Wild-type P. gingivalis strain ATCC33277 and Fusobacterium nucleatum strain ATCC10953 were grown on blood agar anaerobically at 37°C as described previously (Gawron et al. 2014 (link)). Bacteria were next inoculated into brain–heart infusion (BHI) broth (Becton Dickinson) containing 0.5 mg/mL L-cysteine, 10 µg/mL hemin and 0.5 µg/mL vitamin K. Following anaerobic (85% N2, 10% CO2 and 5% H2) overnight (o/n) culture, bacteria were resuspended in fresh BHI broth at optical density (OD)600nm = 0.1 and cultured for an additional 20 h. After centrifugation and washing, bacteria were suspended in PBS to obtain a final concentration of 109 colony-forming units (CFU)/mL and used for cell infection. GFs were treated with dimethyl sulfoxide (DMSO) (BioShop) or HDACi: SAHA (Abcam), ITF2357 (Italfarmaco), MS-275 (TargetMol) or a panel of selective HDACi synthesized at Italfarmaco: HDAC3/6i, HDAC6i(a), HDAC6i(b) or HDAC8(i). Details of HDACi specificity are shown in Appendix Table 2 . After 30 min incubation with HDACi, cells were stimulated with TNFα (BioLegend) or infected with P. gingivalis or F. nucleatum at the indicated multiplicity of infection (MOI). In some experiments, protein synthesis was blocked with cycloheximide (TargetMol) before TNFα stimulation.
Cycloheximide (chx)
Manufactured by Targetmol
Sourced in China
Cycloheximide (CHX) is a chemical compound commonly used as a laboratory tool. It functions as a potent inhibitor of protein synthesis in eukaryotic organisms, effectively blocking the translocation step in protein translation.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Targetmol (3 mentions)
Z vad fmk, by Targetmol (2 mentions)
Bhi broth, by BD (1 mentions)
Tnf α, by BioLegend (1 mentions)
Ab72139, by Abcam (1 mentions)
Ab209384, by Abcam (1 mentions)
Ab184718, by Abcam (1 mentions)
Ab187091, by Abcam (1 mentions)
Chloroquine, by Targetmol (1 mentions)
Ferrostatin 1, by Targetmol (1 mentions)
Necrostatin 1, by Targetmol (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Dual luciferase reporter gene assay kit, by Promega (1 mentions)
Pericyte medium pm, by ScienCell (1 mentions)
Hrp conjugated anti rabbit igg 7074, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 conjugated donkey anti rabbit igg ab150064, by Abcam (1 mentions)
Chip pcr kit, by Merck Group (1 mentions)
Pkh26, by Merck Group (1 mentions)
Pkh67, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
7 protocols using cycloheximide (chx)
1
Anaerobic Culture of Oral Pathogens
2
Investigating Apoptosis and Necroptosis Pathways
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Antibodies against GAPDH (#97166S, 1 : 1000), PARP (#9542S, 1 : 1000), caspase-3 (#9662S, 1 : 1000), cleaved caspase-3 (#9664S, 1 : 1000), caspase-8 (#9746S, 1 : 1000), cleaved caspase-8 (#9496S, 1 : 1000), Bax (#5023S, 1 : 1000), Bcl-2 (#15071S, 1 : 1000), p-RIP1 (Ser166; #65746S, 1 : 1000), RIP3 (#95702S, 1 : 1000), anti-rabbit IgG, HRP-linked antibody (#7074S, 1 : 3000), and anti-mouse IgG, HRP-linked antibody (#7076S, 1 : 3000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MLKL (#ab184718, 1 : 1000), p-MLKL (Ser358; #ab187091, 1 : 1000), RIP1 (#ab72139, 1 : 1000), and p-RIP3 (Ser227; #ab209384, 1 : 1000) were purchased from Abcam (Cambridge, UK). Cladribine, homoharringtonine, Z-VAD-FMK, cycloheximide, chloroquine, ferrostatin-1, and necrostatin-1 were purchased from TargetMol (Shanghai, China). Aclarubicin was obtained from APExBIO (Houston, TX). A stock solution containing cladribine, homoharringtonine, and aclarubicin was prepared in DMSO and kept at -80°C. TNF-alpha was purchased from ABclonal (Wuhan, China).
3
Angiogenesis Regulation in Endothelial Cells
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Endothelial cell medium (ECM) and pericyte medium (PM) were products of ScienCell Research Laboratories (San Diego, CA, USA). Antibodies against Ki67 (#12,202), α-SMA (#19,245), bFGF (#20,102), FGFR1 (#76,123), FGFR2 (#23,328), β-actin (#23,328) and HRP-conjugated anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Gli1 (AF3455) and CD31 (AF3628), and a Proteome Profiler Human Angiogenesis Array Kit (ARY007) were obtained from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 594-conjugated donkey anti-rabbit IgG (ab150064) and Alexa Fluor488- conjugated donkey anti-mouse IgG (ab150073) were obtained from Abcam (Cambridge, UK). GANT-61, cycloheximide (CHX) and MG132 were purchased from TargetMol (Shanhai, China). Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ). Recombinant human bFGF was purchased from BioVision (Palo Alto, CA, USA). A ChIP PCR kit was obtained from Merck Millipore (Billerica, MA, USA). A dual luciferase reporter gene assay kit was provided by Promega (Madison, WI, USA). Rhodamine-labeled lysinated dextran (70-kDa) was product from Thermo Fisher Scientific (Waltham, MA, USA). PKH 26, PKH 67 and other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4
Antibody Detection Protocol for Signaling Proteins
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Antibodies against USP7, BCR-ABL, Lyn, p-Lyn (Tyr507), STAT5, p-STAT5 (Tyr94), PARP, CRKL, and p-CRKL (Tyr207) were purchased from Cell Signaling Technologies, Inc., (Danvers, MA, USA). The antibodies against USP25 and Caspase-3 were obtained from Proteintech (Wuhan, China). The monoclonal antibodies including anti-Flag, anti-HA, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Medical and Biological Laboratories Co., Ltd (Nagoya, Japan). The anti-Ub antibody was purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA). HRP-labeled goat anti-mouse and goat anti-rabbit IgG (H + L) antibodies were purchased from Beyotime Institute of Biotechnology (Nantong, China). ART and cycloheximide (CHX) were purchased from TargetMol (Boston, MA, USA), Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. P5091 and imatinib were purchased Selleck Chemicals Inc. (Houston, TX, USA).
5
AML Cell Lines for Therapeutic Screening
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Three human AML cell lines, Kasumi-1 (ATCC, Manassas, VA) with t(8;21) and heterozygous KIT N822K mutation, SKNO-1 (JCRB, Osaka, Japan) with t(8;21) and homozygous KIT N822K mutation, and OCI-AML3 (DSMZ, Brauschweig, Germany) with NPM1 mutation and DNMT3A mutation, were used in this study. Kasumi-1 cells were maintained in RPMI 1640 medium (ThermoFisher Scientific/GIBCO, Waltham, MA) containing 20% fetal bovine serum (FBS), SKNO-1 cells in RPMI 1640 medium with 10% FBS containing 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D systems, Minneapolis, MN), and OCI-AML3 cells in αMEM medium (GIBCO) containing 20% FBS. Cultures were grown at 37 °C in humidified atmosphere containing 5% CO2. These cell lines were authenticated (16-Markers STR) by the Food Industry Research and Development Institute, Hsinchu, Taiwan. Their genetic profiles were identical to reported information.
Cabozantinib-malate was purchased from Selleck Chemicals (Houston, TX) and dissolved in DMSO for storage at −20 °C. Cycloheximide (CHX), MG-132, Z-VAD-FMK, and puromycin were purchased from TargetMol (Boston, MA), Tocris Bioscience (Abingdon, UK), Abcam (Cambridge, MA), and GIBCO, respectively.
Cabozantinib-malate was purchased from Selleck Chemicals (Houston, TX) and dissolved in DMSO for storage at −20 °C. Cycloheximide (CHX), MG-132, Z-VAD-FMK, and puromycin were purchased from TargetMol (Boston, MA), Tocris Bioscience (Abingdon, UK), Abcam (Cambridge, MA), and GIBCO, respectively.
6
Cell Lines and Experimental Treatments
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A549, 293T, HeLa, NCI-H3122, NCI-H125, NCI-H1975 and 293FT cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and A549, 293T, HeLa and 293FT were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, 26400044) and 1% penicillin/streptomycin. NCI-H3122, NCI-H125 and NCI-H1975 were maintained in Roswell Park Memorial Institute 1640 (RPMI 1640). MG132 (Sigma-Aldrich, C2211) was used at 10 μM, cycloheximide (CHX; Targetmol, T1225) was used at 100 μg/mL unless otherwise specified, nocodazole (Noc; Sigma-Aldrich, M1404) at 100 ng/mL, WP1130 (SelleckChem, S2243) at 5 μM unless otherwise specified, and Bortezomib (SelleckChem, S1013) at 10 μM. For synchronization in mitosis, A549 and HeLa cells were treated with nocodazole for 16 h.
7
Protein Analysis using Diverse Antibodies
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Primary antibodies used were as follows: Anti-HA (#3724), anti-p-YAPSer127 (#13008), anti-LATS1 (#3477), anti-p-LATS1Ser909 (#9157), and anti-β-Trcp (#4394) were from Cell Signaling Technology. Anti-YAP (WH0010413M1), anti-Flag (F1804), anti-PCNA (05-347), and anti-Siah1 (AV38212) were from Sigma. Anti-p-YAPTyr357 (ab62751), anti-YAP (ab52771), and anti-SKP1 (ab76502) were from abcam. Anti-Phosphotyrosine (#05-321) was from Millipore. Anti-FRK (sc-166478), anti-CYR61 (sc-374129), and anti-CTGF (sc-101586) were from Santa Cruz Biotechnology. Anti-LATS2 (A16249), anti-p-LATS1T1079/LATS2T1041 (AP0912), anti-β-actin (AC026), and anti-Siah1 (A2494) were from ABclonal. Cycloheximide (CHX) and MG132 were from TargetMol. Puromycin, Protein A/G agarose and verteporfin (VP) were from MedChemExpress. PolyJet was from Signagen. Polybrene was from Sigma.
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