hdac8 np 060956 1  (Addgene inc)

Journal: Scientific reports

Article Title: The hydroxamate based HDAC inhibitor WMJ-J-09 induces colorectal cancer cell death by targeting tubulin and downregulating survivin.

doi: 10.1038/s41598-025-04714-w

Figure Lengend Snippet: Fig. 2. WMJ-J-09 disrupted microtubule assembly through HDAC inhibition. (A) Microtubule formation in HCT116 cells treated with colchicine, paclitaxel, WMJ-J-09, or vehicle, detected by confocal immunofluorescence analysis with β-tubulin-staining images in the upper panel, DAPI-staining images in the middle panel, and merged images in the bottom panel. (B) Immunoblotting result of polymerized tubulin in HCT116 cells treated with colchicine, paclitaxel, WMJ-J-09, or vehicle (C, D) Immunoblot result of α-tubulin acetylation induced by WMJ-J-09 in either a concentration-dependent (C) or time-dependent manner (D). (E) Immunoblot result of α-tubulin acetylation in WMJ-J-09-treated HCT116 cells with HDAC6 or HDAC8 overexpression. Each band intensity was quantified, and the total α-tubulin level normalized the fold changes of α-tubulin acetylation. Error bars, mean ± S.E.M. (shown only for independent replicate experiments with n ≥ 5).

Article Snippet: HDAC8 Flag was a gift from Eric Verdin (Addgene plasmid # 13825; http://n2t.net/addgene:13825; RRID:Addgene_13825) 64.

Techniques: Inhibition, Immunofluorescence, Staining, Western Blot, Concentration Assay, Over Expression

Journal: Genes & Cancer

Article Title: HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1

doi: 10.18632/genesandcancer.197

Figure Lengend Snippet: A. and B. Cell viability determined by CCK-8 assay after treatment with 30 and 40 µM PCI34051 for 4 days. C. T98G cell viability determined with the CCK-8 assay after 3 days’ treatment with either 30 µM PCI34051 or 250 µM TMZ. n = 3 * p < 0.05, ** p < 0.005. D. T98G cell (Empty vector or TRCN0000350469 shHDAC8) viability determined with the CCK-8 assay after 4 days’ treatment with 250 µM TMZ. E. Extracts of U87 and T98G cells expressing empty vector (EV) or shHDAC8 (1- TRCN0000350469, 2-TRCN0000004852 and 3-TRCN0000314874) were subject to Western blotting with tubulin and HDAC8 antibodies.

Article Snippet: The FLAG-HDAC8 vector, which was published previously [ ], was subcloned into pLenti CMV Puro DEST (Addgene plasmid # 17452) with the same procedure as for FLAG MGMT, using TAFLAGHDAC8-F and TAFLAGHDAC8-R. HDAC8 was subcloned into pcDNA3.1 HA and pcDNA3.1/Myc-His (Invitrogen) vectors using HAHDAC8-EcoRIF and HAHDAC8-XhoIR primers and pcDNA3.1mycHisHDAC8XhoI-F and pcDNA3.1mycHisHDAC8KpnI-R primers, respectively, and the FLAG-HDAC8 vector as a template. pcDNA5-Adrm1-Flag (plasmid # 19418) and pcDNA3 HA-UCH37 (plasmid #19415) were obtained from Addgene.

Techniques: CCK-8 Assay, Plasmid Preparation, Expressing, Western Blot

Journal: Genes & Cancer

Article Title: HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1

doi: 10.18632/genesandcancer.197

Figure Lengend Snippet: A. Extracts from T98G cells treated with 15, 20 and 30 µM PCI34051 for 24 hours were subject to Western blotting with MGMT, tubulin, γH2AX and H3 antibodies. B. Extracts from T98G cells expressing stable shHDAC8 (1- TRCN0000350469, 2-TRCN0000004852 and 3-TRCN0000314874) and treated with TMZ for 48 hours were subject to Western blotting with MGMT, tubulin and HDAC8 antibodies. C. Quantitative RT-PCR analysis of mRNA from T98G cells expressing stable shHDAC8 (TRCN0000350469). D. Extracts from T98G and U87 cells expressing stable FLAG-MGMT and treated with PCI34051 for 24 hours were subject to Western blotting with FLAG and tubulin antibodies. E. Extracts from U87 cells were subject to Western blotting with MGMT, vinculin and HA antibodies.

Article Snippet: The FLAG-HDAC8 vector, which was published previously [ ], was subcloned into pLenti CMV Puro DEST (Addgene plasmid # 17452) with the same procedure as for FLAG MGMT, using TAFLAGHDAC8-F and TAFLAGHDAC8-R. HDAC8 was subcloned into pcDNA3.1 HA and pcDNA3.1/Myc-His (Invitrogen) vectors using HAHDAC8-EcoRIF and HAHDAC8-XhoIR primers and pcDNA3.1mycHisHDAC8XhoI-F and pcDNA3.1mycHisHDAC8KpnI-R primers, respectively, and the FLAG-HDAC8 vector as a template. pcDNA5-Adrm1-Flag (plasmid # 19418) and pcDNA3 HA-UCH37 (plasmid #19415) were obtained from Addgene.

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: Genes & Cancer

Article Title: HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1

doi: 10.18632/genesandcancer.197

Figure Lengend Snippet: A. Extracts from T98G cells expressing shADRM1 (1-TRCN0000286432, 2-TRCN0000293817) were subject to Western blotting with MGMT, actin, tubulin and ADRM1 antibodies. B. Quantitative RT-PCR analysis of mRNA from T98G cells stably expressing shADRM1 (TRCN0000286432). C. Extracts from T98G cells expressing FLAG-MGMT, shHDAC8 (TRCN0000350469) and shADRM1 (TRCN0000286432) were subject to Western blotting with FLAG and tubulin antibodies. D. Extracts from T98G cells either expressing HA-ADRM1, FLAG-HDAC8 or treated with 15 µM PCI34051 for 24 hours were subject to Western blotting with MGMT, HA, FLAG, PSMD1 and tubulin antibodies.

Article Snippet: The FLAG-HDAC8 vector, which was published previously [ ], was subcloned into pLenti CMV Puro DEST (Addgene plasmid # 17452) with the same procedure as for FLAG MGMT, using TAFLAGHDAC8-F and TAFLAGHDAC8-R. HDAC8 was subcloned into pcDNA3.1 HA and pcDNA3.1/Myc-His (Invitrogen) vectors using HAHDAC8-EcoRIF and HAHDAC8-XhoIR primers and pcDNA3.1mycHisHDAC8XhoI-F and pcDNA3.1mycHisHDAC8KpnI-R primers, respectively, and the FLAG-HDAC8 vector as a template. pcDNA5-Adrm1-Flag (plasmid # 19418) and pcDNA3 HA-UCH37 (plasmid #19415) were obtained from Addgene.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Stable Transfection

Journal: Genes & Cancer

Article Title: HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1

doi: 10.18632/genesandcancer.197

Figure Lengend Snippet: A. Extracts from U87 cells treated with TMZ for 48 hours were immunoprecipitated with a FLAG antibody and subject to Coomassie blue staining. B. HA immunoprecipitation of extracts from U87 and T98G cells expressing FLAG-HDAC8 and HA-ADRM1 and treated with TMZ for 48 hours. C. Schematics of the different FLAG-ADRM1 constructs used in D. D. FLAG immunoprecipitation of extracts from HeLa cells transfected with different constructs of FLAG-ADRM1 and HA-HDAC8. E. FLAG immunoprecipitation of extracts from HeLa cells transfected with HA-UCH37, myc-HDAC8 and FLAG-ADRM1. F. HA immunoprecipitation of extracts from T98G cells expressing HA-ADRM1 and FLAG-HDAC8.

Article Snippet: The FLAG-HDAC8 vector, which was published previously [ ], was subcloned into pLenti CMV Puro DEST (Addgene plasmid # 17452) with the same procedure as for FLAG MGMT, using TAFLAGHDAC8-F and TAFLAGHDAC8-R. HDAC8 was subcloned into pcDNA3.1 HA and pcDNA3.1/Myc-His (Invitrogen) vectors using HAHDAC8-EcoRIF and HAHDAC8-XhoIR primers and pcDNA3.1mycHisHDAC8XhoI-F and pcDNA3.1mycHisHDAC8KpnI-R primers, respectively, and the FLAG-HDAC8 vector as a template. pcDNA5-Adrm1-Flag (plasmid # 19418) and pcDNA3 HA-UCH37 (plasmid #19415) were obtained from Addgene.

Techniques: Immunoprecipitation, Staining, Expressing, Construct, Transfection

Journal: Genes & Cancer

Article Title: HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1

doi: 10.18632/genesandcancer.197

Figure Lengend Snippet: A. Extracts from U87 cells treated with PCI34051 for 24 hours were subject to Western blotting with γH2AX and H3 antibodies. B. Extracts from T98G cells treated with 15 µM PCI34051 for 24 hours were subject to Western blotting with phosphorylated ATM S1981, ATM, vinculin, γH2AX and H3 antibodies. C. Immunofluorescence demonstrating γH2AX expression in U87 cells treated with TMZ and 15 µM PCI34051 for 24 hours. D. Immunofluorescence showing 53BP1 and γH2AX expression in T98G cells treated with PCI34051 for 24 hours. E. Alkaline comet assay analysis of T98G cells treated with 20, 30 and 40 µM PCI34051 for 24 hours, n > 20, *** p < 0.0005. F. Extracts from U87 cells expressing shHDAC8 (1- TRCN0000350469, 2-TRCN0000004852) and treated with TMZ for 48 hours were subject to Western blotting with γH2AX, H3, HDAC8 and tubulin antibodies. G. Extracts of U87 cells expressing FLAG-HDAC8 and treated with TMZ for 48 hours were subject to Western blotting with γH2AX and H3 antibodies.

Article Snippet: The FLAG-HDAC8 vector, which was published previously [ ], was subcloned into pLenti CMV Puro DEST (Addgene plasmid # 17452) with the same procedure as for FLAG MGMT, using TAFLAGHDAC8-F and TAFLAGHDAC8-R. HDAC8 was subcloned into pcDNA3.1 HA and pcDNA3.1/Myc-His (Invitrogen) vectors using HAHDAC8-EcoRIF and HAHDAC8-XhoIR primers and pcDNA3.1mycHisHDAC8XhoI-F and pcDNA3.1mycHisHDAC8KpnI-R primers, respectively, and the FLAG-HDAC8 vector as a template. pcDNA5-Adrm1-Flag (plasmid # 19418) and pcDNA3 HA-UCH37 (plasmid #19415) were obtained from Addgene.

Techniques: Western Blot, Immunofluorescence, Expressing, Alkaline Single Cell Gel Electrophoresis

Journal: Genes & Cancer

Article Title: HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1

doi: 10.18632/genesandcancer.197

Figure Lengend Snippet: HDAC8 regulates MGMT protein levels via its interaction with ADRM1. In TMZ-sensitive U87 cells, TMZ treatment causes the dissociation of HDAC8 from ADRM1, which results in the downregulation of MGMT together with accumulated DNA damage in cells. In TMZ-resistant T98G cells, the HDAC8 and ADRM1 interaction cannot be disrupted by TMZ treatment. The high level of MGMT expression confers resistance against TMZ treatment.

Article Snippet: The FLAG-HDAC8 vector, which was published previously [ ], was subcloned into pLenti CMV Puro DEST (Addgene plasmid # 17452) with the same procedure as for FLAG MGMT, using TAFLAGHDAC8-F and TAFLAGHDAC8-R. HDAC8 was subcloned into pcDNA3.1 HA and pcDNA3.1/Myc-His (Invitrogen) vectors using HAHDAC8-EcoRIF and HAHDAC8-XhoIR primers and pcDNA3.1mycHisHDAC8XhoI-F and pcDNA3.1mycHisHDAC8KpnI-R primers, respectively, and the FLAG-HDAC8 vector as a template. pcDNA5-Adrm1-Flag (plasmid # 19418) and pcDNA3 HA-UCH37 (plasmid #19415) were obtained from Addgene.

Techniques: Expressing