The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells
- Department of Chemistry, University of Montreal, Canada
- Department of Biochemistry and Molecular Medicine, CR-CHUM and Montreal Cancer Institute, University of Montreal, Canada
- Faculté de Pharmacie, University of Montreal, Canada
- Institute for Research in Immunology and Cancer, University of Montreal, Canada
Figures
Figure 1 with 3 supplements
Biguanide pharmacophore interacts with ATP synthase subunit e (ATP5I).
(A) Design of bio-inspired probe biotin functionalized biguanide (BFB) based on the structure of metformin (Met). (B) Immunoblots for the phosphorylation of AMPK (Thr172) and ACC (Ser79) in extracts from KP-4 pancreatic cancer cells treated with 2.5 mM Met or BFB for 16 hr. β-ACTIN was used as loading control. (C) Representative quantification of cell viability and growth with corresponding EC50 values of 3-day treatments with metformin (Met) or BFB in KP-4 cells. Values represent the mean ± standard deviation of N = 3. (D) Representative images of mitochondria and BFB localization in cells as in (B). Cells were treated with 1 mM of metformin (Met) or BFB for 16 hr and mitochondrial signal and BFB localization were analyzed by co-immunofluorescence using streptavidin fluorophore conjugate and anti-TOMM20 antibody, scale bar = 10 μm. Cells untreated (-) and treated with 1 mM Met were used as negative controls. (E) Colocalization between TOMM20 (TOMM20-568) and streptavidin (Strep-488) fluorophores was analyzed for the BFB condition from (D) through job plot intensity profile. (F) Pull-down validation experiments with streptavidin beads alone (-), D-biotin (B), biotin functionalized amine (BFA) and BFB using antibody followed by immunoblot against ATP5I in cells as in in extracts from HEK-293T embryonic kidney cells. The whole cell lysate (WCL) was added as control. (G) Binding interactions studies of BFB with recombinant purified ATP5I (rATP5I) using Surface Plasmons of Resonance (SPR). Representative sensorgrams show affinity kinetics of BFB and rATP5I. BFB was exposed onto streptavidin immobilized sensor chip and several concentrations of rATP5I were added until saturation of the signal. RU: Resonance Units. (H) Binding affinity curve obtained from each steady state from (G). KD refers to the dissociation equilibrium constant and Rmax represent the theoretical maximum response.
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Figure 1—source data 1
Contain details of chemical synthesis and structural characterization of the compounds made for this article.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig1-data1-v1.docx
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Figure 1—source data 2
PDF file containing original western blots for Figure 1B, F.
Many of our source data contains cut blotting membranes. Blotting membranes were cut after transfer to allow probing for proteins with different molecular weights. Each membrane section was incubated with the appropriate antibody corresponding to the target protein size.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig1-data2-v1.zip
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Figure 1—source data 3
Contain original TIF files used to make Figure 1B, F.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig1-data3-v1.zip
Figure 1—figure supplement 1
Synthetic routes for biotin-NHS, biotin-functionalized biguanide, and biotin-functionalized amine probes.
(A) Synthesis of biotin-NHS (1); (a) NHS, EDC, DMF, room temperature (r.t.) overnight (o/n). (B) Synthesis of biotin functionalized biguanide (BFB) chloride salt (3); (a) Dicyandiamide, TMSCl, MeCN, 160°C, 3 hr, 2: 6-aminohexylbiguanide hydrochloride salt; (b) Biotin-NHS, DIPEA, DMF, r.t., o/n. (C) Synthesis of biotin functionalized amine (BFA) chloride salt (5); (a) N-Boc-1,6-hexanediamine, DMF, r.t., o/n, 4: biotin functionalized N-Boc-amine; (b) HCl/MeOH, MeOH, r.t., o/n.
Figure 1—figure supplement 2
Proteins were isolated from streptavidin-coated beads after affinity purification using the biotinylated biguanide probe (BFB), followed by competitive elution with metformin (50 mM).
Mass spectrometry (MS/MS) analysis was performed to identify proteins potentially involved in BFB-specific interactions. The table reports the approximate molecular weight and total number of peptides identified for each protein.
Figure 1—figure supplement 3
Purification and size-exclusion chromatography characterization of recombinant ATP5I.
(A) SDS–PAGE analysis of recombinant ATP5I (rATP5I) after the purification process. Purity is estimated at ≥90%. MM: molecular weight marker. (B) Gel filtration profile analysis of rATP5I using Superose 12 10/300 GL. DV represents column dead volume. mAU: milli-Absorbance Unit. (C) Calibration of the Superose 6 10/300 GL size-exclusion column for conformational analysis of the Nter-6×His-ATP5I construct. The calibration curve was generated by plotting the logarithm of the molecular weight (log(MW)) of standard proteins (thyroglobulin, γ-globulin, myoglobin) and vitamin B12 as a function of their partition coefficient (Kav). Ve: elution volume; V₀: void volume (1.12 ml); Vt: total column volume (2.8 ml).
Figure 2 with 2 supplements
ATP5I knockout in pancreatic cancer cells alters the organization of the mitochondrial network.
(A) Immunoblot for the indicated proteins in extracts from clones of KP-4 cells expressing a control small guide RNA against GFP (sgGFP: control) or two different sgRNAs against ATP5I (sgATP5I #1 and sgATP5I #2). GAPDH antibody was used as a loading control. (B) Representative images of mitochondrial morphologies visualized by TOMM20 immunofluorescence (scale bar = 10 μm). A magnified inset (yellow box) is shown for each image to highlight mitochondrial structural details. All images were analyzed using the Mitochondria Analyzer plugin in Fiji (ImageJ). Quantitative analysis of key mitochondrial parameters: (C) number of branches, (D) total branch length, (E) number of branch endpoints, and (F) mean branch diameter. Data represent mean ± standard deviation from N = 3 independent clones, with 50–100 cells analyzed per clone. ns: not significant, **p < 0.01, and ****p < 0.0001 using an unpaired Student’s t-test. (G) Representative Blue Native-PAGE followed by western blotting using an antibody against the β-subunit of the F₁ domain of ATP synthase in KP-4 or U2OS cells treated with metformin (10 mM, 16 hr or 3 days), or in ATP5I knockout cells (ATP5I KO). Monomer* indicates the assembly intermediates of the F₁F₀-ATP synthase known to accumulate after disabling ATP5I.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2A, G.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig2-data1-v1.zip
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Figure 2—source data 2
Contain original TIF files used to make Figure 2A, G.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig2-data2-v1.zip
Figure 2—figure supplement 1
Loss of ATP5I disrupts OXPHOS complex protein expression in KP-4 cells.
(A) Immunoblot for the indicated proteins in extracts from clones of KP-4 cells expressing a control small guide RNA against GFP (sgGFP: control 1 and control 2) or two clones for each of the two different guides targeting ATP5I (sgATP5I #1: sgATP5I 1 and sgATP5I 2, and sgATP5I #2: sgATP5I 3 and sgATP5I 4). * is a nonspecific band that migrates like 40 KD MTCO1 from Complex IV. GAPDH was used as loading control. (B) Relative qPCR quantification of the mRNAs encoding proteins representative of the five OXPHOS complexes (in Figure 2A) in clones of KP-4 cells expressing a control small guide RNA against GFP (sgGFP: control 1 and control 2) or two clones for each of the two different guides targeting ATP5I (sgATP5I #1: sgATP5I 1 and sgATP5I 2, and sgATP5I #2: sgATP5I 3 and sgATP5I 4). Values represent the mean ± standard deviation of three biological replicates. (C) qPCR quantification of mitochondrial genomic DNA (Mt) over cellular nuclear genomic DNA (Nu) in cells as in (A). Values represent the mean ± standard deviation of N = 3.
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Figure 2—figure supplement 1—source data 1
PDF file containing original western blot for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Contain original TIF files used to make Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Quantification of immunoblot in Figure 2G.
(A) The PVDF membrane was stained with Ponceau S to visualize total protein and subsequently imaged on Bio-Rad ChemiDoc XRS+ system. (B) Quantification. Using Bio-Rad Image Lab software, total band intensity was quantified for the band corresponding to the vestigial form of ATP synthase (monomer*) in Figure 2G and normalized to the total band intensity of the corresponding lane in the Ponceau image. Values for each cell line were normalized to the ‘untreated’ condition.
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Figure 2—figure supplement 2—source data 1
PDF file containing original western blot for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Contain original TIF files used to make Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig2-figsupp2-data2-v1.zip
Figure 3 with 1 supplement
ATP5I knockout desensitizes pancreatic cancer cells to biguanides.
(A) Quantification of NAD+/NADH ratio in KP-4 cells expressing a control small guide RNA against GFP (control) or a representative clone of two different guides targeting ATP5I (sgATP5I #1 or sgATP5I #2). Values represent the mean ± standard deviation of N = 3. ***p < 0.001 using an unpaired Student’s t-test. (B) Relative quantification of oxygen consumption rate (OCR) over extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N = 3. ***p < 0.001 using a paired Student’s t-test. (C) Immunoblot for total and phosphorylated levels of AMPK (Thr172) protein in extracts from cells as in (A). ATP5I confirms loss of expression in KO, and GAPDH was used as loading control. (D) Growth curves of cells as in (A) measuring the relative number of cells over 6 days. Media was changed every 2 days. (E) Representative kinetic curves of OCR in cells as in (A) treated with 5 mM of metformin (Met) relative to control treated cells using Seahorse. (F) Representative kinetic curves of ECAR in cells as in (A) treated with 5 mM metformin (Met) relative to control treated cells (dashed line) using Seahorse. (G) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hr from kinetic curves (E, F). Values represent the mean ± standard deviation of N = 3. ns: not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (H) Representative growth of cells as in (A) exposed to different concentrations of metformin for 3 days with corresponding EC50 values of metformin. Values represent the mean ± standard deviation of N = 3. ***p < 0.001 and ****p < 0.0001 using an unpaired Student’s t-test. (I) Representative kinetic curves of OCR in cells as in (A) treated with 100 μM phenformin (Phen) relative to control treated cells using Seahorse. (J) Representative kinetic curves of ECAR in cells as in (A) treated with 100 μM of phenformin (Phen) relative to control treated cells (dashed line) using Seahorse. (K) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hr from kinetic curves (I, J). Values represent the mean ± standard deviation of at least three biological replicates. ns: not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 using an RM one-way ANOVA with Sidak’s multiple comparison test. (L) Representative growth of cells as in (A) exposed to different concentrations of phenformin for 3 days with corresponding EC50 values. Values represent the mean ± standard deviation of N = 3. ****p < 0.0001 using an unpaired Student’s t-test.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig3-data1-v1.zip
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Figure 3—source data 2
Contain original TIF files used to make Figure 3.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig3-data2-v1.zip
Figure 3—figure supplement 1
ATP5I loss disrupts NAD metabolism, mitochondrial respiration, glycolytic dependence, and metformin sensitivity in KP-4 cells.
(A) Quantification of NAD+ concentration in KP-4 cells expressing a control small guide RNA against GFP (sgGFP) or a representative clone of two different guides targeting ATP5I (sgATP5I #1 or sgATP5I #2). Values represent the mean ± standard deviation of N = 3. ***p < 0.001, ****p < 0.0001 using an unpaired Student’s t-test. (B) Quantification of NADH concentration in cells as in (A). Values represent the mean ± standard deviation of N = 3. ns: not significant, **p < 0.01 using an unpaired Student’s t-test. (C) Relative quantification of oxygen consumption rate (OCR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N = 3. ns: not significant, *p < 0.05 using a paired Student’s t-test. (D) Relative quantification of extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N = 3. ***p < 0.001 using a paired Student’s t-test. (E) Representative cell growth of cells as in (A) treated with different concentrations of 2-d-deoxyglucose with corresponding EC50 values of 2-d-deoxyglucose treatment in cells as in (A). Values represent the mean ± standard deviation of N = 3. **p < 0.01 using an unpaired Student’s t-test. (F) Representative cell viability curves with corresponding EC50 values of treatments of 2-d-deoxyglucose in combination without (-) and with different concentrations (1, 2.5, and 5 mM) of metformin (Met) in KP-4 cells expressing control sgGFP. Values represent the mean ± standard deviation of three biological replicates. **p < 0.01 using an unpaired Student’s t-test. (G) Total and phosphorylated levels of AMPK protein extracts from cells as in (A) treated with 2.5 or 5 mM of Met for 16 hr. β-ACTIN was used for loading control. (H) Growth curves of cells as in (A) supplemented with 100 μg/ml sodium pyruvate (Pyr) and 50 μg/ml uridine (Uri) by measuring the percentage of confluency over 6 days. Media was changed every 2 days.
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Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Contain original TIF files used to make Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig3-figsupp1-data2-v1.zip
Figure 4 with 1 supplement
Exogenous ATP5I enables the reorganization of mitochondrial network in ATP5I knockout pancreatic cancer cells.
(A) A representative immunoblots for the indicated proteins in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control cells expressing a small guide RNA against GFP (sgGFP) or in ATP5I KO cells (clones of two different small guide RNAs: sgATP5I #1 sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). GAPDH antibody was used as loading control. (B) Representative threshold images of mitochondrial morphologies visualized by TOMM20 immunofluorescence (scale bar = 10 μm) of ATP5I KO cells and their derivative re-expressing ATP5I. A magnified inset (yellow box) is shown for each image to highlight mitochondrial structural details. All images were analyzed using the Mitochondria Analyzer plugin in Fiji (ImageJ). Quantitative analysis of key mitochondrial parameters: (C) number of branches, (D) total branch length, (E) number of branch endpoints, and (F) mean branch diameter. Data represent mean ± standard deviation from N = 3 independent clones, with 50–100 cells analyzed per clone. ns: not significant, **p < 0.01, ****p < 0.0001 using an unpaired Student’s t-test.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig4-data1-v1.zip
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Figure 4—source data 2
Contain original TIF files used to make Figure 4.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig4-data2-v1.zip
Figure 4—figure supplement 1
Exogenous ATP5I restores mitochondrial networks in ATP5I-deficient KP-4 cells.
(A) Representative images of mitochondrial localization of ATP5I in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control (clones with sgGFP) or a representative clone of each of two small guide RNAs against ATP5I (sgATP5I #1 or sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). Mitochondrial localization was analyzed by co-immunofluorescence using ATP5I and TOMM20 antibodies, scale bar = 10 μm. DAPI was used as a DNA counterstain. (B) Colocalization between TOMM20 (TOMM20-568) and ATP5I (ATP5I-488) signals was analyzed with job plot intensity profile.
Figure 5 with 2 supplements
Re-expression of ATP5I rescues metabolic profile and resensitizes ATP5I knockout pancreatic cancer cells to biguanides.
(A) Quantification of NAD+/NADH ratio in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control sgGFP or a representative clone of two different small guide RNAs (sgATP5I #1 and sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). Values represent the mean ± standard deviation of three biological replicates. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (B) Relative quantification of oxygen consumption rate (OCR) over extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least three biological replicates. ns: not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (C) Immunoblot of total and phosphorylated levels of AMPK (Thr172) protein in extracts from cells as in (A). GAPDH was used as loading control. (D) Intracellular ATP levels measured in cell lines as in (A). Data are presented as mean ± standard deviation. N = 2. ns: not significant. (E) Growth curves of cells as in (A) by measuring the relative number of cells over 6 days. Media was changed every 2 days. (F) EC50 values of metformin (Met) treatments in cells as in (A). Values represent the mean ± standard deviation of N = 3. ns: not significant, **p < 0.01, ****p < 0.0001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (G) EC50 values of phenformin (Phen) treatment in cells as in (A). Values represent the mean ± standard deviation of three biological replicates. ns: not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig5-data1-v1.zip
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Figure 5—source data 2
Contain original TIF files used to make Figure 5.
- https://cdn.elifesciences.org/articles/102680/elife-102680-fig5-data2-v1.zip
Figure 5—figure supplement 1
Exogenous ATP5I rescues NAD metabolism, mitochondrial respiration, glycolytic compensation, and 2-deoxyglucose sensitivity in ATP5I-deficient KP-4 cells.
(A) Quantification of NAD+ concentration in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control sgGFP clones or a representative clone of two different small guide RNAs against ATP5I (sgATP5I #1 and sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). Values represent the mean ± standard deviation of three biological replicates. ns: not significant, *p < 0.05, ****p < 0.0001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (B) Quantification of NADH concentration in cells as in (A). Values represent the mean ± standard deviation of three biological replicates. ns: not significant using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (C) Relative quantification of oxygen consumption rate (OCR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least three biological replicates. ns: not significant, **p < 0.01 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (D) Relative extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N = 3. ns: not significant, *p < 0.05, ****p < 0.0001 using an RM one-way ANOVA with Sidak’s multiple comparison test. (E) EC50 values of 2-d-deoxyglucose treatment in cells as in (A). Values represent the mean ± standard deviation of N = 3. **p < 0.01, ***p < 0.001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test.
Figure 5—figure supplement 2
ATP5I is required for biguanide-induced remodeling of mitochondrial respiration and glycolytic flux in KP-4 cells.
(A) Representative kinetic curves of oxygen consumption rate (OCR) fold change in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control sgGFP clones or a representative clone of each of two small guide RNA against ATP5I (sgATP5I #1 and sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-) treated with 5 mM metformin (Met) using Seahorse. (B) Representative kinetic curves of extracellular acidification rate (ECAR) fold change in cells as in (A) treated with 5 mM Met using Seahorse. (C) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hr from kinetic curves in (A) and (B). Values represent the mean ± standard deviation of at least three biological replicates. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (D) Representative kinetic curves of OCR fold change in cells as in (A) but treated with 100 μM phenformin (Phen) using Seahorse. (E) Representative kinetic curves of ECAR fold change in cells as in (A) but treated with 100 μM Phen using Seahorse. (F) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hr from kinetic curves (D) and (E). Values represent the mean ± standard deviation of at least three biological replicates. ns: not significant, ****p < 0.0001 using an RM one-way ANOVA with Sidak’s multiple comparison test.
Figure 6
ATP5I deletion mimics biguanide-induced bioenergetic remodeling.
(A) Representative oxygen consumption rate (OCR) profiles following sequential injection of oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/AA) in control, ATP5I knockout (sgATP5I #1 and #2), and Rescue (sgATP5I+exoATP5I) cells treated with vehicle, 5 mM metformin (Met), or 100 µM phenformin (Phen). (B) Representative extracellular acidification rate (ECAR) profiles under the same conditions as in (A). (C) Quantification of basal mitochondrial respiration corresponding to the conditions in (A), N = 3. (D) Representative confocal images of cells stained with the membrane potential sensitive dye TMRE (100 nM, 30 min at 37°C in complete DMEM without phenol red) under control conditions, following ATP5I knockout (ATP5I KO), or after depolarization with FCCP (10 µM, 30 min prior to TMRE incubation), scale bar = 10 µm. (E) Quantification of TMRE fluorescence intensity per cell. Data are expressed as mean ± standard deviation and normalized to control levels. Results are from three independent experiments performed on separate days (n = 431 cells for control; n = 371 for FCCP; n = 536 for ATP5I KO). ns: not significant, ***p < 0.001, ****p < 0.0001 using an unpaired Student’s t-test.
Figure 7
Chemogenomic screening of metformin reveals an imprint on F1Fo-ATP synthase.
(A) Results of the pooled genome-wide CRISPR/Cas9 KO screen made in NALM-6 cells treated with 16 mM metformin or control. Data are represented as a Volcano plot of gene enrichment/depletion scores vs p-values from using the CRANKS algorithm. Some genes of interest are labeled. Enhancers of metformin growth inhibition with negative CRANKS scores below 2.5 (dashed line) are labeled blue, while suppressors with positive CRANKS scores above 2.5 (dashed line) are labeled red. (B, C) Pairwise comparison of gene CRANKS scores obtained from screening metformin 16 mM in NALM-6 cells against that from screening either 70 nM rotenone or (C) 2 μM oligomycin A. (D) Model for metformin action triggering the OMA1–DELE1–HRI pathway.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | ATP5ME/ATP5I | GenBank | Gene ID: 521 | Cloned by RT-PCR |
| Strain, strain background (Escherichia coli) | Rosetta E. coli BL21 | Addgene | Bacterial strain #176583 | Competent cells |
| Cell line (Homo sapiens) | KP-4 | Dr. N. Bardeesy | RRID:CVCL_1338 | Pancreatic ductal carcinoma |
| Cell line (Homo sapiens) | U2OS | ATCC | RRID:CVCL_0042 | Osteosarcoma |
| Cell line (Homo sapiens) | NALM-6 | Dr. M. Tyers | RRID:CVCL_UJ05 | Acute lymphoblastic leukemia (CRISPR screen) |
| Chemical compound, drug | Metformin hydrochloride | Combi-Blocks | Cat# ST-9194 | |
| Chemical compound, drug | Phenformin hydrochloride | Sigma-Aldrich | Cat# P7045 | |
| Chemical compound, drug | 2-deoxy-D-glucose | BioShop | Cat# DXG498.5 | |
| Chemical compound, drug | D-biotin | Thermo Fisher Scientific | Cat# B1595 | |
| Chemical compound, drug | Rotenone | Sigma-Aldrich | Cat# R8875 | Mitochondrial inhibitor |
| Chemical compound, drug | Oligomycin A | Tocris Bioscience | Cat# 4110 | Mitochondrial inhibitor |
| Chemical compound, drug | TMRE | Thermo Fisher Scientific | Cat# T669 | Mitochondrial membrane potential dye |
| Chemical compound, drug | MitoTracker Green | Thermo Fisher Scientific | Cat# M46750 | Mitochondrial mass control |
| Chemical compound, drug | FCCP | Abcam | Cat# NC0474145 | Mitochondrial uncoupler |
| Chemical compound, drug | Coomassie Brilliant Blue G-250 | Bio-Rad | Cat# 1610406 | Native-PAGE |
| Chemical compound, drug | Coomassie Brilliant Blue R-250 | Thermo Fisher Scientific | Cat# 33445225GM | Protein staining |
| Chemical compound, drug | Crystal violet stain | Sigma-Aldrich | Cat# B21932.14 | Cell viability assay |
| Chemical compound | Vectashield mounting medium with DAPI | Vector Laboratories | Cat# H-1200-10 | Nuclear staining |
| Antibody | anti-phospho-ACC S79 (Rabbit polyclonal) | Cell Signaling | Cat# 3661S RRID:AB_330337 | WB (1:1000) |
| Antibody | anti-AMPK (Rabbit polyclonal) | Cell Signaling | Cat# 2532 RRID:AB_330331 | WB (1:1000) |
| Antibody | anti-phospho-AMPK T172 (Rabbit polyclonal) | Cell Signaling | Cat# 2531 RRID:AB_330330 | WB (1:1000) |
| Antibody | anti-ATP5I (Rabbit polyclonal) | Proteintech | Cat# 16483-1-AP RRID:AB_2062052 | WB (1:500–1000), IF (1:100) |
| Antibody | anti-OXPHOS cocktail (Mouse monoclonal) | Abcam | Cat# ab110411 RRID:AB_2756818 | WB (1:750) |
| Antibody | anti-F1-ATPase β-subunit (Mouse monoclonal) | Sigma-Aldrich | Cat# MABS1304 | WB (1:1000), BN-PAGE |
| Antibody | anti-OSCP (Mouse monoclonal) | Abcam | Cat# ab110276 RRID:AB_10887942 | WB (1:1000) |
| Antibody | anti-ATP5L (Rabbit polyclonal) | Abcam | Cat# ab126181 RRID:AB_11129974 | WB (1:1000) |
| Antibody | anti-GAPDH (Goat polyclonal) | Novus Biologicals | Cat# NB300-320 RRID:AB_10001796 | WB (1:3000) |
| Antibody | anti-α-Tubulin (Mouse monoclonal) | Sigma-Aldrich | Cat# T6074 RRID:AB_477582 | WB |
| Antibody | anti-β-Actin (Mouse monoclonal) | Cell Signaling | Cat# 3700 RRID:AB_2242334 | WB (1:10000) |
| Antibody | anti-TOMM20 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-17764 RRID:AB_628381 | IF (1:100) |
| Antibody | anti-rabbit IgG HRP (Goat polyclonal) | Bio-Rad | Cat# 170-6515 RRID:AB_11125142 | Secondary WB (1:3000) |
| Antibody | anti-mouse IgG HRP (Goat, clonality unknown) | Bio-Rad | Cat# 170-6516 RRID:AB_11125547 | Secondary WB (1:3000) |
| Antibody | anti-goat IgG HRP (Donkey polyclonal) | Santa Cruz Biotechnology | Cat# sc-2020 RRID:AB_631728 | Secondary WB |
| Antibody | anti-mouse AF488 (Goat polyclonal) | Invitrogen | Cat# A-11029 RRID:AB_2534088 | IF (1:2000) |
| Antibody | anti-rabbit AF568 (Goat, clonality unknown) | Invitrogen | Cat# A-11011 RRID:AB_143157 | IF (1:2000) |
| Other | Streptavidin AF488 | Invitrogen | Cat# S11223 RRID:AB_2336881 | Fluorescent detection of biotinylated proteins |
| Recombinant DNA reagent | pET-TEV-ATP5I (plasmid) | Dr. J.G. Omichinski | N/A | N-terminal 6xHis-tag expression |
| Recombinant DNA reagent | lentiCRISPRv2 (plasmid) | Addgene | Cat# 52961 | CRISPR/Cas9 vector |
| Recombinant DNA reagent | MSCV-ATP5I (plasmid) | This paper | N/A | Retroviral expression |
| Recombinant DNA reagent | pCW-Cas9 (plasmid) | Addgene | Cat# 50661 | Inducible Cas9 expression |
| Transfected construct (human) | sgATP5I #1 | This paper | N/A | CRISPR guide RNA targeting ATP5I |
| Transfected construct (human) | sgATP5I #2 | This paper | N/A | CRISPR guide RNA targeting ATP5I |
| Transfected construct (human) | sgATP5I #3 | This paper | N/A | CRISPR guide RNA targeting ATP5I |
| Transfected construct (human) | sgATP5I #4 | This paper | N/A | CRISPR guide RNA targeting ATP5I |
| Transfected construct (human) | sgGFP | This paper | N/A | Control guide RNA |
| Peptide, recombinant protein | ATP5I (6xHis-tagged) | This paper | N/A | Recombinant purified protein |
| Commercial assay or kit | Mitochondria Isolation Kit | Abcam | Cat# ab110170 | |
| Commercial assay or kit | NAD+/NADH assay kit | Sigma-Aldrich | Cat# MAK460 | |
| Commercial assay or kit | ATP Determination Kit | Thermo Fisher Scientific | Cat# A22066 | |
| Commercial assay or kit | BCA Protein Assay | Pierce | Cat# 23225 | Protein quantification |
| Software, algorithm | ImageJ / Fiji | NIH | RRID:SCR_003070 | Image analysis |
| Software, algorithm | GraphPad Prism | GraphPad | RRID:SCR_002798 | Statistical analysis |
| Software, algorithm | PEAKS 7 | Bioinformatics Solutions | N/A | Proteomics |
| Software, algorithm | Wave software | Agilent | RRID:SCR_024491 | Seahorse analysis |
| Other | Zeiss Axio-Observer Z1 spinning disk confocal microscope | Zeiss | N/A | 63× objective, Z-stack imaging |
| Other | Zeiss Axio Imager Z2 upright microscope | Zeiss | N/A | Immunofluorescence imaging |
| Other | SPARK 10 M plate reader | TECAN | N/A | Fluorescence detection |
| Other | Q-Exactive Plus | Thermo Fisher Scientific | N/A | Mass spectrometry |
| Other | Seahorse XFe96 Analyzer | Agilent | N/A | Metabolic flux |
| Other | P4SPR | Affinité Instruments | N/A | Surface plasmon resonance |
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The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells
eLife 13:RP102680.
https://doi.org/10.7554/eLife.102680.3
