Fig 1.
ATP9A knock-down in HepG2 cells enhances EV release.
HepG2 cells were transduced with shATP9A #33, #34 or sh-control. Cells and medium were harvested 116 hours after transduction. (A) Ratio of ATP9A mRNA / geometric mean of HRPT and 36B4. Data were presented as mean of 8 independent experiments ± standard error of mean (SEM). (B) Number of EVs/ml/cell in medium of ATP9A knock-down and sh-control cells. Values were expressed as mean of 8 independent experiments ± standard error of mean (SEM). (C) Size distribution of EVs in culture medium of sh control cells and ATP9A knock-down cells. For statistical significance One way ANOVA with Dunnett’s multiple comparison test was done. * P<0.05, **p<0.005,***P < 0.0005.
Fig 2.
Enhanced EV release in ATP9A knock-down cells is independent of caspase-3 activation.
(A) 116 hours after transduction, sh control and ATP9A knock-down cells were incubated with lactadherin-FITC and binding was analysed by FACS. Mean fluorescent intensity (MFI) of ATP9A knock-down cells compared to sh-control cells of two independent experiments performed in six replicates (n = 12) is presented. Statistical significance was tested by one-way ANOVA with Dunnett’s correction for multiple testing: **p<0.005,***p< 0.0005. (B) Caspase activity in ATP9A knock-down and Chenodeoxycholate (CDC) treated cells compared to sh-control cells in presence and absence of caspase inhibitor. Approximately 48 hours after transduction, sh-control and ATP9A knock-down cells were incubated with or without 5 μM caspase inhibitor (Q-VD-Oph) and 66 hours later caspase activity was determined. CDC was added as a positive control for apoptosis 1 hour before harvesting cells. (C) Number of EVs/cell in medium of ATP9A knock-down cells and CDC treated cells compared to sh-control cells. Values are expressed as the mean ± Standard deviation (SD) of 3 independent experiments. Statistical analysis was done by two-way ANOVA with Tukey’s correction for multiple testing: * represents the significant difference between the control and ATP9A knock-down or CDC treated cells in the vehicle treated groups;*p < 0.05, **, ***<0.0005. # shows the significant difference between the Q-VD-Oph treated and untreated groups; ##p<0.005, ###p<0.0005, n.s p≥0.05.+ represents the nonsignificant difference between control and ATP9A knock-down or CDC treated cells in the Q-VD-Oph treated groups; +++p <0.0005.
Fig 3.
ATP9A localizes to intracellular vesicles and to the plasma membrane.
Immunostaining and surface biotinylation was performed in ATP9A-flag overexpressing HepG2 cells to study the localization of ATP9A. (A) Immunostaining of ATP9A-flag (red) in HepG2. Nuclear DAPI staining in blue. (B) Surface biotinylation of ATP9A-flag in over-expressing HepG2 cells. Immunoblot showing ATP9A and GAPDH in the total lysate and eluate fraction.
Fig 4.
Ceramide dependent release of EVs in ATP9A knock-down cells.
ATP9A knock-down and sh-control cells were incubated for 66 hours with GW4869 (10μM) or vehicle (1% DMSO). The mean of excreted EVs/cell of a representative of 3 independent experiments all performed in triplo (n = 9) ± SD is shown. Statistical significance was tested by two-way ANOVA with Tukey’s correction for multiple testing: ***p<0.0005 and n.s p≥0.05.+ represents the nonsignificant difference.
Fig 5.
ATP9A knock-down in HepG2 cells affects endocytic pathways.
RNA was isolated from sh-control and ATP9A knock-down HepG2 cells at 72 hours after transduction and used for microarray analysis. (A) Ingenuity pathway analysis of significantly up-regulated and down-regulated genes enriched for multiple pathways related to endocytosis. (B) Gene Ontology (GO) molecular function analysis of significantly altered genes enriched for the indicated pathways affecting cell migration, cell growth and proliferation, and cell death and survival. Pathways are arranged in order of their significance [-log(p-value)].
Fig 6.
ATP9A knock-down affects ARPC3 and CORO1A expression and leads to changes in the actin cytoskeleton.
(A) Relative mRNA expression of ARPC3 in ATP9A knock-down cells compared to sh-control cells. (B) Relative mRNA expression of CORO1A in ATP9A knock-down cells compared to sh-control cells. (C) Phalloidin staining for actin filaments (green) and DAPI staining for nuclei (blue) in control and ATP9A knock-down cells. Statistical significance was tested by a student’s t-test: *p<0.05, **p<0.005.
Fig 7.
ATP9A does not transport NBD-labeled phospholipids.
(A) UPS-1 cells overexpressing ATP9A-flag (UPS-ATP9Aflag) and control cells were incubated with 1 mg/ml sulfur-NH-ss-biotin for 1 hour. Biotinylated proteins were isolated and the presence of ATP9A-flag and GAPDH was analyzed by Western blot. (B and C) UPS-ATP9Aflag cells were incubated with NBD-PS and NBD-PE respectively for 15, 30 and 60 minutes and internalization of NBD–phospholipids was analyzed by FACS. Mean fluorescent intensity of (B) NBD-PS, (C) NBD-PE was plotted over 60 minutes. Statistical significance was determined by one-way ANOVA with Bonferroni correction for multiple testing. *p<0.05,**p<0.005.