Fig. 5

ATP6V1A ubiquitination by FBXO9 impairs V-ATPase assembly. A–C Assessment of lysosomal acidity in A549 cells after FBXO9 knockdown. A549 cells were transfected with FBXO9 siRNA and stained with LysoTracker Red. Plots show the overall LysoTracker intensity per cell (Scale bar = 100 μm, n = 6–8 images from three independent experiments, ****P < 0.0001). D, E Representative images demonstrate LysoTracker Red staining in negative control and FBXO9-knockout H1299 cells. FBXO9-knockout H1299 cells treated with 200 nM bafilomycin A1 (BAF) for 12 h were used as a rescue control. The average staining intensity was quantified as in C. Scale bar = 100 μm, ***P < 0.0001 and ****P < 0.0001. F, G Representative images demonstrate LysoTracker Red staining in H1299 cells ectopically expressing V1A-23 aa. The average staining intensity was quantified using the method described in C. H, I Analysis of V-ATPase assembly in FBXO9-knockdown H1299 cells. FBXO9 was depleted using shRNA, followed by subcellular fractionation. Immunoblot analysis was conducted using antibodies targeting ATP6V1A and ATP6VoD (H). LAMP1 and GAPDH were used as loading controls for membrane and cytosolic proteins, respectively. Ratio of ATP6V1A (n = 3 independent experiments, *P < 0.05, **P < 0. 01) to ATP6VoD in the membrane fraction represents the assembly of the V-ATPase (I). J, K Analysis of V-ATPase assembly in FBXO9-knockout H1299 cells. sgCtrl and FBXO9-knockout H1299 cells were fractionated, followed by immunoblot analysis of ATP6V1A and ATP6VoD (J). Levels of assembled ATP6V1A (K) (n = 3 independent experiments, **P < 0.01) were normalized against ATP6VoD in the membrane fraction. L, M Analysis of V-ATPase assembly in H1299 cells ectopically expressing V1A-23 aa or EGFP is depicted. Representative immunoblots for ATP6V1A, ATP6VoD, LAMP1, and GAPDH are presented (L). The levels of assembled ATP6V1A (M) (n = 3 independent experiments, **P < 0. 01) are measured plotted, and normalized against ATP6VoD in the membrane fraction (M). N, O HEK293T cells were treated with MLN4924 at indicated concentrations for 12 h. Immunoblotting using antibodies against ATP6V1A and ATP6VoD was performed on cytosolic and membrane fractions (N). The levels of assembled ATP6V1A (O) (n = 3 independent experiments, **P < 0. 01) were determined as described in panel (M)