Fig 2: VPS34 inhibition decreases intracellular levels of cholesteryl-esters, blocks cholesterol uptake and enhances lysosomal degradation of LDLR.(A) Lipidomic analysis of RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III for 24 hours and lipids were extracted and subjected to liquid chromatography mass spectrometry analysis. The table shows PIK-III-induced log2 fold change for aggregated and averaged lipid classes in RKO cells. The adjusted p-values were calculated between PIK-III and vehicle treatment group. Data represents the mean of three biological replicates and the entire dataset is reported in S4 Table. (B) RKO.Cas9 cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with BODIPY FL LDL probe for 2 hours at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (C) BODIPY FL LDL probe cellular uptake in (B) was quantified as percent average intensity of total cells. Data in the form of technical replicates were averaged and presented as the mean ± SD from 6 wells. (D) RKO CTRL cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours. (M) stands for mature protein and (P) stands for precursor protein. (E, F) RKO CTRL cells were treated with the indicated concentrations of PIK-III with or without 50 nM bafilomycin A1 (E) or 30 μM chloroquine (F) for 12 and 24 hours. Lysates were immunoblotted with the specified antibodies. (M) and (P) indicate the mature and precursor protein, respectively. (G) RKO CTRL cells were treated with indicated concentrations of PIK-III along with vehicle, 2 μg/ml and 10 μg/ml soluble cholesterol (Chol) for 5 days and cell viability was assessed using the CellTiter-Glo assay. Data presented is the mean from three independent experiments ± SD. NS, not significant (two-way ANOVA).

Fig 3: Vps34 protein and ubiquitination levels are not consistently altered in the brain of APP/PS1 mice. (A) Western blots (left) and quantification (right) of Vps34 in the hippocampus and cortex of wild-type (WT) and APP/PS1 mice at 6 mon (n = 6 in each). (B) Western blots (left) and quantification (right) of Vps34 in the hippocampus and cortex of WT and APP/PS1 mice at 12 mon (n = 8 in each). (C) Representative Western blots of ubiquitin and Vps34 in the input from the cortex of WT and APP/PS1 mice at 12 mon are shown on the left panel, whereas quantification of Vps34 protein levels is shown on the right (n = 8). (D) Representative Western blots of ubiquitin (top) and VPS34 (bottom) in cell extracts from the cortex of WT and APP/PS1 mice at 12 mon immunoprecipitated by Vps34 antibody are shown on the left panel, whereas quantification of Vps34 relative protein levels is shown on the right (n = 8).

Fig 4: Involvement of PIK3C3 in miR-338-5p mediated cell migration and invasion. (a) miR-338-5p or negative control (N.C.) (100 nM) was transiently transfected into HCT116 cells, put into 96-well plates (4000/well) and cultured for 24 h. The number of cells was calculated every 24 h using MTT assays for four consecutive days. Data are expressed as means (n = 6) (P values were analysed using ANOVA). (b, c) Cell migration was analysed using transwell migration assays 48 h after transfection with miR-338-5p or anti-miR-338-5p into HCT116 or SW480. The transfected cells were seeded on transwell plates, and migrated cells at the bottom of membrane were calculated. The scale bar represents 50 μm. The number of migrated HCT116 cells are shown as means ± SEM (n = 8) (P values were analysed using t tests).The number of migrated SW480 cells are shown as median (IQR) (n = 5) (P values were analysed using Mann-Whitney tests). (d) miR-338-5p or pCMV-Vps34 plasmid was transiently transfected into SW480 cells and analysed for PIK3C3 protein expression 48 h later. Cell migration was evaluated using transwell assay. Forty-eight hours after transfection, SW480 cells were seeded on transwell plates, and those cells appeared at the bottom of membrane were counted. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (e) Both miR-338-5p and pCMV-Vps34 plasmids were transiently transfected into SW480 cells. Transwell columns were coated with a Matrigel membrane and seeded with transfected cells 48 h later. The invaded cells at the bottom of membrane were counted after 96 h. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (f) SW480 cells were transiently transfected with anti-miR-338-5p (100 nM) and shVps34 lentivirus and assayed for PIK3C3 protein expression. Migration of SW480 cells was assayed using transwell assay. Number of migrated CRC cells was counted after 48 h. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (g) Transwell invasion assays were used to analyse SW480 cells transfected with anti-miR-338-5p or shVps34 lentivirus and counted for invaded cells after 96 h. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). The scale bar represents 50 μm.

Fig 5: PIK3C3 was one of the target genes of miR-338-5p in CRC. (a) QPCR was used to evaluate the miR-338-5p expression in CRC cell lines (SW480, SW620 and HCT116). The data are shown as means ± SEM (n = 5). miR-338-5p (50 nM or 100 nM) or N.C. (100 nM) was transiently transfected into HCT116 cells and analysed using QPCR. The anti-miR- 338-5p (50 nM or 100 nM) or anti-N.C. (100 nM) was transiently transfected into SW480 cells and analysed using QPCR. Data are presented as median (IQR) (n = 3) (P values were analysed using Mann-Whitney tests). (b) RNA expression of miR-338-5p target genes (SPRY2, HEMGN, NDFIP1, ID1, ADM, PPP2R5A, DDX5, SCN9A, PIK3C3, and HOXA5) was measured using RT-PCR after transfection of miR-338-5p or anti-miR-338-5p into SW480 cells. (c) The target sequence of PIK3C3 was constructed into 3´-UTR of p-miR-reporter luciferase plasmid downstream of luciferase gene. Wild-type (WT) and mutant-type (mutant) target sequences are shown. The SW480 cells were transfected with WT or mutant p-miR-report-PIK3C3 plasmid (5 μg/ml) and co-transfected with miR-338-5p, anti-miR-338-5p, N.C. or anti-N.C. (100 nM), respectively. Data are presented as means ± SEM (n = 5) (P values were analysed using Mann-Whitney tests). (d) RIP assay was performed to precipitate the Ago2 complexe from stable miR-338-5p overexpression or shGFP control cells. Western blot was used to confirm the quality of RIP. Expression of miR-338-5p and PIK3C3 RNA in Ago2 RIP fractions was measured by QPCR assay. Data are presented as median (IQR) (n = 5) (P values were analysed using Mann-Whitney tests). (e) Expression of PIK3C3 protein in CRC cell lines was evaluated using Western blotting. miR-338-5p or its N.C. was transiently transfected into HCT116 cells, and anti-miR-338-5p or anti-N.C. was transiently transfected into SW480 cells. Expression of PIK3C3 protein was assessed using Western blotting. β-actin was used as an internal control.