Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann-Pick C1 mutant cells
2017; Elsevier BV; Volume: 58; Issue: 4 Linguagem: Inglês
10.1194/jlr.m072140
ISSN1539-7262
AutoresNina H. Pipalia, Kanagaraj Subramanian, Shu Mao, Harold Ralph, Darren M. Hutt, Samantha M. Scott, William E. Balch, Frederick R. Maxfield,
Tópico(s)Adenosine and Purinergic Signaling
ResumoNiemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat. Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat. ERRATUMJournal of Lipid ResearchVol. 58Issue 9PreviewRegarding "Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells" (J. Lipid Res. 58: 695–708): Full-Text PDF Open Access Lipoprotein-derived cholesterol is normally transported out of late endosomes and lysosome (LE/Ly) by a process that requires the Niemann-Pick C1 (NPC1) and NPC2 proteins (1Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Crossref PubMed Scopus (700) Google Scholar, 2Watari H. Blanchette-Mackie E.J. Dwyer N.K. Glick J.M. Patel S. Neufeld E.B. Brady R.O. Pentchev P.G. Strauss 3rd, J.F. Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization.Proc. Natl. Acad. Sci. USA. 1999; 96: 805-810Crossref PubMed Scopus (126) Google Scholar). A current model (3Infante R.E. Wang M.L. 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In a previous study (19Pipalia N.H. Cosner C.C. Huang A. Chatterjee A. Bourbon P. Farley N. Helquist P. Wiest O. Maxfield F.R. Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts.Proc. Natl. Acad. Sci. USA. 2011; 108: 5620-5625Crossref PubMed Scopus (148) Google Scholar), we showed that histone deacetylase inhibitors (HDACis), including vorinostat (also called suberoylanilide hydroxamic acid, or SAHA) and panobinostat (LBH589), are remarkably effective in correcting the NPC1 phenotype in human fibroblast cells that have an NPC1I1061T mutation. The pharmacological profile was most consistent with the effects being attributed to inhibition of HDACs 1, 2, or 3 (20Helquist P. Maxfield F.R. Wiech N.L. Wiest O. Treatment of Niemann–pick type C disease by histone deacetylase inhibitors.Neurotherapeutics. 2013; 10: 688-697Crossref PubMed Scopus (45) Google Scholar). Treatment of patient-derived fibroblasts with HDACi reduced the accumulation of cholesterol in lysosomal storage organelles (LSOs) and restored other aspects of cholesterol homeostasis, including normal processing of sterol regulatory element-binding protein 2 and reduction of the expression of LDL receptors (19Pipalia N.H. Cosner C.C. Huang A. Chatterjee A. Bourbon P. Farley N. Helquist P. Wiest O. Maxfield F.R. Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts.Proc. Natl. Acad. Sci. USA. 2011; 108: 5620-5625Crossref PubMed Scopus (148) Google Scholar, 21Munkacsi A.B. Chen F.W. Brinkman M.A. Higaki K. Gutierrez G.D. Chaudhari J. Layer J.V. Tong A. Bard M. Boone C. An "exacerbate-reverse" strategy in yeast identifies histone deacetylase inhibition as a correction for cholesterol and sphingolipid transport defects in human Niemann-Pick type C disease.J. Biol. Chem. 2011; 286: 23842-23851Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). HDACi treatment did not correct the cholesterol storage defect of patient-derived cells expressing NPC2 mutations (19Pipalia N.H. Cosner C.C. Huang A. Chatterjee A. Bourbon P. Farley N. Helquist P. Wiest O. Maxfield F.R. Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts.Proc. Natl. Acad. Sci. USA. 2011; 108: 5620-5625Crossref PubMed Scopus (148) Google Scholar), indicating that the HDACis do not bypass the need for the NPC1/NPC2 transport system as HPBCD does (22Rosenbaum A.I. Zhang G. Warren J.D. Maxfield F.R. Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells.Proc. Natl. Acad. Sci. USA. 2010; 107: 5477-5482Crossref PubMed Scopus (203) Google Scholar). This indicated that the HDACi might work by allowing the mutant NPC1 proteins to function sufficiently well to correct the cholesterol transport out of LSOs. Vorinostat and panobinostat do enter the CNS, although the levels achieved in the brain are much lower than in the plasma (20Helquist P. Maxfield F.R. Wiech N.L. Wiest O. Treatment of Niemann–pick type C disease by histone deacetylase inhibitors.Neurotherapeutics. 2013; 10: 688-697Crossref PubMed Scopus (45) Google Scholar, 23Palmieri D. Lockman P.R. Thomas F.C. Hua E. Herring J. Hargrave E. Johnson M. Flores N. Qian Y. Vega-Valle E. Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA double-strand breaks.Clin. Cancer Res. 2009; 15: 6148-6157Crossref PubMed Scopus (125) Google Scholar, 24.Food and Drug Administration. 2014. Application no. 205353Orig1s000; Farydak (panobinostat) http://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/205353Orig1s000PharmR.pdf.Google Scholar). Nevertheless, there is some evidence that vorinostat has effects on tumors in brains (23Palmieri D. Lockman P.R. Thomas F.C. Hua E. Herring J. Hargrave E. Johnson M. Flores N. Qian Y. Vega-Valle E. Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA double-strand breaks.Clin. Cancer Res. 2009; 15: 6148-6157Crossref PubMed Scopus (125) Google Scholar). Some other HDACis do cross the blood-brain barrier more efficiently and have been shown to have neurological effects in animal studies (25Schroeder F.A. Chonde D.B. Riley M.M. Moseley C.K. Granda M.L. Wilson C.M. Wagner F.F. Zhang Y-L. Gale J. Holson E.B. FDG-PET imaging reveals local brain glucose utilization is altered by class I histone deacetylase inhibitors.Neurosci. Lett. 2013; 550: 119-124Crossref PubMed Scopus (12) Google Scholar). The mechanism by which HDACi might restore the function of mutant NPC1 proteins has not been determined. It has been observed that there is more rapid degradation of the NPC1I1061T protein as compared with WT NPC1 protein, and it was proposed that this is because of enhanced endoplasmic reticulum-associated degradation (ERAD) of the mutant protein (26Gelsthorpe M.E. Baumann N. Millard E. Gale S.E. Langmade S.J. Schaffer J.E. Ory D.S. Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding.J. Biol. Chem. 2008; 283: 8229-8236Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Treatment of cells expressing NPC1I1061T with HDACi such as panobinostat or vorinostat increased the expression of the mutant NPC1 protein (19Pipalia N.H. Cosner C.C. Huang A. Chatterjee A. Bourbon P. Farley N. Helquist P. Wiest O. Maxfield F.R. Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts.Proc. Natl. Acad. Sci. USA. 2011; 108: 5620-5625Crossref PubMed Scopus (148) Google Scholar). Correction of the NPC phenotype would require that this mutant protein retains adequate functional capability and that a sufficient amount is delivered to the LE/Ly. Other data are consistent with the hypothesis that some mutant NPC1 proteins can function in LE/Ly if they are delivered to those organelles. Simply overexpressing NPC1I1061T in mutant cells leads to partial correction of the phenotype (26Gelsthorpe M.E. Baumann N. Millard E. Gale S.E. Langmade S.J. Schaffer J.E. Ory D.S. Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding.J. Biol. Chem. 2008; 283: 8229-8236Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Some indirect treatments also increase the abundance of NPC1 and lead to correction of the phenotype in cultured cells. These include treatment with ryanodine receptor antagonists (27Yu T. Chung C. Shen D. Xu H. Lieberman A.P. Ryanodine receptor antagonists adapt NPC1 proteostasis to ameliorate lipid storage in Niemann-Pick type C disease fibroblasts.Hum. Mol. Genet. 2012; 21: 3205-3214Crossref PubMed Scopus (28) Google Scholar), treatment with oxysterols that bind to NPC1 (28Ohgane K. Karaki F. Dodo K. Hashimoto Y. Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.Chem. Biol. 2013; 20: 391-402Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), or reduced expression of TMEM97, an NPC1-binding protein (29Ebrahimi-Fakhari D. Wahlster L. Bartz F. Werenbeck-Ueding J. Praggastis M. Zhang J. Joggerst-Thomalla B. Theiss S. Grimm D. Ory D.S. Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells.Hum. Mol. Genet. 2016; 25: 3588-3599Crossref PubMed Scopus (55) Google Scholar). These studies have indicated that alterations in the proteostasis environment (30Calamini B. Silva M.C. Madoux F. Hutt D.M. Khanna S. Chalfant M.A. Saldanha S.A. Hodder P. Tait B.D. Garza D. Small-molecule proteostasis regulators for protein conformational diseases.Nat. Chem. Biol. 2011; 8: 185-196Crossref PubMed Scopus (173) Google Scholar, 31Hutt D.M. Powers E.T. Balch W.E. The proteostasis boundary in misfolding diseases of membrane traffic.FEBS Lett. 2009; 583: 2639-2646Crossref PubMed Scopus (74) Google Scholar, 32Rauniyar N. Subramanian K. Lavallée-Adam M. Martínez-Bartolomé S. Balch W.E. Yates J.R. Quantitative proteomics of human fibroblasts with I1061T mutation in Niemann–Pick C1 (NPC1) protein provides insights into the disease pathogenesis.Mol. Cell. Proteomics. 2015; 14: 1734-1749Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) by various mechanisms leads to reduced degradation of mutant forms of NPC1. As described here, we found that treatment of some NPC1 mutant cells with vorinostat led to a longer lifetime of the NPC1I1061T protein and increased delivery of the protein to LE/Ly. A mouse knock-in model of NPC1I1061T has been described recently, and mouse embryo fibroblasts from these mice respond to vorinostat similarly to the human NPC1I1061T fibroblasts (33Praggastis M. Tortelli B. Zhang J. Fujiwara H. Sidhu R. Chacko A. Chen Z. Chung C. Lieberman A.P. Sikora J. A murine Niemann-Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele.J. Neurosci. 2015; 35: 8091-8106Crossref PubMed Scopus (72) Google Scholar). Another recent study in Npc1nmf164 mice, which have a D1005G mutation in the Npc1 protein, reported that a combination therapy with vorinostat, HPBCD, and polyethylene glycol led to slowed neuronal degeneration and improved lifespan in Npc1nmf164 mutant animals (34Alam M.S. Getz M. Haldar K. Chronic administration of an HDAC inhibitor treats both neurological and systemic Niemann-Pick type C disease in a mouse model.Sci. Transl. Med. 2016; 8: 326ra23Crossref PubMed Scopus (47) Google Scholar). Approximately 95% of NPC cases are due to mutations in the NPC1 protein, and the NPC1I1061T mutation, which occurs in approximately 15–20% of NPC1 patients, is the most commonly observed mutation (35Davies J.P. Ioannou Y.A. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.J. Biol. 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It would be very difficult to test drug treatments in hundreds of different human NPC1 mutant fibroblast cell lines, and the large number of compound heterozygous mutations would make it nearly impossible to evaluate the ability of HDACis to correct a specific mutation. In order to evaluate the effectiveness of HDACis as a potential therapy for NPC patients, we developed an efficient screening system using an engineered cell line. Human U2OS osteosarcoma cells were stably transfected with scavenger receptor type A (SRA), and the endogenous NPC1 expression in the cells was stably silenced with an shRNA. The U2OS-SRA-shNPC1 cells were then transiently transfected with a bicistronic vector expressing green fluorescent protein (GFP) (to identify transfected cells) and one of 60 NPC1 mutations found in patients. This system was used to test the effect of HDACi treatments on multiple NPC1 mutations simultaneously. After treatment with vorinostat or panobinostat, a high fraction of NPC1 mutant proteins were effective in reducing cholesterol accumulation. This suggests that HDACi therapy might be effective for a large majority of NPC1 patients. Because vorinostat and panobinostat are FDA-approved drugs for treatment of some cancers, and other HDACis have been in large scale clinical trials, HDACis may be considered as a potential therapy for NPC1 disease (20Helquist P. Maxfield F.R. Wiech N.L. Wiest O. Treatment of Niemann–pick type C disease by histone deacetylase inhibitors.Neurotherapeutics. 2013; 10: 688-697Crossref PubMed Scopus (45) Google Scholar). Gibco® McCoy's 5A medium (Gibco, Tokyo, Japan), MEM, FBS, HBSS, penicillin/streptomycin (P/S), Geneticin (G418), Alexa Fluor 546, Cy5 goat anti-rat (A10525), and Alexa Fluor 546 goat anti-rabbit and Alexa Fluor 488 goat anti-rat antibody, 1,1′-dioctadecyl-3,3,3′3′-tetramethyindocarbocyanine perchlorate [DiIC18 (3Infante R.E. Wang M.L. Radhakrishnan A. Kwon H.J. Brown M.S. Goldstein J.L. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes.Proc. Natl. Acad. Sci. USA. 2008; 105: 15287-15292Crossref PubMed Scopus (338) Google Scholar)] were purchased from Invitrogen Life Technologies Corp. (Carlsbad, CA). Effectene Transfection Reagent Kit and DNA purification kit was purchased from Qiagen Inc. (Valencia, CA). HDACis (vorinostat and panobinostat) were stocked at a concentration of 5 mM in DMSO and stored at −20°C. Vorinostat and panobinostat were a generous gift from Dr. Paul Helquist (University of Notre Dame, South Bend, IN). Acetylated LDL (AcLDL) was prepared by acetylation of LDL with acetic anhydride (38Basu SK Goldstein JL Anderson GW Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts.Proc. Natl. Acad. Sci. USA. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar). Alexa Fluor 546-labeled human LDL was prepared as described (39Grosheva I Haka AS Qin C Pierini LM Maxfield FR Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar, 40Havel RJ Eder HA Bragdon JH The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. T.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6486) Google Scholar). Rabbit polyclonal anti-NPC1 antibody and rabbit polyclonal anti-lysosomal associated membrane protein 1 (anti-LAMP1) antibody (ab24170) were purchased from Abcam (Cambridge, MA). A rat monoclonal anti-NPC1 antibody has been described previously (32Rauniyar N. Subramanian K. Lavallée-Adam M. Martínez-Bartolomé S. Balch W.E. Yates J.R. Quantitative proteomics of human fibroblasts with I1061T mutation in Niemann–Pick C1 (NPC1) protein provides insights into the disease pathogenesis.Mol. Cell. Proteomics. 2015; 14: 1734-1749Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The specificity of the rabbit anti-NPC1 antibody was verified by immunostaining U2OS-SRA-shNPC1 cells, which are stably silenced for NPC1 expression in parallel with WT U2OS cells. In WT U2OS cells, NPC1 staining was observed in punctate LE/Ly, but no staining was observed in U2OS-SRA-shNPC1 cells, indicating that antibody specifically binds to NPC1 protein in cells (supplemental Fig. S1). Similar specificity of labeling was observed for the rat monoclonal anti-NPC1. All other chemicals, including DMSO, 99% fatty-acid free BSA, filipin, paraformaldehyde (PFA), and HEPES were purchased from Sigma-Aldrich (St. Louis, MO). Draq5 was from Biostatus (Leicestershire, UK). Metamorph image-analysis software was from Molecular Devices (Downington, PA). Human NPC1 fibroblasts GM05659, GM18453 (homozygous NPC1 mutant I1061T), and GM03123 (heterozygous NPC1 mutations P237S and I1061T) were from Coriell Institute (Camden, NJ). [The P237S allele has been found recently to be in linkage with a pathogenic splice mutation that may be responsible for its defect (F.D. Porter, unpublished observations)]. Patient-derived mutant NPC1 skin fibroblasts were from Forbes Porter's laboratory at the National Institutes of Health (Bethesda, MD) as listed in Table 1. All human skin fibroblasts were maintained in MEM supplemented with 10% FBS. For drug treatment, cells were maintained in MEM supplemented with 5.5% FBS and 20 mM HEPES. The use of human fibroblasts was approved by an Institutional Review Board at Weill Cornell Medical College for the research performed.TABLE 1List of mutations in NPC1 patient fibroblastsGM03123Heterozygous I1061T/ P237SGM18453Homozygous I1061TNPC1-2V1165M/ 3741-44 del ACTCNPC1-5I1061T/ R1186GNPC1-16P887L/ 3741-44 del ACTCNPC1-17I1061T, 10 bp deletion in exon 19 at codon 962 = fs(exon19)NPC1-191920delG/ IVS9-1009G>ANPC1-22R978C, IVS21-2 A>GNPC1-25N701K/ C2979 dupANPC1-31G46V/ L491P Open table in a new tab Human osteosarcoma U2OS (HTB-96™) cells from ATCC (Manassas, VA) were transfected with murine SRA-II cDNA [kind gift from Dr. Monty Krieger (Massachusetts Institute of Technology, Boston, MA)]. Briefly, the 1.0 kb coding region of murine scavenger receptor type II was cut from a larger 4.0 kb plasmid encoding the coding region and the 3′ untranslated region (UTR) in a pcDNA3.1 expression vector (41Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. Structures and high and low affinity ligand binding properties of murine type I and type II macrophage scavenger receptors.J. Lipid Res. 1993; 34: 983-1000Abstract Full Text PDF PubMed Google Scholar). The coding sequence was amplified by using PCR and inserted into a neomycin-resistant pcDNA 3.1 TOPO plasmid by using pcDNA 3.1 Directional TOPO Expression kit (Invitrogen), according to the manufacturer's instructions. The insertion of SRA-II in the pcDNA3.1 TOPO plasmid was confirmed by band size of full-length plasmid and also after digestion with appropriate restriction enzymes using gel electrophoresis. The pcDNA 3.1 TOPO plasmid encoding SRA-II was purified by using the Qiagen DNA purification kit and transfected in U2OS cells by using Lipofectamine (Invitrogen) as a transfection reagent. U2OS cells expressing murine SRA-II were grown in McCoy's 5A medium supplemented with 10% FBS, 1% P/S, and selection antibiotic G418 (1 mg/ml) in a humidified incubator with 5% CO2 at 37°C for five passages. To select a population of U2OS cells expressing the SRA-II gene, cells were incubated with DiIC18 (3Infante R.E. Wang M.L. Radhakrishnan A. Kwon H.J. Brown M.S. Goldstein J.L. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes.Proc. Natl. Acad. Sci. USA. 2008; 105: 15287-15292Crossref PubMed Scopus (338) Google Scholar) labeled AcLDL and sorted by using flow cytometry cell sorting. Murine macrophage cells J774 were used as a positive control, and the pool of cells with equivalent DiI intensity, indicative of SRA-II expression, were collected. The cells were expanded, and stock cultures were frozen for future experiments. The dose dependence of two HDACis (vorinostat and panobinostat) was determined after 48 h treatment of NPC1 fibroblasts from several patients carrying different mutations. The assay is based on the use of filipin, a fluorescent dye that binds to unesterified cholesterol. Human fibroblasts were seeded in 384-well plates at 450 cells/well in growth medium on day one. Four cell lines were seeded in different wells of a plate, and GM03123 fibroblasts were used as a control in each plate. After overnight incubation, 2× concentrated compounds were added at six different doses, such that the final concentration ranged from 40 nM to 10 μM for vorinostat and 5 nM to 1 μM for panobinostat diluted in growth medium supplemented with 20 mM HEPES buffer and 5.5% FBS. DMSO was used as a control in each plate for each concentration and for each cell line. After 48 h, the plate was washed with PBS three times, fixed with 1.5% PFA, and stained with 50 μg/ml filipin
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