Mol. Cells 2022; 45(4): 231-242
Published online March 30, 2022
https://doi.org/10.14348/molcells.2022.5005
© The Korean Society for Molecular and Cellular Biology
Correspondence to : bochoi77@hanmail.net(BOC); jieun.lee@skku.edu(JEL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The neuromuscular junction (NMJ), which is a synapse for signal transmission from motor neurons to muscle cells, has emerged as an important region because of its association with several peripheral neuropathies. In particular, mutations in GARS that affect the formation of NMJ result in Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. These disorders are mainly considered to be caused by neuronal axon abnormalities; however, no treatment is currently available. Therefore, in order to determine whether the NMJ could be targeted to treat neurodegenerative disorders, we investigated the NMJ recovery effect of HDAC6 inhibitors, which have been used in the treatment of several peripheral neuropathies. In the present study, we demonstrated that HDAC6 inhibition was sufficient to enhance movement by restoring NMJ impairments observed in a zebrafish disease model. We found that CKD-504, a novel HDAC6 inhibitor, was effective in repairing NMJ defects, suggesting that treatment of neurodegenerative diseases via NMJ targeting is possible.
Keywords CKD-504, GARS, HDAC6 inhibitor, Neuromuscular Junction, Zebrafish
The neuromuscular junction (NMJ) is a chemical synapse established by motor neurons in the spinal cord and muscle fibers in the peripheral nervous system (PNS). In the NMJ, the presynaptic axons of motor neurons release acetylcholine, which binds to acetylcholine receptors (AChRs) present on the surface of the postsynaptic muscle fibers. Thus, the NMJ is important for transmitting neuronal signals to innervated muscles involved in peripheral movements. Impairments in the NMJ lead to muscle weakness due to disrupted neuronal transmission, which results in several diseases, such as Lambert-Eaton syndrome (presynaptic) and myasthenia gravis (postsynaptic) (Howard, 2018; Kesner et al., 2018; Rodriguez Cruz et al., 2020).
Among peripheral neuropathies, Charcot–Marie–Tooth (CMT) disease is one of the most commonly inherited neuropathies in both sexes and all ethnic groups (Morena et al., 2019). The disease includes numerous subtypes (types 1-7 and X-linked forms), with common symptoms of progressive muscle weakness and atrophy in the early stage and deformities of the foot and hand in the late stage (Morena et al., 2019). CMT type 2 is common, and most causative genes are mainly involved in the axonal development of motor neurons (Borg and Ericson-Gripenstedt, 2002; Loprest et al., 1992). CMT type 2D results from mutations in
Notably, several cases of
In contrast, mutations in
In this study, we investigated the efficacy of HDAC6 inhibition in repairing NMJ defects in peripheral neuropathies. We first generated an NMJ disease model using zebrafish (
Adult zebrafish were maintained with a cycle of 13-h light and 11-h dark in an automatic system (Genomic-Design, Korea) at 28.5°C and pH of 7.0-7.9. The zebrafish embryos were collected via natural breeding and incubated in clean petri dishes containing E3 medium (297.7 mM NaCl, 10.7 mM KCl, 26.1 mM CaCl2, and 24.1 mM MgCl2), with 1% methylene blue (M2662; Samchun Chemicals, Korea), at 28.5°C. To inhibit the production of melanin, which interferes with immunostaining, zebrafish larvae were raised in E3 medium containing 0.2 mM N-phenylthiourea (P7629; Sigma-Aldrich, USA). Animal research was reviewed and approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute/Samsung Medical Center and Sungkyunkwan University (IACUC#20201008001 and IACUC#20200916001).
To block the expression of zebrafish
Zebrafish larvae at 84 h post-fertilization (hpf) were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4°C overnight. The fixed zebrafish larvae were permeabilized with PBST (0.5% TritonX-100 in 0.1 M PBS) for 15 min. The larvae were then washed with 1× PBS three times and blocked in PBDT (1% DMSO and 1% BSA in PBST) containing 4% normal goat serum at room temperature for 1 h. The larvae were incubated with the following primary antibodies: mouse anti-SV2 (1:50; Developmental Studies Hybridoma Bank, USA) and Alexa 647-conjugated α-BTX (B35450, 1:150; Molecular Probes, USA) antibodies at 4°C overnight in blocking solution. After washing three times with PBST, the larvae were incubated with Alexa Fluor 488-conjugated secondary antibodies (mouse A11001, 1:250; Life Technologies, USA) at RT for 2 h. The larvae were mounted on slides with PBS containing 70% glycerol. To generate imaging data, the mounted larvae were imaged using a confocal microscope (LSM 700; Carl Zeiss, Germany) and analyzed using ImageJ (NIH, USA) or Zeiss ZEN imaging software. The formed presynapse, postsynapse, and NMJ were analyzed by measuring the proportion of areas with green, red, and merged yellow signals within the region of interest (ROI) of the zebrafish trunk, respectively. To assess the extent of NMJ innervation, the proportion of areas with yellow signals within each area of green and red signals was measured.
At 3 days post-fertilization (dpf), zebrafish larvae were washed with PBS and lysed with T-per tissue protein extraction buffer (78510; Thermo Scientific, USA). Protein samples (30 μg) were denatured at 100°C for 5 min, and then separated using SDS-PAGE. The proteins were transferred onto a 0.45 μm PVDF membrane (IPVH00010; Millipore, USA), and the membrane was immersed in a blocking solution (TBST [Tris-buffered saline pH 7.5, containing 0.5% Tween-20] containing 5% skim milk [232100; BD Biosciences, USA]) at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C: anti-GARS (sc-365442; Santa Cruz Biotechnology, USA), anti-HDAC6 (07-732; Millipore), anti-acetylated tubulin (T7451; Sigma-Aldrich), and anti-β-actin (sc-47778; Santa Cruz Biotechnology). The membranes were washed three times with TBST buffer and incubated with secondary antibodies for 1 h at room temperature. Finally, the membrane was enhanced with a chemiluminescence substrate (NEL104001EA; PerkinElmer, USA) to visualize the specific proteins. The expression level of each protein was normalized to that of β-actin in each blot and quantified using ImageJ.
The velocities of the zebrafish larvae were analyzed using light stimulation using with DanioVision (Noldus, Netherlands). Individual larvae injected with MOs or mRNAs were transferred onto 24-well plates, with each well containing 1 ml of E3 medium. The movements of the larvae that responded to light were recorded for 30 min and analyzed using EthoVision XT software (Noldus).
All drugs and chemicals tested in this study were used twice in zebrafish at 48 and 72 hpf until fixation at 84 hpf. Vorinostat and pomiferin, selected from the Pharmakon-1760 collection (MicroSource Discovery Systems, USA), were dissolved in DMSO and used at a concentration of 10 μM in larvae submerged in E3 media. Tubastatin A and CKD-504 were dissolved in distilled water and used at a concentration of 20 μM.
All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, USA). Values are presented as mean ± SD or fold change relative to the mean control. Differences between two groups were evaluated using unpaired Student’s
The zebrafish was used to model peripheral neuropathy with NMJ defects
Next, to determine whether the deficit of
Based on the identification of mutations in
To clarify whether GARS-mediated NMJ development is involved in peripheral neuropathology, we investigated the effects of
Previous studies have reported that mutant forms of
To elucidate the efficacy of HDAC6 inhibitors as potential treatments for NMJ disorders, we focused on U.S. Food and Drug Administration (FDA)-approved drugs that have been used to treat cancers, but not peripheral neuropathies. We considered two different drugs, vorinostat and pomiferin, that inhibit HDACs, including HDAC6, in cancer (Licciardi and Karagiannis, 2012; Mariadason, 2008; Namdar et al., 2010). Vorinostat is the first FDA-approved treatment for cutaneous T-cell lymphoma with HDAC6 inhibitory activity (Mann et al., 2007). In contrast, pomiferin is a novel HDAC inhibitor isolated from flavonoid compounds (Son et al., 2007) and has been proven to be effective in inhibiting the growth of colon cancer cells (Mariadason, 2008; Son et al., 2007). We investigated the therapeutic effects of vorinostat and pomiferin on the impaired NMJs of
A recently reported novel HDAC6 inhibitor, CKD-504, which specifically affects the acetylation of α-tubulin (Choi et al., 2020), is effective in treating not only PNS disorders (e.g., CMT type 1A) but also in CNS disorders (e.g., Alzheimer’s disease) (Choi et al., 2020; Ha et al., 2020). Hence, as compared with vorinostat and pomiferin, we determined whether CKD-504 could lead to recovery from NMJ diseases, such as CMT type 2D, by influencing the GARS-related NMJ formation. We injected
Here, we hypothesize that HDAC6 inhibition is key in repairing NMJ damage associated with peripheral neuropathies, including CMTs. We demonstrated that a novel HDAC6 inhibitor, CKD-504, is effective in restoring NMJ deficits in zebrafish larvae lacking
Posttranslational modifications (PTMs) of microtubules, such as acetylation, detyrosination, polyglutamylation, and polyglycylation, are important for the regulation of microtubule polymerization and depolymerization, which are major determinants of microtubule stability (Janke and Bulinski, 2011). In particular, (de)acetylation of Lys40 of α-tubulin in the lumen of microtubules is modulated by α-tubulin acetyltransferase (αTAT) and HDAC6 (Asthana et al., 2013; Kalebic et al., 2013; Zhang et al., 2003). The reciprocal function of αTAT and HDAC6 is also associated with several signaling pathways, such as Rho/ROCK signaling, that regulate axonal growth through the cytoskeletal network (Wong et al., 2018). Previous reports have shown that HDAC6 inhibition in neurodegenerative diseases leads to an increase in microtubule acetylation, which improves neurite growth, axonal transportation, neuroprotection, and mitochondrial movements (Simoes-Pires et al., 2013; Wenzel et al., 2019). It is noteworthy that HDAC6 inhibition was involved not only in enhancing NMJ stability but also in the clustering of AChRs on the postsynaptic side of the NMJ (Osseni et al., 2020; Smith et al., 2022). Correspondingly, our data clearly showed that
CKD-504, a hydroxybenzamide HDAC6 inhibitor that chelates Zn2+, has emerged as an attractive therapeutic agent for Huntington’s disease (HD) and CMT (Ha et al., 2020). The advantages of CKD-504 include its high enzymatic activity and selectivity for HDAC6 (Ha et al., 2020). Intracellular transport along the microtubules in motor neurons is important for axonal guidance via the NMJ to muscle cells (Banerjee and Riordan, 2018; Vilmont et al., 2016), and HDAC6 is a negative regulator of intracellular transport (Valenzuela-Fernandez et al., 2008; Wenzel et al., 2019). A previous study suggested that HDAC6 inhibitors used in the treatment of HD act on mechanisms to enhance the intracellular transport of brain-derive neurotrophic factor and increase microtubule acetylation (Dompierre et al., 2007). Individuals with CMT exhibiting mutations in
This work was supported by the National Research Foundation, funded by the Korean government’s MSIP (2021R1A2C3004572 to J.E.L., 2021R1A6A3A13041249 to H.S.J., 2021R1A4A2001389 to K.W.C., B.O.C., and J.E.L.), and was supported by the National Institutes of Health, USA (NIH RO1 NS094388 to B.O.C. and D.H.K.).
H.S.J. conducted the experiments and analyzed the data with substantial contributions from H.J.K., D.H.K., K.W.C., and B.O.C. who advised the experimental designs and commented on the manuscript. J.E.L. designed the experiments and wrote the manuscript with substantial contributions from H.S.J. and B.O.C.
D.H.K. is a scientific founder and equity holder of Curi Bio. The other authors have no potential conflicts of interest to disclose.
Mol. Cells 2022; 45(4): 231-242
Published online April 30, 2022 https://doi.org/10.14348/molcells.2022.5005
Copyright © The Korean Society for Molecular and Cellular Biology.
Hui Su Jeong1 , Hye Jin Kim1
, Deok-Ho Kim2,3,4
, Ki Wha Chung5
, Byung-Ok Choi1,6,*
, and Ji Eun Lee1,7,*
1Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences & Technology, Sungkyunkwan University, Seoul 06351, Korea, 2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA, 3Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, 4Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, 5Department of Biological Sciences, Kongju National University, Gongju 32588, Korea, 6Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea, 7Samsung Biomedical Research Institute, Samsung Medical Center, Seoul 06351, Korea
Correspondence to:bochoi77@hanmail.net(BOC); jieun.lee@skku.edu(JEL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The neuromuscular junction (NMJ), which is a synapse for signal transmission from motor neurons to muscle cells, has emerged as an important region because of its association with several peripheral neuropathies. In particular, mutations in GARS that affect the formation of NMJ result in Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. These disorders are mainly considered to be caused by neuronal axon abnormalities; however, no treatment is currently available. Therefore, in order to determine whether the NMJ could be targeted to treat neurodegenerative disorders, we investigated the NMJ recovery effect of HDAC6 inhibitors, which have been used in the treatment of several peripheral neuropathies. In the present study, we demonstrated that HDAC6 inhibition was sufficient to enhance movement by restoring NMJ impairments observed in a zebrafish disease model. We found that CKD-504, a novel HDAC6 inhibitor, was effective in repairing NMJ defects, suggesting that treatment of neurodegenerative diseases via NMJ targeting is possible.
Keywords: CKD-504, GARS, HDAC6 inhibitor, Neuromuscular Junction, Zebrafish
The neuromuscular junction (NMJ) is a chemical synapse established by motor neurons in the spinal cord and muscle fibers in the peripheral nervous system (PNS). In the NMJ, the presynaptic axons of motor neurons release acetylcholine, which binds to acetylcholine receptors (AChRs) present on the surface of the postsynaptic muscle fibers. Thus, the NMJ is important for transmitting neuronal signals to innervated muscles involved in peripheral movements. Impairments in the NMJ lead to muscle weakness due to disrupted neuronal transmission, which results in several diseases, such as Lambert-Eaton syndrome (presynaptic) and myasthenia gravis (postsynaptic) (Howard, 2018; Kesner et al., 2018; Rodriguez Cruz et al., 2020).
Among peripheral neuropathies, Charcot–Marie–Tooth (CMT) disease is one of the most commonly inherited neuropathies in both sexes and all ethnic groups (Morena et al., 2019). The disease includes numerous subtypes (types 1-7 and X-linked forms), with common symptoms of progressive muscle weakness and atrophy in the early stage and deformities of the foot and hand in the late stage (Morena et al., 2019). CMT type 2 is common, and most causative genes are mainly involved in the axonal development of motor neurons (Borg and Ericson-Gripenstedt, 2002; Loprest et al., 1992). CMT type 2D results from mutations in
Notably, several cases of
In contrast, mutations in
In this study, we investigated the efficacy of HDAC6 inhibition in repairing NMJ defects in peripheral neuropathies. We first generated an NMJ disease model using zebrafish (
Adult zebrafish were maintained with a cycle of 13-h light and 11-h dark in an automatic system (Genomic-Design, Korea) at 28.5°C and pH of 7.0-7.9. The zebrafish embryos were collected via natural breeding and incubated in clean petri dishes containing E3 medium (297.7 mM NaCl, 10.7 mM KCl, 26.1 mM CaCl2, and 24.1 mM MgCl2), with 1% methylene blue (M2662; Samchun Chemicals, Korea), at 28.5°C. To inhibit the production of melanin, which interferes with immunostaining, zebrafish larvae were raised in E3 medium containing 0.2 mM N-phenylthiourea (P7629; Sigma-Aldrich, USA). Animal research was reviewed and approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute/Samsung Medical Center and Sungkyunkwan University (IACUC#20201008001 and IACUC#20200916001).
To block the expression of zebrafish
Zebrafish larvae at 84 h post-fertilization (hpf) were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4°C overnight. The fixed zebrafish larvae were permeabilized with PBST (0.5% TritonX-100 in 0.1 M PBS) for 15 min. The larvae were then washed with 1× PBS three times and blocked in PBDT (1% DMSO and 1% BSA in PBST) containing 4% normal goat serum at room temperature for 1 h. The larvae were incubated with the following primary antibodies: mouse anti-SV2 (1:50; Developmental Studies Hybridoma Bank, USA) and Alexa 647-conjugated α-BTX (B35450, 1:150; Molecular Probes, USA) antibodies at 4°C overnight in blocking solution. After washing three times with PBST, the larvae were incubated with Alexa Fluor 488-conjugated secondary antibodies (mouse A11001, 1:250; Life Technologies, USA) at RT for 2 h. The larvae were mounted on slides with PBS containing 70% glycerol. To generate imaging data, the mounted larvae were imaged using a confocal microscope (LSM 700; Carl Zeiss, Germany) and analyzed using ImageJ (NIH, USA) or Zeiss ZEN imaging software. The formed presynapse, postsynapse, and NMJ were analyzed by measuring the proportion of areas with green, red, and merged yellow signals within the region of interest (ROI) of the zebrafish trunk, respectively. To assess the extent of NMJ innervation, the proportion of areas with yellow signals within each area of green and red signals was measured.
At 3 days post-fertilization (dpf), zebrafish larvae were washed with PBS and lysed with T-per tissue protein extraction buffer (78510; Thermo Scientific, USA). Protein samples (30 μg) were denatured at 100°C for 5 min, and then separated using SDS-PAGE. The proteins were transferred onto a 0.45 μm PVDF membrane (IPVH00010; Millipore, USA), and the membrane was immersed in a blocking solution (TBST [Tris-buffered saline pH 7.5, containing 0.5% Tween-20] containing 5% skim milk [232100; BD Biosciences, USA]) at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C: anti-GARS (sc-365442; Santa Cruz Biotechnology, USA), anti-HDAC6 (07-732; Millipore), anti-acetylated tubulin (T7451; Sigma-Aldrich), and anti-β-actin (sc-47778; Santa Cruz Biotechnology). The membranes were washed three times with TBST buffer and incubated with secondary antibodies for 1 h at room temperature. Finally, the membrane was enhanced with a chemiluminescence substrate (NEL104001EA; PerkinElmer, USA) to visualize the specific proteins. The expression level of each protein was normalized to that of β-actin in each blot and quantified using ImageJ.
The velocities of the zebrafish larvae were analyzed using light stimulation using with DanioVision (Noldus, Netherlands). Individual larvae injected with MOs or mRNAs were transferred onto 24-well plates, with each well containing 1 ml of E3 medium. The movements of the larvae that responded to light were recorded for 30 min and analyzed using EthoVision XT software (Noldus).
All drugs and chemicals tested in this study were used twice in zebrafish at 48 and 72 hpf until fixation at 84 hpf. Vorinostat and pomiferin, selected from the Pharmakon-1760 collection (MicroSource Discovery Systems, USA), were dissolved in DMSO and used at a concentration of 10 μM in larvae submerged in E3 media. Tubastatin A and CKD-504 were dissolved in distilled water and used at a concentration of 20 μM.
All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, USA). Values are presented as mean ± SD or fold change relative to the mean control. Differences between two groups were evaluated using unpaired Student’s
The zebrafish was used to model peripheral neuropathy with NMJ defects
Next, to determine whether the deficit of
Based on the identification of mutations in
To clarify whether GARS-mediated NMJ development is involved in peripheral neuropathology, we investigated the effects of
Previous studies have reported that mutant forms of
To elucidate the efficacy of HDAC6 inhibitors as potential treatments for NMJ disorders, we focused on U.S. Food and Drug Administration (FDA)-approved drugs that have been used to treat cancers, but not peripheral neuropathies. We considered two different drugs, vorinostat and pomiferin, that inhibit HDACs, including HDAC6, in cancer (Licciardi and Karagiannis, 2012; Mariadason, 2008; Namdar et al., 2010). Vorinostat is the first FDA-approved treatment for cutaneous T-cell lymphoma with HDAC6 inhibitory activity (Mann et al., 2007). In contrast, pomiferin is a novel HDAC inhibitor isolated from flavonoid compounds (Son et al., 2007) and has been proven to be effective in inhibiting the growth of colon cancer cells (Mariadason, 2008; Son et al., 2007). We investigated the therapeutic effects of vorinostat and pomiferin on the impaired NMJs of
A recently reported novel HDAC6 inhibitor, CKD-504, which specifically affects the acetylation of α-tubulin (Choi et al., 2020), is effective in treating not only PNS disorders (e.g., CMT type 1A) but also in CNS disorders (e.g., Alzheimer’s disease) (Choi et al., 2020; Ha et al., 2020). Hence, as compared with vorinostat and pomiferin, we determined whether CKD-504 could lead to recovery from NMJ diseases, such as CMT type 2D, by influencing the GARS-related NMJ formation. We injected
Here, we hypothesize that HDAC6 inhibition is key in repairing NMJ damage associated with peripheral neuropathies, including CMTs. We demonstrated that a novel HDAC6 inhibitor, CKD-504, is effective in restoring NMJ deficits in zebrafish larvae lacking
Posttranslational modifications (PTMs) of microtubules, such as acetylation, detyrosination, polyglutamylation, and polyglycylation, are important for the regulation of microtubule polymerization and depolymerization, which are major determinants of microtubule stability (Janke and Bulinski, 2011). In particular, (de)acetylation of Lys40 of α-tubulin in the lumen of microtubules is modulated by α-tubulin acetyltransferase (αTAT) and HDAC6 (Asthana et al., 2013; Kalebic et al., 2013; Zhang et al., 2003). The reciprocal function of αTAT and HDAC6 is also associated with several signaling pathways, such as Rho/ROCK signaling, that regulate axonal growth through the cytoskeletal network (Wong et al., 2018). Previous reports have shown that HDAC6 inhibition in neurodegenerative diseases leads to an increase in microtubule acetylation, which improves neurite growth, axonal transportation, neuroprotection, and mitochondrial movements (Simoes-Pires et al., 2013; Wenzel et al., 2019). It is noteworthy that HDAC6 inhibition was involved not only in enhancing NMJ stability but also in the clustering of AChRs on the postsynaptic side of the NMJ (Osseni et al., 2020; Smith et al., 2022). Correspondingly, our data clearly showed that
CKD-504, a hydroxybenzamide HDAC6 inhibitor that chelates Zn2+, has emerged as an attractive therapeutic agent for Huntington’s disease (HD) and CMT (Ha et al., 2020). The advantages of CKD-504 include its high enzymatic activity and selectivity for HDAC6 (Ha et al., 2020). Intracellular transport along the microtubules in motor neurons is important for axonal guidance via the NMJ to muscle cells (Banerjee and Riordan, 2018; Vilmont et al., 2016), and HDAC6 is a negative regulator of intracellular transport (Valenzuela-Fernandez et al., 2008; Wenzel et al., 2019). A previous study suggested that HDAC6 inhibitors used in the treatment of HD act on mechanisms to enhance the intracellular transport of brain-derive neurotrophic factor and increase microtubule acetylation (Dompierre et al., 2007). Individuals with CMT exhibiting mutations in
This work was supported by the National Research Foundation, funded by the Korean government’s MSIP (2021R1A2C3004572 to J.E.L., 2021R1A6A3A13041249 to H.S.J., 2021R1A4A2001389 to K.W.C., B.O.C., and J.E.L.), and was supported by the National Institutes of Health, USA (NIH RO1 NS094388 to B.O.C. and D.H.K.).
H.S.J. conducted the experiments and analyzed the data with substantial contributions from H.J.K., D.H.K., K.W.C., and B.O.C. who advised the experimental designs and commented on the manuscript. J.E.L. designed the experiments and wrote the manuscript with substantial contributions from H.S.J. and B.O.C.
D.H.K. is a scientific founder and equity holder of Curi Bio. The other authors have no potential conflicts of interest to disclose.
Hyunju Ro, Kyungchull Soun, Eun-jung Kim, Myungchull Rhee
Mol. Cells 2004; 17(2): 373-376