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. 2020 Aug 24;15(8):e0235551. doi: 10.1371/journal.pone.0235551

Fig 5. VPS34 inhibition depletes intracellular iron by blocking iron uptake through enhanced degradation of TFR and excess iron rescues the growth defect in RKO cells.

Fig 5

(A) Aconitase activity in RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III, 30 μM deferasirox (DFX) or vehicle control for 24 hours. The aconitase activity assay was used to quantify the isocitrate present in the cell extracts. All data was normalized to cells treated with vehicle, transformed to represent the percent isocitrate generated and averaged from three biological replicates ± SD; ****, p < 0.0001; NS, not significant (two-way ANOVA). (B) RKO CTRL cells were treated with vehicle or 0.5 μM PIK-III for 24 hours and lysates were immunoblotted with the indicated antibodies. (C) Quantitation of immunoblot in (B) as fold change versus vehicle treated cells. All data were normalized to the β-actin loading control. Data is averaged from three independent experiments ± SD, ****, p < 0.0001 (two-way ANOVA). (D) RKO.Cas9 cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with Transferrin Alexa Fluor 488 probe for 30 min at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (E) Cellular uptake of Transferrin Alexa Fluor 488 probe from panel (D) was quantified as percent average intensity of total cells. Data were averaged and presented as the mean ± SD from 6 wells. (F) RKO CTRL cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours and lysates were immunoblotted with the indicated antibodies. (G, H) RKO CTRL cells were treated with the indicated concentrations of PIK-III with or without 50 nM Bafilomycin A1 (G) or 30 μM chloroquine (H) for 12 and 24 hours and lysates were immunoblotted with the indicated antibodies. (I) RKO CTRL cells were treated with indicated concentrations of PIK-III along with vehicle, 1 μM or 50 μM Ferric Ammonium Citrate (FAC) for 5 days and cell viability was assessed using the CellTiter-Glo assay. Data is derived from three independent experiments and presented as mean ± SD, ***, p < 0.001; NS, not significant (two-way ANOVA). (J) RKO CTRL cells were treated with the indicated concentrations of PIK-III along with vehicle or 50 μM FAC for 24 hours. Lysates were immunoblotted with the specified antibodies. (K) Mitochondrial respiration defect due to PIK-III treatment. RKO CTRL cells were treated with the indicated concentrations of PIK-III along with vehicle for 24 hours with or without 50 μM FAC and mitochondrial respiration was assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in response to the specified mitochondrial inhibitors using the Seahorse XFe96 analyzer. Oligomycin (Oligo) inhibits ATP synthase (Complex V) and decreases OCR; FCCP uncouples oxidative phosphorylation and increases OCR; and rotenone/antimycin A (ROT & AA) inhibits Complex I and III, respectively and decreases OCR. OCR and ECAR readouts from a single 96-well plate were normalized to the total DNA content measured in each well by DRAQ5 staining. Relative magnitude of mitochondrial oxidative phosphorylation and glycolysis is depicted as a ratio between the basal oxygen consumption rate and basal extracellular acidification rate (OCR/ECAR) and presented as the mean ± SD (n = 11 or 12 wells). Individual OCR and ECAR graphs are shown in S5 Fig.