Journal: Scientific Reports

Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

doi: 10.1038/s41598-017-11445-0

Figure Lengend Snippet: Effects of ADI on the regulation of HIF-1α stability by p300, HDAC2, and Sin3A. ( a ) ChIP assay of ASS1 promoter associations of p300, HDAC2 and Sin3A in A2058 cells treated with ADI for 15 min. ( b to d ) Effects of p300, Sin3A, and HDAC2 knockdown by siRNA as indicated on the expression of other proteins in the presence and absence of ADI (0.5 μg/ml, 1 hr). ( e to g ) ChIP assays of effects of p300, Sin3A, and HDAC2 knockdown on the ASS1 promoter associations of HIF1α, PHD2, HDAC2, p300, and Sin3A as indicated Cells were transfected with given siRNA as specified for 24 hr followed by ADI treatment (0.5 μg/ml) for 15 min.

Article Snippet: PCBP1(PA5–19409) antibody from Thermo Fisher Scientific; anti-LIMID1 antibody from Millipore Co.; rabbit anti-HDAC2, Sin3A, and p300 antibodies from Bethyl Laboratories; hydroxy-HIF-1α (Pro564) antibody and acetyl-Histone H3 Antibody Sampler Kit (#9927); antibodies of eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α(S51), mammalian target of rapamycin (mTOR), phosphor-mTOR(S2448), p70S6K (T389), elf-4E-binding protein (4EBP1) and phosphor-4EBP1 (S65) were from Cell Signaling Technology; HDAC inhibitor (SAHA, suberoylanilide hydroxamic acid) was from Sigma-Aldrich; and HAT inhibitors PU139 was prepared as described previously , and I-CBP112 was obtained from Sigma-Aldridge.

Techniques: Expressing, Transfection

Journal: Scientific Reports

Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

doi: 10.1038/s41598-017-11445-0

Figure Lengend Snippet: Effects of histone H3 deacetylation by ADI. ( a ) A2058 cells were treated with ADI for the time intervals as indicated, or with SAHA (20 μM for 1 hr as positive control), and lamin B expression (as control for sample loading). Acetylation status of various modified histones H3 were determined. ( b ) Reduction of ASS1-promoter associations of H3K27ac and H3K14ac by ADI (0.5 μg/ml, 15 min.). ( c to e ), Effects of p300, Sin3A, and HDAC2 knockdown by siRNAs, respectively, on the expression levels of H3K14ac and H3K27ac in A2058 cells treated with or without ADI. ( f ) Western blotting analyses of H3K14ac, H3K27ac, H3K9ac, and H3K18ac expression in A2058 and ADI-resistant (ADI R variants, 58R1 to 58R3). ( g ) ChIP assay of promoter-associations of p300, HDAC2, and HIF-1α in A2058 and ADI R cells (R1 to R3). ( h ) Western blotting analyses of HIF-1α, ASS1, H3K14ac, H3K27ac, and H3 expression in 4 matched pairs of primary cell lines derived from melanoma patients before (−) and after failed (+) by ADI treatments.

Article Snippet: PCBP1(PA5–19409) antibody from Thermo Fisher Scientific; anti-LIMID1 antibody from Millipore Co.; rabbit anti-HDAC2, Sin3A, and p300 antibodies from Bethyl Laboratories; hydroxy-HIF-1α (Pro564) antibody and acetyl-Histone H3 Antibody Sampler Kit (#9927); antibodies of eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α(S51), mammalian target of rapamycin (mTOR), phosphor-mTOR(S2448), p70S6K (T389), elf-4E-binding protein (4EBP1) and phosphor-4EBP1 (S65) were from Cell Signaling Technology; HDAC inhibitor (SAHA, suberoylanilide hydroxamic acid) was from Sigma-Aldrich; and HAT inhibitors PU139 was prepared as described previously , and I-CBP112 was obtained from Sigma-Aldridge.

Techniques: Positive Control, Expressing, Modification, Western Blot, Derivative Assay

Journal: Scientific Reports

Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

doi: 10.1038/s41598-017-11445-0

Figure Lengend Snippet: Effects of ROS on ADI-induced HIF-1α degradation. ( a ) ADI-induced HF-1α degradation is inhibited by antioxidant, NAC. A2058 cells were treated with 10 μM MG-132 in the absence or presence of ADI or NAC (1 mM) for 4-h. Expression levels of HIF-1α, hydroxylated HIF-1α, and actin were determined by Western blotting. ( b ) The antioxidant NAC suppresses ADI-induced PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding HA-PHD2. Cells were treated with NAC or ADI as indicated for 1 hr. PHD2 enzymatic activity was measured using GST-ODDD (100 ng) as a substrate and production of HO-HIF-1α (p564). ( c ) Similar to those described in ( b ) was performed using anti-oxidants Mito-TEMPO (40 μM) and TEMPO (100 μM) for 1 hr. ( d , e ) Inhibitions of HDAC2 and PHD2 interaction by TEMPO in reciprocal co-IP assays. ( f , g ), Effects of antioxidants NAC (N, 1 mM) or TEMPO (T, 100 μM)) on ADI (A)-induced ASS1 promoter association of HIF-1 α , PHD2, p300, HDAC2 and H3K27ac in A2058 cells treated with or without ADI (A, 0.5 μg/ml, 1 hr) as determined by ChIP assay.

Article Snippet: PCBP1(PA5–19409) antibody from Thermo Fisher Scientific; anti-LIMID1 antibody from Millipore Co.; rabbit anti-HDAC2, Sin3A, and p300 antibodies from Bethyl Laboratories; hydroxy-HIF-1α (Pro564) antibody and acetyl-Histone H3 Antibody Sampler Kit (#9927); antibodies of eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α(S51), mammalian target of rapamycin (mTOR), phosphor-mTOR(S2448), p70S6K (T389), elf-4E-binding protein (4EBP1) and phosphor-4EBP1 (S65) were from Cell Signaling Technology; HDAC inhibitor (SAHA, suberoylanilide hydroxamic acid) was from Sigma-Aldrich; and HAT inhibitors PU139 was prepared as described previously , and I-CBP112 was obtained from Sigma-Aldridge.

Techniques: Expressing, Western Blot, Activity Assay, Transfection, Recombinant, Co-Immunoprecipitation Assay