EMBO/SourceData huggingface dataset:
Figure 1HeLa cells co-transfected with EMTB-CFP-FRB and YFP-FKBP were treated with 100 nM rapamycin (Rapa). Addition of rapamycin rapidly translocated YFP-FKBP onto EMTB-CFP-FRB-labeled microtubules and increased the FRET signal. The scale for the FRET/CFP intensity ratio is shown. Scale bar, 10 μm. The normalized intensity of FRET/CFP in cells before and after rapamycin (blue) and 0.1% DMSO (control; red) treatment. n = 6 and 10 cells in the rapamycin and DMSO groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.HeLa cells co-transfected with the indicated constructs were treated with nocodazole (Noc, 3.3 µM; left) or rapamycin (Rapa, 100 nM; right) for 0, 30, and 60 min. Transfected cells were then fixed and labeled with anti-acetylated tubulin (red). Dotted lines indicate the boundary of transfected cells. Scale bars, 10 μm. The intensity of acetylated tubulin in cells transfected with EMTB-CFP-FRB (orange) or cotransfected with EMTB-CFP-FRB and dNSpastin3Q-YFP-FKBP (blue) after addition of nocodazole (Noc, 3.3 µM) or rapamycin (Rapa, 100 nM) for the indicated times. n = 123 and 81 cells in the nocodazole and dNSpastin3Q groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed, with p-values indicated.
Figure 2NIH3T3 fibroblasts co-transfected with 5HT6-mCherry (5HT6-mCh; a ciliary membrane marker; red), CFP-FRB-MAP4m (blue), and dNSpastin3Q-YFP-FKBP (green) were serum-starved for 24 hr to induce ciliogenesis. Ciliated cells were then treated with rapamycin (Rapa, 100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to axonemal microtubules. Dotted lines indicate the cell boundaries. Scale bar, 5 μm. The normalized length of axonemes (blue), primary cilia (red), and dNSpastin3Q-YFP-FKBP in cilia (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.HeLa cells co-transfected with H2B-mCherry (H2B-mCh; a chromosome marker; red), CFP-FRB-MAP4m (a marker of mitotic spindles; blue), and dNSpastin3Q-YFP-FKBP (green) were synchronized in metaphase and treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to mitotic spindles. Dotted lines indicate the cell boundaries. Scale bar, 10 μm. The normalized area of the mitotic spindle (blue) and intensity of dNSpastin3Q-YFP-FKBP in the mitotic spindle (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.HeLa cells co-transfected with H2B-mCherry (red), CFP-FRB-MAP4m (intercellular bridges; blue), and dNSpastin3Q-YFP-FKBP (green) were treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to intercellular bridges. Arrows indicate the intercellular bridges. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.   The normalized area of intercellular bridges (blue) and intensity of dNSpastin3Q-YFP-FKBP at intercellular bridges (green) upon rapamycin treatment. n = 5 cells from three independent experiments. Data are shown as the mean ± S.E.M.
Figure 3COS7 cells transfected with TagRFP-FRB-A1AY1 (red) were labeled by anti-α-tubulin antibody (green; upper panel), anti-tyrosinated tubulin antibody (green; middle panel), or anti-detyrosinated tubulin antibody (green; lower panel), respectively. Scale bar, 10 µm. The intensity profiles of TagRFP-FRB-A1AY1 (red) and tubulin with the indicated PTMs (green) along dotted lines drawn in (A). The Pearson's correlation coefficients for TagRFP-FRB-A1AY1 and indicated tubulin (tub) subtypes were calculated, and data are shown as the mean ± S.E.M. n = 16, 13, and 15 cells from left to right, respectively; three independent experiments.COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and TagCFP-FKBP (green) were treated with rapamycin (100 nM). The addition of rapamycin rapidly recruits TagCFP-FKBP from cytosol onto A1AY1-labeled microtubules (arrows). Scale bar, 10 µm. The normalized intensity of TagCFP-FKBP at A1AY1-labeled microtubules upon rapamycin treatments. n=7 cells from three independent experiments. Data are shown as the mean ± S.E.M.COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and dNSpastin3Q-TagCFP-FKBP (cyan) were treated with 0.1% DMSO or rapamycin (100 nM) for 1 hr and followed by immunostaining with anti-tyrosinated tubulin antibody (green). Dotted lines highlight the transfected cells. Scale bar, 20 μm. The normalized intensity of tyrosinated microtubules in TagRFP-FRB-A1AY1 and dNSpastin3Q-TagCFP-FKBP co-transfected cells upon 0.1% DMSO or rapamycin treatment for 1 hr. n=31 and 26 cells in DMSO and rapamycin treated groups, respectively, from three independent experiments. Data (blue) represent as mean ± S.E.M. Student's t-tests were performed with p values indicated.
Figure 4COS7 cells co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red) were incubated with SPY650-tubulin (SPY650-tub) to visualize microtubules. The cells were illuminated by blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm. The normalized intensity of mCh-Cry2 at microtubules in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.COS7 cells co-transfected with EMTB-YFP-CIBN and dNSpastin3Q-mCh-Cry2 were incubated with SPY650-tubulin to visualize microtubules. The cells were illuminated by blue light as in (A). Scale bar, 10 μm. The normalized intensity of dNSpastin3Q-mCh-Cry2 and mCh-Cry2 at microtubules and the normalized area of SPY650-tubulin in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastin3Q-mCh-Cry2 and mCh-Cry2 groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.
Figure 5E-F. COS7 cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon were treated with rapamycin (100 nM) for acute microtubule disruption. The morphology of mitochondria at different levels of microtubule disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.G-H. The normalized intensity of MitoTracker Red in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB and dNSpastin3Q-YFP-FKBP or dNSpastin3QED-YFP-FKBP after rapamycin (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.
Figure 6COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Lifeact-Neon (heatmap and green) were treated with rapamycin (Rapa, 100 nM) to induce microtubule disassembly. Arrows and arrowheads indicate lamellipodia and stress fibers, respectively. Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals. Scale bar, 10 µm. The normalized level of microtubule area, lamellipodia length, and filopodia density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.
Figure 1B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.F, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).
Figure 2A. The most effective one among the candidate compounds, Carfilzomib, significantly promotes the expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage markers. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the expression of M1 (Il-6/Inos) or M2 (Cd206/Arg1) macrophage markers were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).D. Statistics for proportion of CD86, CD80, MHC-II or CD206 positive cells in BMDMs. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).E. Carfilzomib promotes the phagocytosis of macrophages. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM) for 12 hours. After starving for 2 hours and stained with Red membrane dye, BMDMs (red) were incubated with L1210-GFP cells (green) in serum-free medium for another 2 hours. Phagocytosis effect was observed and photographed under fluorescence microscope. Scale bars, 50 μm (up), 20 μm (down). The yellow arrows indicated L1210 that is phagocytosed by macrophages. F. Statistics represent the number of phagocytosis L1210 in 100 macrophages. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).M. Carfilzomib promotes the M1-polarization of Tumor associated macrophages in vitro. BMDMs were cultured with tumor culture supernatant (TSN) produced by L1210 cells, followed by activation with DMSO or Carfilzomib (1 μM). RNA was extracted from cells and the expression of Il-1β, Il-6, Inos were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).
Figure 3A, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes (Bip, Chop) were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).F. ER stress agonists, TUN and TG, promote the expression of M1 macrophage markers, as well as reduce the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).
Figure 4A, B Deficiency of Ern1 reduces the expression of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). The mRNA of Il-1β and Il-6 were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).E. Kira6 inhibits the expression of ER stress related genes and inflammatory related genes. BMDMs was treated by IL-4 (20 ng/mL) for 24 hours, treated with Kira6 (100 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression of Bip, Chop, Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test). F, G. Carfilzomib has no effect on splicing of XBP1 in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).J. Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages. Raw264.7 cells were pretreated by IL-4 (20ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.
Figure 5C. Representative images of Hematoxylin and eosin (H&E) staining of the lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. D. Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression of CD80 and CD206 by flow cytometry. F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test). G. Carfilzomib treatment promotes the infiltration and activation of CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry. H. Statistics about the proportion of CD8+ or CD69+ positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).
Figure 6D. CD4+ and CD8+ T cells play a role in mediating cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by saline, Carfilzomib, anti-mouse CD4 antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment. E. Representative images of H&E staining of the treated lung tissues. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice were treated by saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning was performed after two-week treatment. G. Representative images of H&E staining of the lung tissues. Expression of Ki-67 in lung tissue was detected by immunohistochemistry method.
Figure 7A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).
Figure 1(A,B) Effect of MPC inhibition on cellular lipid droplet content. (A) Live-cell super-resolution confocal imaging of primary brown adipocytes pre-stained overnight with the fatty acid tracer BODIPY C12 558/568 (Bodipy C12, red). Cells were stained with mitotracker green (MTG, green) prior to imaging. Cells were imaged before and 120 minutes after treatment with either vehicle (DMSO) or 100 nM UK5099. As a positive control, cells were imaged 30 min after treatment with 1 µM norepinephrine. LD, lipid droplet; N, nucleus. Scale bar=10 µm. (B) Quantification of changes in lipid droplet cross-sectional area with indicated treatments from images shown in (A). Data are represented as percentage change in LD area at time=120 min compared to time=0 min.(C,D) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides (TAGs) (C) Representative thin-layer chromatography (TLC) plate of lipids extracted from primary brown adipocytes and cell culture media. Cells were incubated with Bodipy C12 in presence of vehicle (V), 100 nM UK5099 (UK) or 1 µM norepinephrine (NE) for 24 h and triacylglyceride (TAG) synthesis (Lipids in cells) and fatty acid uptake (Lipids in media) were detected. The relative polarity of the lipid species determines the motility, with nonpolar TAG migrating the furthest. (D) Quantification of C12 uptake from media, TAG in cells and TAG per amount of free fatty acid uptake shown in (C), n=5 individual experiments. UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake, similarly to NE.(E-G) Effect of UK5099 treatment on cellular energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or UK5099 at indicated concentrations for 2 h. Oxygen consumption rates (OCR) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK-5099. (E) Quantification of non-stimulated OCR from n=3-7 individual experiments. Data were normalized to vehicle for each individual experiment. UK5099 increases OCR in non-stimulated brown adipocytes. vehicle. (F) Brown adipocytes were treated with 100 nM UK5099 or vehicle. Etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. Representative OCR traces averaging 6 technical replicates. (G) Quantification of non-stimulated OCR, etomoxir sensitive, and etomoxir insensitive OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. *(H,I) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid. (H) Schematic representation of metabolite tracing using [U-13C16] palmitate. (I) [U-13C16] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h. Data shows mole percent enrichment (MPE) of isotope labeled substrate into respective metabolite.
Figure 2(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment.Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (C) Representative Western Blot analysis of MPC1, MPC2 and actin. (D) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in (C) from n=3 individual experiments.Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (E) mRNA levels of MPC1 and MPC2 from n=3 individual experiments.Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment.