below is quantitation. (G) Proximity ligation assay for the mTOR – Rheb interaction in WT PPT1 and KO PPT1 cells. Shown below is quantitation. (H) Membrane fractions generated from WT PPT1 and KO PPT1 cells were subsequently immunoblotted. All data is representative of at least 2 experiments. Standard deviation is shown in (D), (F), and (G). * indicates p<0.05. Students T test was used.