Journal: Drug Design, Development and Therapy

Article Title: Targeted inhibition of mammalian target of rapamycin (mTOR) enhances radiosensitivity in pancreatic carcinoma cells

doi: 10.2147/DDDT.S42390

Figure Lengend Snippet: Scatter plot of the gene differential expression in PC-2 cells induced by RPM. Notes: The Cy3 (RPM treated group) and Cy5 (untreated control) channel intensities from the two-color DNA microarray experiments were shown in the scatter plot. The variables appear in a linear relationship, and the linear correlation between them is 0.935. The red plots represented upregulated genes, and the green plots represented downregulated genes. Abbreviation: RPM, rapamycin.

Article Snippet: We used an Agilent Whole Human Genome Microarray chip to assess the expression profiles in RPM-treated cells.

Techniques: Expressing, Microarray

Journal: Drug Design, Development and Therapy

Article Title: Targeted inhibition of mammalian target of rapamycin (mTOR) enhances radiosensitivity in pancreatic carcinoma cells

doi: 10.2147/DDDT.S42390

Figure Lengend Snippet: ( A ) RT-PCR analysis for the DNA-damage repair and transcription genes after the treatment with RPM; ( B ) Difference between the cDNA microarray and RT-PCR in DNA-damage repair and transcription genes. Notes: The genes examined here are: (1) DDB1(NM_001923); (2) RAD51 (NM_002876); (3) XRCC5 (NM_021141); (4) PCNA (NM_002592); and (5) ABCC4 (NM_005845). The values represent the mean ± SD of the data from three independent experiments. Abbreviations: RPM, rapamycin; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation.

Article Snippet: We used an Agilent Whole Human Genome Microarray chip to assess the expression profiles in RPM-treated cells.

Techniques: Reverse Transcription Polymerase Chain Reaction, Microarray, Standard Deviation

Journal: Nature Communications

Article Title: H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

doi: 10.1038/ncomms14799

Figure Lengend Snippet: ( a ) Summary of H3 ubiquitination sites identified in various large-scale quantitative proteomics studies. ( b ) Glucose deprivation abolished H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid (His-Ub) for 36 h and treated with various stresses for 4 h before in vivo ubiquitination assay to access the H3 ubiquitination (see experimental procedures for details). ( c ) Add-back of glucose recovered H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid for 36 h, then glucose-starved for 4 h and added-back glucose for indicated times (see experimental procedures for detail) before in vivo ubiquitination assay. ( d ) Screening of E3 ligases for H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid and various E3 ligases constructs for in vivo ubiquitination assay. ( e ) NEDD4 E3 ligase dead mutant (CS mutant) failed to trigger H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid and WT NEDD4 or NEDD4 CS mutant construct for in vivo ubiquitination assay. ( f ) NEDD4 knockdown abolished H3 ubiquitination. Control and NEDD4 knockdown 293T cells were transfected with his-ubiquitin plasmid for in vivo ubiquitination assay. ( g ) NEDD4 ubiquitinated H3 in vitro . In vitro ubiquitination assay was performed for recombinant NEDD4 and histone octamer (see experimental procedures for details). Reaction products were then assessed by western blotting using anti H3 antibody. H3 mono- and di-ubiquitination have predicted molecular weights of ∼25 kDa and ∼33 kDa. S.E. and L.E. are abbreviations for shorter exposure time and longer exposure time, respectively. ( h ) NEDD4 knockdown abolished glucose-induced H3 ubiquitination. Hep3B cells were glucose starved for 4 h and added-back glucose for 2 h before immunoprecipitation assay for endogenous ubiquitinated proteins (see experimental procedures for details). H3 ubiquitination was then visualized by western blotting. ( i ) Add-back of glucose recovered NEDD4 overexpression induced H3 ubiquitination. 293T cells were transfected with his-ubiquitin and NEDD4 plasmids for 36 h, then glucose-starved for 4 h and added-back glucose for indicated times before in vivo ubiquitination assay. ( j ) NEDD4 triggered monoubiquitination on H3. 293T cells were transfected with Flag-H3, HA-NEDD4, His-Ub WT and His-Ub K0 as indicated before in vivo ubiquitination assay. ( k ) Glucose-induced NEDD4 phosphorylation at Y43 and Y585. 293T cells transfected with WT or Y43/585F NEDD4 plasmids were treated with glucose and harvested for IP. ( l ) NEDD4 phosphorylation is required for H3 ubiquitination. 293T cells transfected with WT, Y43585F or Y43/585E NEDD4 plasmids were harvested for in vivo ubiquitination assay.

Article Snippet: The following antibodies were used in this study: anti-H3 (Abcam, ab12079, 1:5,000), anti-H3 pan-ac (Active Motif, 39139, 1:2,000), anti-H3 K4ac (Active Motif, 39381, 1:2,000), anti-H3 K9ac (Active Motif, 39917, 1:3,000), anti-H3 K14ac (Active Motif, 39697, 1:2,000), anti-H3 K18ac (Active Motif, 39755, 1:2,000), anti-H3 K23ac (Active Motif, 39131, 1:2,000), anti-H3 K27ac (Active Motif, 39133, 1:2,000), anti-H3 K36ac (Active Motif, 39379, 1:2,000), anti-H3 K56ac (Active Motif, 61061, 1:2,000), anti-H3 K4me3 (39915, 1:5,000), anti-H3 K9me3 (Active Motif, 39765, 1:5,000), anti-H3 K27me3 (Active Motif, 39155, 1:5,000), anti-H3 K4me2, K9me2, K27me2, K36me2, K79me2 (Cell Signaling Technology, 9847, 1:5,000), anti-H3s10p (Abcam, ab5176, 1:1,000), anti-H3.3 (EMD Millipore, 09-838, 1:1,000), anti-NEDD4 (Novus, NBP1-40112, 1:4,000), anti-GCN5 (Active Motif, 39975, 1:1,000), anti-USP22 (Abcam, ab4812, 1:2,000), anti-Flag (Sigma, 1:2,000), anti-HA (Covance, 1:2,000), anti-Actin (Sigma, 1:10,000), anti-IL1α (Abcam, ab17281), anti-IL1β (Abcam, ab2105) and anti-IgG heavy chain HRP (Sigma Aldrich, a1949, 1:1,000).

Techniques: Transfection, Plasmid Preparation, In Vivo, Ubiquitin Assay, Construct, Mutagenesis, In Vitro, Recombinant, Western Blot, Immunoprecipitation, Over Expression

Journal: Nature Communications

Article Title: H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

doi: 10.1038/ncomms14799

Figure Lengend Snippet: ( a – c ) NEDD4 regulates H3 K9ac at TSS of NEDD4 target genes. Shown were Venn diagram of genes with differential expression or differential H3 K9ac at TSS. GSEA was performed to evaluate the distribution of genes that show down-regulation of H3K9ac at TSS in NEDD4 knockdown cells in microarray-derived gene list, which is rank ordered either by T -test or fold change. ( d ) Heat map view of top and bottom gene list of microarray data sets. Microarray analysis for total RNA was performed for control and NEDD4 knockdown Hep3B cells. ( e ) NEDD4 knockdown impaired IL1α, IL1β and GCLM expression. qPCR was performed to analyse the mRNA level in control and NEDD4 knockdown Hep3B cells ( n =3, mean±s.e.m.). ( f ) IL1α, IL1β and GCLM were induced by glucose. Hep3B cells were glucose starved for 4 h and added-back glucose for 6 h before qPCR analysis ( n =3, mean±s.e.m.). ( g ) UCSC genome browser view of ChIP-seq H3 K9ac signals along the IL1B gene. ( h ) NEDD4 knockdown impaired H3 K9ac at TSS of IL1α IL1β and GCLM genes. ChIP-qPCR using anti-H3 K9ac antibody was performed for control and NEDD4 knockdown Hep3B cells ( n =3, mean±s.e.m.). ( i ) H3 K9ac was induced at TSS of IL1 α IL1β and GCLM genes by glucose. Hep3B cells were glucose-starved for 4 h and added-back glucose for 6 h before ChIP-qPCR analysis using anti-H3 K9ac antibody ( n =3, mean±s.e.m.). ( j ) NEDD4 knockdown impaired glucose-induced polymerase II (pol II) binding at TSS of IL1A and IL1B genes. Control and NEDD4 knockdown Hep3B cells were glucose-starved for 4 h and added-back glucose for 6 h before ChIP-qPCR analysis using anti-pol II antibody ( n =3, mean±s.e.m.). All asterisks (*) represent P <0.05, using Student's T -test.

Article Snippet: The following antibodies were used in this study: anti-H3 (Abcam, ab12079, 1:5,000), anti-H3 pan-ac (Active Motif, 39139, 1:2,000), anti-H3 K4ac (Active Motif, 39381, 1:2,000), anti-H3 K9ac (Active Motif, 39917, 1:3,000), anti-H3 K14ac (Active Motif, 39697, 1:2,000), anti-H3 K18ac (Active Motif, 39755, 1:2,000), anti-H3 K23ac (Active Motif, 39131, 1:2,000), anti-H3 K27ac (Active Motif, 39133, 1:2,000), anti-H3 K36ac (Active Motif, 39379, 1:2,000), anti-H3 K56ac (Active Motif, 61061, 1:2,000), anti-H3 K4me3 (39915, 1:5,000), anti-H3 K9me3 (Active Motif, 39765, 1:5,000), anti-H3 K27me3 (Active Motif, 39155, 1:5,000), anti-H3 K4me2, K9me2, K27me2, K36me2, K79me2 (Cell Signaling Technology, 9847, 1:5,000), anti-H3s10p (Abcam, ab5176, 1:1,000), anti-H3.3 (EMD Millipore, 09-838, 1:1,000), anti-NEDD4 (Novus, NBP1-40112, 1:4,000), anti-GCN5 (Active Motif, 39975, 1:1,000), anti-USP22 (Abcam, ab4812, 1:2,000), anti-Flag (Sigma, 1:2,000), anti-HA (Covance, 1:2,000), anti-Actin (Sigma, 1:10,000), anti-IL1α (Abcam, ab17281), anti-IL1β (Abcam, ab2105) and anti-IgG heavy chain HRP (Sigma Aldrich, a1949, 1:1,000).

Techniques: Expressing, Microarray, Derivative Assay, ChIP-sequencing, Binding Assay