1. Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor 
Israa Sharkia, MSc, Tal Hadad Erlich, PhD, Nadine Landolina, PhD, Miri Assayag, PhD, Alex Motzik, MSc, Inbal Rachmin, PhD, Gillian Kay, PhD, Ziv Porat, PhD, Sagi Tshori, MD, PhD, Neville Berkman, MD, PhD, Francesca Levi-Schaffer, PharmD, PhD, Ehud Razin, PhD 
Journal of Allergy and Clinical Immunology 
Volume 140, Issue 1, Pages e8 (July 2017)
DOI: /j.jaci
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Journal of Allergy and Clinical Immunology 2017 140, 204-214
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Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 1
Inhibition of PDH decreases the degranulation and cytokine secretion levels in RBL cells and CBMCs. A and B, RBL cells (Fig 1, A) and BMMCs (Fig 1, B) were sensitized with IgE and triggered with DNP-BSA for 5, 15, or 30 minutes. Afterward, levels of total and phospho-S293 PDH were determined by using Western blot analysis. One representative of 3 independent experiments is shown. C and D, RBL cells were sensitized with IgE, followed by a 30-minute DNP-BSA challenge. One hour before addition of DNP-BSA, 300 μmol/L CPI-613 was added to medium. β-Hexosamindase (Fig 1, C) or TNF-α (Fig 1, D) release were then measured. Results represent means ± SEMs (n = 3). *P < .05. E, RBL cells were treated as in Fig 1, C, and phospho-S293 PDH levels were determined by using Western blot analysis. One representative of 3 independent experiments is shown. F and G, RBL cells were transfected with siRNA targeted to PDH-E1α, and 48 hours later, β-hexosamindase release was measured (Fig 1, F). Western blot shows the levels of PDH-E1α in cells transfected with small interfering PDH-E1α compared with cells transfected with nontargeted siRNA (NT). H and I, IgE-sensitized CBMCs were incubated with 300 μmol/L CPI-613 for 3 hours and then activated with 5 μg/mL rabbit α-human IgE for 30 minutes (β-hexosaminidase) or overnight (IL-6 release). Percentage of β-hexosamindase release (Fig 1, H) and IL-6 (in picograms per milliliter) release (Fig 1, I) were assayed. Results represent means ± SEMs (n = 3). *P < .05.
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4. Fig 2
Inhibition of PDH decreases histamine secretion and lung resistance but not inflammation in mice. A, Mice were sensitized with IgE anti-DNP through injection into the tail vein and injected intraperitoneally with 50 mg/kg CPI-163. Saline was used as a control. Twenty-four hours later, mice were injected with DNP-BSA for 1.5 minutes. Plasma was collected, and histamine levels were measured by using competitive ELISA. Results represent means ± SEMs (n [control] = 4; n [CPI-613] = 5). *P < .05. B and C, Increase in lung resistance in response to methacholine (MCH) measured in mice sensitized and challenged with OVA with and without CPI-613 during the challenge periods compared with that in nonsensitized and nonchallenged mice (saline control). Fig 2, B, Response to various doses of MCH. Fig 2, C, Response to maximal dose of MCH. Values represent means ± SEMs (10 mice per group). P < .05 compared with the OVA or saline group. D and E, Inflammatory cell counts in OVA-induced allergic asthmatic mice administered CPI-613 (75 mg/kg) or saline intraperitoneally during the challenge period compared with those in nonsensitized mice (saline control). Fig 2, D, represents total cell number per milliliter in BAL fluid, and Fig 2, E, represents differential BALF cell count. Values represent means ± SEMs (10 mice per group). *P < .05 compared with the OVA or saline group.
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5. Fig 3
Role played by PDH phosphorylation on MITF-PDH interaction in IgE-antigen activation of RBL cells. A, Immunoprecipitation (IP) of MITF in RBL lysates using anti-MITF antibody for IP and anti–PDH-E1α antibody for Western blot analysis. One representative of 3 independent experiments is shown. B, IP of PDH-E1β in 293T cells transfected with MITF-H–flag tag lysates by using anti–PDH-E1β antibody for IP and anti-Flag tag antibody for Western blot analysis. One representative of 3 independent experiments is shown. C, PLA. Fixed and permeabilized RBL cells were incubated with anti-MITF and anti–PDH-E1α antibodies and detected by using the Duolink probe (Sigma-Aldrich, St Louis, Mo). Scale bar = 1 μm. The blue dye (4′-6-diamidino-2-phenylindole dihydrochloride) represents the nucleus, and red dots indicate the positive PLA signal. Red arrows indicate the representative positive PLA signals. One representative of 3 independent experiments is shown. D, IP in quiescent or 5-minute IgE-DNP–activated RBL cell lysates using anti-MITF antibody for IP and PDH-E1α antibody for Western blot analysis. One representative of 3 independent experiments is shown. E, IP in control or CPI-613–treated RBL cell lysates using anti-MITF antibody for IP and anti-PDH antibody for Western blot analysis. One representative of 3 independent experiments is shown.
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6. Fig 4
MITF is localized to the mitochondria of RBL cells. A, RBL cells were fractionated into cytosol and mitochondria. Mitoplasts were prepared by using proteinase K treatment. Levels of MITF, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Bcl-XL were determined by means of Western blot analysis. One representative of 3 independent experiments is shown. B-G, Quantification of mitochondrial MITF localization. To visualize MITF, RBL cells were stained with mouse anti-MITF (C5) antibody, followed by CY2-conjugated anti-mouse secondary antibody. Hoechst staining was used to visualize nuclei. Rabbit anti–complex 1 antibody followed by rhodamine-conjugated anti-rabbit secondary antibody were used to visualize mitochondria. To visualize HDAQ1, a nuclear marker, mouse anti-HDAQ1, and CY2-conjugated anti-mouse secondary antibody were used. To visualize Hsp60, a mitochondrial marker, mouse HSP60 antibody, and anti-mouse CY2-conjugated secondary antibody were used. Afterward, mitochondrial localization of MITF was evaluated by using ImageStream flow cytometry. Localization of HDAQ1 and HSP60 was used as a validity of the staining method. For each treatment, 30,000 cells were collected. Cells that were included in the quantification were not in the mitotic state. Positive single cells were gated, and mitochondrial expression of MITF was plotted. The nuclei staining area was omitted to reduce unspecific plotting. Fig 4, B, Quantification of the similarity between the CY2-stained MITF and Hoechst-stained nuclei, which represents the localization of MITF to the nuclei. Fig 4, C, Quantification of the similarity between the CY2-stained MITF and rhodamine-stained mitochondria, which represents the localization of MITF in the mitochondria. Fig 4, D, Quantification of the similarity between the CY2-stained HDAQ1 (nuclear marker) and Hoechst-stained nuclei, which represents the validity of nuclear staining. Fig 4, E, Quantification of the similarity between the CY2-stained HDAQ1 and rhodamine-stained mitochondria, which represents the validity of the mitochondrial staining. Fig 4, F, Quantification of the similarity between the CY2-stained HSP60 and Hoechst-stained nuclei, which represents the validity of mitochondrial staining. Fig 4, G, Quantification of the similarity between the CY2-stained HSP60 (mitochondrial marker) and the rhodamine-stained mitochondria.
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7. Fig 5
Overexpression of MITF-H and MITF-CE decreased PDH's activity and increased pyruvate levels in RBL cells. A, RBL cells were transfected with EV, MITF-H, or MITF-CE. Forty-eight hours later, cells were lysed, and PDH activity was determined based on PDH activity. An ELISA kit was used to determine PDH activity in the different lysates. Results represent means ± SEMs (n = 3). *P < .05. B, RBL cells were transfected with EV, MITF-H, or MITF-CE, and 48 hours later, cells were lysed and pyruvate levels were determined by using an ELISA kit. Results represent means ± SEMs (n = 3). *P < .05.
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8. Fig 6
Overexpression of MITF-H and MITF-CE decreased ATP and oxygen consumption levels in RBL cells. A, RBL cells were transfected with EV, MITF-H, or MITF-CE. Forty-eight hours later, the cells were lysed, and then total cell ATP levels were measured. Results represent means ± SEMs (n = 3). *P < .05. B, Oxygen consumption analysis. Cells were transfected as for Fig 6, A, and then intracellular oxygen consumption was determined with the Seahorse Extracellular Flux Analyzer. Results represent means ± SEMs (n = 3). *P < .05.
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9. Fig E1
A, Densitometry of pS293PDH/PDH-E1α levels in quiescent (0 minutes) RBL cells and BMMCs or RBL cells and BMMCs that were immune activated for 5, 15, or 30 minutes. Results represent means ± SEMs (n = 3). *P < .05. B, RBL cells were incubated with DMSO or 50, 200, or 300 μmol/L CPI-613 for 15, 30, or 60 minutes, and then degranulation levels were estimated by measuring β-hexosamindase release. Results represent means ± SEMs (n = 3). *P < .05. C, Densitometry of pS293PDH/PDH-E1α levels in RBL cells that were treated with 300 μmol/L CPI-613 for 1 hour or DMSO as a control. Results represent means ± SEMs (n = 3). *P < .05. D, RBL cells were cultured in OXPHOS-dependent medium for 24 hours and then incubated with DMSO or 50, 200, or 300 μmol/L CPI-613 for 15, 30, or 60 minutes, and then mitochondrial ATP levels were measured by using an ATP-lite kit. Results represent means ± SEMs (n = 3). *P < .05. E, RBL cells were cultured in complete medium for 24 hours and then incubated with DMSO or 50, 200, or 300 μmol/L CPI-613 for 15, 30, or 60 minutes, and then ATP levels were measured by using the ATP-lite kit. Results represent means ± SEMs (n = 3). *P < .05. F, RBL cells were incubated with 300 μmol/L CPI-613 for 1 hour or with DMSO, cells were lysed, and levels of β-actin, caspase-3, or cleaved caspase-3 were determined by using Western blot analysis. One representative experiment of 3 in shown. G, Densitometry of PDH-E1α/β-actin levels in RBL cells that were transfected with either siPDH-E1a or nontargeted siRNA and cultured for 48 hours. Results represent means ± SEMs (n = 3). *P < .05. H, Degranulation levels in RBL cells activated with different concentrations (1.25, 2.5, 5, or 40 μg/mL) of calcium ionophore A23187 for 30 minutes. One hour before activation, the cells were incubated with 300 μmol/L CPI-613. DMSO was used as a control. Results represent means ± SEMs (n = 3). *P < .05.
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10. Fig E2
A, Immunoprecipitation (IP) of PDH-E1β in BMMC lysates using anti–PDH-E1β antibody for IP and anti-MITF antibody for Western blot analysis. Corresponding lysates in RIPA buffer were analyzed by using Western blot analysis with MITF and PDH antibodies. One representative of 3 independent experiments is shown. B, Densitometry of precipitated PDH-E1α/precipitated MITF levels in quiescent (0 minutes) or immune-activated (5 minutes) RBL cells. Results represent means ± SEMs (n = 3). *P < .05. C, Densitometry of precipitated PDH-E1α/precipitated MITF levels in RBL cells that were treated with 300 μmol/L CPI-613 for 1 hour or DMSO as a control. Results represent means ± SEMs (n = 3). P = .12. D, IP of MITF in 293T lysates. Twenty-four hours after transfection with EV, MITF-H, or MITF-CE, each was overexpressed with PDH-E1β plasmid. For IP, anti-flag tag antibody was used, and for Western blot analysis, anti-flag tag and anti–PDH-E1β antibodies were used. Corresponding lysates in RIPA buffer were analyzed by means of Western blot analysis of MITF and PDH. One representative of 3 independent experiments is shown.
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11. Fig E3
A, 293T cells were transfected with MITF-H–flag or MITF-CE–flag or EV as a control. Twenty-four hours later, the cells were fractionated to cytosolic and mitochondrial fractions, and Western blot analysis was conducted. B, Anti-flag tag antibody was used to show the different constructs of MITF in the cellular fractions, and caspase-3 was used as a cytosolic marker to show the purity of the mitochondrial fraction. One representative experiment of 3 is shown.
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12. Fig E4
A, RBL cells were treated with 300 μmol/L CPI-613 or DMSO for 1 hour. Then cells were lysed and protein levels were measured by using Bradford analysis, and the samples were diluted for equal protein concentration. The PDC activity ELISA kit was used to determine PDH activity in the different lysates. Results represent means ± SEMs (n = 3). *P < .05. B, RBL cells were treated with dichloroacetic acid or nuclease-free water for 30 minutes. Then cells were lysed and protein levels were measured by using Bradford analysis, and the samples were diluted for equal protein concentration. The PDC activity ELISA kit was used to determine PDH activity in the different lysates. Results represent means ± SEMs (n = 3).
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