Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Experimental design to dissect TGFβ-regulated genes in CCA cells and LINC00313 expression in HuCCT1 and Huh28 in response to TGFβ1, as identified by gene expression profiling. ( B ) LINC00313 expression in response to TGFβ1 stimulation for 16 h, in normal human cholangiocytes (NHC) and CCA cell lines. ( C ) LINC00313 expression in response to the indicated doses of TGFβ1 in HuCCT1 and Huh28 cell lines. ( D ) LINC00313 expression in response to TGFβ1 for the indicated time periods in HuCCT1 and Huh28 cell lines. ( E ) MALAT1 , RNU48 , GAPDH and LINC00313 RNA levels in nuclear and cytoplasmic fractions of HuCCT1 and Huh28 cell lines. Data information: Data in graphs are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test), n.s. not significant. .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Expressing, Gene Expression

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) LINC00313 expression in HuCCT1 and Huh28 cells treated with LY2157299 or DMSO, and stimulated with TGFβ1 or BSA/HCl for 16 h. ( B ) LINC00313 and SERPINE1 expression in HuCCT1 and Huh28 treated with SIS3 or DMSO and stimulated with TGFβ1 or BSA/HCl for 16 h. ( C ) LINC00313 expression in HuCCT1 transiently transfected with siRNA targeting SMAD2 , SMAD3 , or SMAD4 , alone or in combination, with or without TGFβ1. ( D ) LINC00313 expression in Huh28 transiently transfected with siRNA targeting SMAD2 , SMAD3 , or SMAD4 , alone or in combination, with or without TGFβ1. ( E ) Snapshot of the UCSC genome browser showing predicted SMAD-binding regions in LINC00313 gene locus, using the JASPAR CORE 2022 collection. Active histone mark (H3K27ac) is also shown around LINC00313 TSS. Data information: Data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test), n.s.: not significant. In panel ( A ) the experiment in HuCCT1 cells was performed twice ( n = 2 biological replicates) and data are presented as single data points. .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Expressing, Transfection, Binding Assay

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) LINC00313 levels in HuCCT1 or Huh28 treated with MEK, p38, JNK or PI3K inhibitors, with or without TGFβ1 stimulation for 16 h ( n = 3 biological replicates). ( B ) Immunoblotting for detection of phosphorylated p44/42 (Thr202/Tyr204), phosphorylated p-SAPK/JNK (Thr183/Tyr185), phosphorylated AKT (Ser473), phosphorylated p38 (Thr180/Tyr182), phosphorylated MAPKAPK-2 (Thr334), total p44/42, SAPK/JNK and AKT protein levels and β-tubulin (used as a loading control) in HuCCT1 cells treated with MEK, p38, JNK or PI3K inhibitors, with or without TGFβ1 stimulation for 16 h. DMSO was used as a vehicle treatment. ( C ) Immunoblotting for detection of the p38 isoforms p38α, p38β, p38γ and p38δ in HuCCT1 cells transiently silenced for MAPK11 or MAPK12 or MAPK13 or MAPK14 and treated or not with TGFβ1 for 16 h. β-tubulin was used as a loading control. ( D ) Real-time qPCR analysis of LINC00313 , MAPK11 , MAPK12 , MAPK13 and MAPK14 expression in HuCCT1 and Huh28 cell lines transiently transfected with siRNAs targeting MAPK11 or MAPK12 or MAPK13 or MAPK14 and treated or not with TGFβ1 for 16 h. Data information: In Panel ( A ) data are presented as mean ± SD ( n = 3 biological replicates). In panel ( D ) data are presented as single data points ( n = 2 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, n.s.: not significant (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Western Blot, Control, Expressing, Transfection

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Experimental approach to identify LINC00313 transcriptional targets. ( B ) Number of differentially expressed genes in pcLINC00313 versus pcDNA3 HuCCT1 cells (shrunken log2FC > 1, adjusted p -value < 0.1). Volcano plot shows differentially expressed genes (green colour: upregulated genes, red colour: downregulated genes). ( C ) KEGG pathway analysis of up or downregulated genes upon LINC00313 over-expression. ( D ) Example of GSEA of differentially expressed genes in response to LINC00313 over-expression. Shown is the enrichment of a Hippo signalling signature in the gene expression of cells overexpressing LINC00313. ( E ) WNT5A , AXIN2 and SULF2 mRNA levels in HuCCT1 expressing LINC00313 (pcLINC) or empty vector (pcDNA3). ( F ) WNT5A , AXIN2 and SULF2 expression in HuCCT1 transfected with siLINC00313 and stimulated with TGFβ1 or BSA/HCl for 16 h. Data information: In panel ( B ) statistically significant differential expressed genes were identified using DEseq2. In panel ( C ) adjusted p -value was calculated using the Benjamini-Hockberg method for correction for multiple hypotheses testing. In panel ( D ) GSEA results are displayed as a normalized enrichment score (NES) and presented as an enrichment plot. The Kolmogorov-Smirnov test was used for statistical analysis of GSEA. In ( E , F ) data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Over Expression, Gene Expression, Expressing, Plasmid Preparation, Transfection

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Efficient ligation of LINC00313 insert to pcDNA3.1 plasmid vector was verified by double digestion with restriction enzymes, followed by agarose gel electrophoresis. Molecular size (in kbp) marker ladder is shown in the first lane. ( B ) Real-time qPCR analysis of LINC00313 expression in HuCCT1 cells stably over-expressing pcDNA3.1 or pcLINC00313 expression vectors stimulated or not with TGFβ1 for 16 h. ( C ) Subcellular fractionation followed by RT-qPCR analysis of LINC00313 RNA levels in nuclear and cytoplasmic fractions of HuCCT1 cells transiently over-expressing an empty vector (pcDNA3.1) or LINC00313 and HuCCT1 clones, stably over-expressing pcDNA3.1 or LINC00313. ( D ) smiFISH for LINC00313 RNA in HuCCT1 cells stably over-expressing LINC00313 and stimulated with TGFβ for 16 h. DAPI was used to stain nuclei. Scale bars: 15 μm. ( E ) Heatmap presenting the top 40 differentially regulated genes in HuCCT1 pcLINC00313 versus pcDNA3-expressing cells (shrunken log2FC > 1, adjusted p -value < 0.1). Biological triplicates (1-3) were used per condition. ( F ) GSEA analysis of differentially expressed genes in pcLINC00313 versus pcDNA3 HuCCT1 cells. ( G ) Real-time qPCR analysis of LINC00313 , TCF7 , WNT5A , AXIN2 , and SULF2 expression in a monoclonal HuCCT1 cell line stably expressing pcDNA3.1 and in two monoclonal HuCCT1 cell lines stably expressing pcLINC00313, in the presence or not of TGFβ1 for 16 h. Data information: Data in panels ( B ), ( C ) and ( G ) are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). In panel ( F ) the Kolmogorov-Smirnov test was used for statistical analysis of GSEA. .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Ligation, Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Expressing, Stable Transfection, Fractionation, Quantitative RT-PCR, Clone Assay, Staining

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Venn diagram showing merged regions with overlapping and unique ATAC-seq peaks between pcDNA3 and pcLINC00313 expressing HuCCT1 cells. ( B ) Bar plot illustrating the top differential regions. ( C ) Venn diagram highlighting the 44 genes, whose chromatin accessibility and expression are altered. ( D ) GO analysis for biological process of the 44 genes highlighted in panel ( C ) using Enrichr. ( E ) Snapshot of the UCSC genome browser with ATAC-seq peaks at human TCF7 gene locus, in pcDNA3 versus pcLINC00313 over-expressing HuCCT1 cells. ( F ) TCF7 mRNA and protein levels in HuCCT1 cells over-expressing LINC00313 (pcLINC) or pcDNA3 control. ( G ) TCF7 RNA and protein levels in HuCCT1 cells, transfected with siLINC00313 and stimulated with TGFβ1 for 16 h. Data information: In panel ( D ) p -value was calculated by Enrichr using Fisher’s exact test or hypergeometric test. In ( F , G ) qPCR data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Expressing, Control, Transfection

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Schematic representation of the TCF/LEF-luciferase reporter assay. Activators (Wnt ligands, CHIR) and inhibitors (XAV939) of TCF/LEF-dependent responses are also shown. ( B ) TCF/LEF-luciferase reporter assay in HuCCT1 cells transiently transfected with TOP-Flash or FOP-Flash luciferase expression vectors and treated with CHIR99021 or DMSO for 24 h. Cells were co-transfected with a Renilla luciferase expression vector for normalization of the firefly luciferase activity. ( C ) TCF/LEF-luciferase reporter assay in HuCCT1 cells stably expressing the pGreenFire 2.0 TCF/LEF reporter construct and treated with CHIR99021 or DMSO for 24 h. TCF/LEF-luciferase reporter assay in HuCCT1 cells stably expressing the pGreenFire 2.0 TCF/LEF reporter construct and treated with the indicated concentrations of TGFβ1 or BSA/HCl (Ctr) for 16 h. ( D ) Subcellular fractionation followed by immunoblotting for β-catenin, lamin B1 (nuclear marker), β-tubulin and 14-3-3 (cytoplasmic markers) using nuclear and cytoplasmic fractions of HuCCT1 cells stably over-expressing an empty vector (pcDNA3.1) or LINC00313 and treated with CHIR99021 or DMSO for 24 h. ( E ) Immunofluorescence to detect β-catenin subcellular localization in control or LINC00313 over-expressing HuCCT1 cells, treated or not with CHIR99021 or DMSO for 24 h. Scale bars: 150 μm. ( F ) Cell viability assays in control or LINC00313 over-expressing HuCCT1 cells treated with CHIR99021 or DMSO for 24 h. ( G ) Colony formation assay in control or LINC00313 over-expressing HuCCT1 cells, treated with CHIR99021 or XAV939 or DMSO for 24 h. Quantification of the number of colonies for each condition is also shown. ( H ) Real-time qPCR analysis of AXIN2 , TCF7 and LINC00313 expression in control or LINC00313 overexpressing HuCCT1 cells, treated with CHIR99021, XAV939, or DMSO for 24 h. Data information: In panel ( B ) data are presented as mean ± SD ( n = 3 biological replicates). In panel ( F ) data are presented as mean ± SD ( n = 6 biological replicates). ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). In panels ( G ) and ( H ) data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay, Stable Transfection, Construct, Fractionation, Western Blot, Marker, Immunofluorescence, Control, Colony Assay

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) In vitro transcribed F-luc and LINC00313 RNAs. ( B ) Overview of interacting proteins identified by mass spectrometry. ( C ) Venn diagram representing the strategy to narrow down LINC0313 -binding proteins. ( D ) Protein network analysis depicting physical and functional interactions between the 51 nuclear interactors, using STRING. ( E ) GO analysis for molecular function of 32 nuclear interactors predicted by CatRAPID. ( F ) Heatmap depicting the probability of interaction between LINC00313 and ACTL6A. ( G ) Immunofluorescence of ACTL6A in control or LINC00313 over-expressing HuCCT1 cells, treated or not with TGFβ1. Scale bars: 150 μm. ( H ) RNA pull-down assays in HuCCT1 cells, stimulated with TGFβ1 or not, using in vitro synthesized F-luc mRNA or LINC00313 lncRNA, followed by immunoblotting for ACTL6A, HuR and β-actin. ( I ) RNA pull-down assay in HEK293T cells, over-expressing an empty vector or HA-tagged ACTL6A using in vitro synthesized LINC00313 lncRNA. An arrow depicts the specific HA-ACTL6A protein band, while an asterisk marks unspecific protein bands. ( J ) RIP assay for endogenous ACTL6A followed by qPCR for LINC00313 in HuCCT1 cells treated with TGFβ or BSA/HCl (vehicle control) for 16 h ( n = 3). An immunoblotting to verify the efficiency of ACTL6A immunoprecipitation is also shown. The arrows depict the specific endogenous ACTL6A protein bands. Data information: In panel J RIP-qPCR data are presented as mean ± SD ( n = 3 technical replicates). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: In Vitro, Mass Spectrometry, Binding Assay, Functional Assay, Immunofluorescence, Control, Expressing, Synthesized, Western Blot, Pull Down Assay, Plasmid Preparation, Immunoprecipitation

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) TCF7 , LINC00313 and ACTL6A mRNA levels in HuCCT1 transfected with siACTL6A or siNC and in the presence or not of TGFβ. ( B ) Immunoblotting for TCF7, ACTL6A and β-actin, upon silencing ACTL6A, using the indicated siRNA concentrations. ( C ) TCF/LEF luciferase reporter assay in siACTL6A or siNC HuCCT1 cells treated with CHIR99021 or DMSO for 24 h. ( D – F ) qPCR analysis of ACTL6A ( D ), AXIN2 ( E ) and TCF7 ( F ) mRNAs in the same conditions as these of panel ( C ). ( G – I ) qPCR analysis of SULF2 ( G ), TCF7 ( H ) and ACTL6A ( I ) mRNA levels in pcDNA3 or LINC00313 over-expressing HuCCT1 transiently transfected with siACTL6A or siNC with or without TGFβ. ( J ) Immunoblotting for TCF7, ACTL6A and β-actin in the same conditions as these of panels G-I. ( K ) qPCR analysis of TCF7 , ACTL6A and LINC00313 RNA levels in HuCCT1 cells transiently transfected with siRNAs against LINC00313 or ACTL6A individually or simultaneously with both siRNAs and treated or not with TGFβ for 16 h. ( L ) RIP assay for endogenous Brg1 followed by qPCR for LINC00313 in HuCCT1 cells treated with TGFβ or BSA/HCl (vehicle control) for 16 h. ( M ) Proposed model for the molecular mechanism of LINC00313 . Data information: In panels ( A ), (D–I) and (K) qPCR data are presented as mean ± SD ( n = 3 biological replicates). In panel ( C ), luciferase assay data are presented as mean ± SD ( n = 6 biological replicates). In panel ( L ), RIP-qPCR data are presented as mean ± SD ( n = 3 technical replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Transfection, Western Blot, Luciferase, Reporter Assay, Expressing, Control

Journal: Biomaterials

Article Title: High Yield, Scalable and Remotely Drug-Loaded Neutrophil-derived Extracellular Vesicles (EVs) for Anti-inflammation Therapy

doi: 10.1016/j.biomaterials.2017.05.003

Figure Lengend Snippet: Proteomics of EVs and their source cell. (a) Protein profile of NC-EVs and their parent cell (activated HL60 cells) by SDS-PAGE. The total proteins (b) and the membrane proteins (c) in NC-EVs, NS-EVs and their parent cell analyzed with mass spectroscopy.

Article Snippet: A HL60 cell line was bought from ATCC (Manassas, VA).

Techniques: SDS Page, Membrane, Mass Spectrometry

Journal: Molecular and Cellular Biology

Article Title: A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene

doi: 10.1128/mcb.25.3.1200-1212.2005

Figure Lengend Snippet: FIG. 1. A TGF- effector protein is purified from hepatic cells in the presence of the p53 protein. (A) Purification of SnoN protein. Solid-phase DNA oligomers, either biotinylated, 31-bp p53RE single-copy (lanes 3 to 6), ligated p53RE multimers (lanes 7 to 10), or single-copy, 1007 control (lanes 11 to 14) sequences, were incubated with 100 g of total protein of adult mouse liver nuclear extract (ML, input extract; lane 2), in the presence of recombinant p53 (p53 alone; lane 1, recombinant protein stabilized by addition of BSA). Following binding, bead-DNA-protein complexes were washed, eluted, and then separated by denaturing gel electrophoresis alongside extract proteins not binding to DNA (unbound, lanes 3, 7, and 11). The gel was silver stained; eluted p53 (dagger, lanes 6 and 9) and a protein enriched in the presence of p53 (asterisk, lanes 5 and 9) were analyzed by MALDI mass spectrometry. (B) In vivo ChIP analysis of wild-type and p53-null mouse liver tissue. PCR analysis of antibody-precipitated genomic fragments was conducted with primers amplifying the region of the SBE/p53RE. Sonicated fragments of formal- dehyde-cross-linked chromatin from 2-month-old wild-type (WT) and p53-null mouse liver tissue were antibody precipitated with tetra-acetylated histone H4 antibody (AcH4), Smad4, p53 (Ab-1), SnoN, P-Smad2, or control IgG. (C) Quantifying in vivo ChIP of Smad4 and P-Smad2 binding to the SBE/p53RE and distal genomic sites in WT and p53-null mouse liver tissue. Chromatin lysates were serially diluted (Input) and PCR amplified as described above to determine a linear range of amplification for all lysates. Antibody-precipitated fragments of DNA bound to Smad4 and SnoN were quantified by comparison to this range of values (% Bound). (D) Semiquantitative RT-PCR analysis of AFP RNA. RNA was isolated from 1- and 2-month-old WT and p53-null mouse liver tissue and analyzed in parallel for determination of GAPDH control RNA levels and AFP RNA levels.

Article Snippet: The primary antibodies used are as follows: p53 (Oncogene Ab1 and Santa Cruz SC-240), SnoN (SC-9141; Santa Cruz), Phospho-Smad2 (07–392; Upstate), Smad4 (06–693; Upstate) (SC-7966; Santa Cruz), AFP (SC C-19; Santa Cruz), and actin (A5316; Sigma).

Techniques: Control, Incubation, Recombinant, Binding Assay, Nucleic Acid Electrophoresis, Staining, Mass Spectrometry, In Vivo, Sonication, Comparison, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Molecular and Cellular Biology

Article Title: A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene

doi: 10.1128/mcb.25.3.1200-1212.2005

Figure Lengend Snippet: FIG. 2. SnoN, but not Ski, mediates repression of AFP expression. (A) SnoN-mediated transcription repression is compromised by dis- ruption of p53 DNA binding. (Top) Hepa 1–6 hepatoma cells were transfected with AFP/lacZ (wild-type sequence for p53 binding) or DelA/lacZ (mutated for p53 binding) reporter constructs (500 ng/ plate) along with the indicated expression vectors (p53 and SnoN; 500 ng/plate [each]). Each plate was also cotransfected with the pGL2 luciferase vector (50 ng/plate) to standardize and control for transfec- tion efficiency. Expression levels relative to baseline are indicated for AFP/lacZ (gray) and DelA/lacZ (black). Cotransfected proteins exhib- ited equivalent levels of protein expression by Western blot analysis (data not shown). (Bottom) The p53/HNF-3 (FoxA) regulatory ele- ment centered at 850 in the AFP promoter (42) overlaps with con- sensus SBEs (89). The DelA/lacZ binding element has a 10-bp deletion within the 5 p53 half site (shaded) and point mutations in the 3 half site. (B) Ski activates AFP expression in the presence of p53. Identical transient transfection analyses were performed with the AFP/lacZ reporter and coexpressed p53, SnoN, and c-Ski constructs, as indi- cated. Expressed Ski protein repressed AFP only slightly and activated expression in the presence of p53. -gal, -galactosidase; luc, lucif- erase.

Article Snippet: The primary antibodies used are as follows: p53 (Oncogene Ab1 and Santa Cruz SC-240), SnoN (SC-9141; Santa Cruz), Phospho-Smad2 (07–392; Upstate), Smad4 (06–693; Upstate) (SC-7966; Santa Cruz), AFP (SC C-19; Santa Cruz), and actin (A5316; Sigma).

Techniques: Expressing, Binding Assay, Transfection, Sequencing, Construct, Luciferase, Plasmid Preparation, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene

doi: 10.1128/mcb.25.3.1200-1212.2005

Figure Lengend Snippet: FIG. 3. TGF- represses endogenous AFP levels in hepatoma cells. (A) TGF- and p53 repress AFP. Serum-starved Hepa 1–6 cells were treated with increasing concentrations of TGF-1 (2 to 20 ng/ml of medium, lanes 3 to 7), vehicle (lanes 8 to 12), or no additions (lanes 1 and 2) and harvested at 0 h (lane 1) or 24 h (lanes 2 to 12) posttreatment. Whole-cell lysates were prepared from these cells, and immunoblot analyses were performed to analyze the effects of TGF-1 treatment on AFP, p53, Smad4, and P-Smad2 protein levels. (B) AFP is a downstream physiological target of TGF-. Hepa 1–6 cells were treated with 2 ng of TGF-1/ml or vehicle control and harvested at the indicated time points posttreatment (0 to 56 h). Total RNA was harvested, and Northern blot analysis was performed to examine the levels of AFP and SnoN mRNA. The graph represents AFP (solid line) and SnoN (broken line) RNA levels in cells treated with ligand relative to those that received only the vehicle, each normalized to an actin RNA loading control. (C) TGF- induces SnoN protein degradation, Smad2 protein phosphorylation, and a decrease in AFP. Hepa 1–6 cell lysates were treated with 4 ng of TGF-1 ligand/ml or vehicle (V) only (as in panel B) over a 24-h interval and harvested at the indicated times for preparation of nuclear and cytoplasmic extracts. Immunoblot analysis of nuclear extracts was performed to determine SnoN, P-Smad2, p53, and actin protein levels. AFP levels were determined in cytoplasmic extracts of the same cells.

Article Snippet: The primary antibodies used are as follows: p53 (Oncogene Ab1 and Santa Cruz SC-240), SnoN (SC-9141; Santa Cruz), Phospho-Smad2 (07–392; Upstate), Smad4 (06–693; Upstate) (SC-7966; Santa Cruz), AFP (SC C-19; Santa Cruz), and actin (A5316; Sigma).

Techniques: Western Blot, Control, Northern Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene

doi: 10.1128/mcb.25.3.1200-1212.2005

Figure Lengend Snippet: FIG. 4. Downstream effectors of TGF- and p53 are recruited to SBE/p53RE in vivo and effect chromatin modifications in response to TGF- treatment. (A) Chromatin immunoprecipitation of proteins bind- ing to the AFP SBE/p53RE. ChIP assays of SBE/p53RE were performed with Hepa 1–6 cells treated with a 2-ng/ml concentration of TGF-1 (black bars) or vehicle control (gray bars) for 24 h. Immunoprecipitation of formaldehyde-cross-linked lysate was performed using 1.5 g of anti- body specific for p53, 3 g of SnoN, 5 g of Smad4, and 5 l of P-Smad2 antibodies followed by PCR using oligonucleotides specific for the SBE/ p53RE. As negative controls, lysis buffer alone was added to the immu- noprecipitation or lysate was incubated with 5 g of anti-IgG (see sup- plementary Fig. S2). PCRs using oligonucleotides specific for the AFP start site were also performed as an additional test for antibody specificity. Graphs indicate the average values of percent input bound calculated from multiple experiments and the standard error of the mean. n-fold changes of ligand versus vehicle control are shown below each data set. (B) Deacetylation and concomitant methylation of histone H3 lysine 9 is targeted at SBE/p53RE in response to TGF- signaling. ChIP assays were performed as described above, except that 5 g of anti-AcH4, 5 g of anti-AcH3K9, and 5 l of anti-diMetH3K9 antibodies were used for ChIP of formaldehyde-cross-linked chromatin lysates. Graphs indicate the av- erage values of percent input bound calculated from multiple experiments and the standard errors of the means. n-fold changes are shown below each data set.

Article Snippet: The primary antibodies used are as follows: p53 (Oncogene Ab1 and Santa Cruz SC-240), SnoN (SC-9141; Santa Cruz), Phospho-Smad2 (07–392; Upstate), Smad4 (06–693; Upstate) (SC-7966; Santa Cruz), AFP (SC C-19; Santa Cruz), and actin (A5316; Sigma).

Techniques: In Vivo, Chromatin Immunoprecipitation, Concentration Assay, Control, Immunoprecipitation, Lysis, Incubation, Methylation

Journal: Molecular and Cellular Biology

Article Title: A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene

doi: 10.1128/mcb.25.3.1200-1212.2005

Figure Lengend Snippet: FIG. 5. Both SnoN and p53 are essential for TGF--induced repression of AFP transcription. (A) Depletion of SnoN and p53 by siRNA methodology. Double-stranded oligomers homologous to specific sites within the coding regions of SnoN and p53, respectively, were cloned into U6 promoter-containing vectors and introduced by transfection of Hepa 1–6 cells. Three targets sequences for each gene were tested, each with various degrees of success in knockdown of protein expression. Left panel: transfection was performed without short, hairpin RNA-expressing constructs (shRNA; mock), in duplicate (a and b) with target 1 and separately with targets 2 and 3. Immunoblotting for SnoN revealed that targets 1 and 3 were most effective in knockdown of SnoN expression. Target 2 oligonucleotides contained sequence mismatches for the SnoN target region. Actin was analyzed as a control for nonspecific effects on total protein. Right panel: analysis of p53 knockdown was conducted on lysates of Hepa 1–6 cells treated with actinomycin D to activate p53 protein as previously described (42). Transfections of shRNA constructs were performed as described above. Lysate of actinomycin D-treated cells that were not transfected are analyzed in the lane labeled as “no siRNA. ” (B) Ligand-mediated repression of AFP transcription requires p53 and SnoN. Plasmid constructs capable of inducing siRNA for SnoN or p53 (target 1 constructs for each) were introduced separately or together into Hepa 1–6 cells by transient transfection. A vector specific for siRNA-mediated knockdown of firefly luciferase was used as a nonspecific, target control for RNA depletion. The “no siRNA” cells were not transfected. RNA was isolated from Hepa 1–6 cells, all of which were treated with TGF-1 ligand (2 ng/ l) or vehicle alone for 24 h, following a 24-h recovery period after transfection. Levels of AFP RNA were determined by Northern blot analysis and comparison to a cyclophilin RNA loading control. Left panel: a graph summarizing the results of four siRNA experiments; RNA levels were determined by Northern blot as described above. Error bars indicate standard deviation. Right panel: representative Northern blot of AFP and cyclophilin RNA isolated from vehicle only (V) or TGF-1 ligand-treated (L) cells, which received siRNA constructs as indicated. (C) Immunoblot analysis of siRNA-treated nuclear extracts. SiRNA and ligand or vehicle-only conditions were exactly as shown in panel B and as described above. Actin protein analysis serves as a loading control and a control for nonspecific effects of siRNA treatment.

Article Snippet: The primary antibodies used are as follows: p53 (Oncogene Ab1 and Santa Cruz SC-240), SnoN (SC-9141; Santa Cruz), Phospho-Smad2 (07–392; Upstate), Smad4 (06–693; Upstate) (SC-7966; Santa Cruz), AFP (SC C-19; Santa Cruz), and actin (A5316; Sigma).

Techniques: Clone Assay, Transfection, Knockdown, Expressing, shRNA, Construct, Western Blot, Sequencing, Control, Labeling, Plasmid Preparation, Luciferase, Isolation, Northern Blot, Comparison, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene

doi: 10.1128/mcb.25.3.1200-1212.2005

Figure Lengend Snippet: FIG. 6. p53 targets histone deacetylation to the SBE/p53RE and mediates SnoN-dependent repression of AFP transcription. (A) Diagram of in vitro chromatin assembly and analysis. AFP bead DNA is preincubated with protein extracts and/or recombinant p53 protein prior to chromatin assembly over a 1-h incubation in X. laevis (Xl) egg extract. Assembled chromatin bead DNA is then washed in an isotonic buffer prior to functional assay by transcription in HeLa extract or structural analysis by ChIP and HDAC assays. (B and C) p53 triggers deacetylation of local and distal chromatin. ChIP assays (two to three independent assays for each data point) were performed in vitro on AFP/lacZ DNA, which was chromatin assembled in the presence of HepG2 whole-cell extract or HepG2 plus 500 ng of activated p53. DNA was purified from protein-chromatin immunoprecipitates and analyzed for percent SBE/p53RE and transcription start site DNA bound to acetylated histone H3 (AcH3) or histone H4 (AcH4) by Southern blotting with labeled (B) SBE/p53RE or start site (4 to 26) oligonucleotides or (C) across the AFP templates with oligonucleotides specific for 10/32, 140/118, 327/305, 520/398, 800/778, 2637/2615, and 3339/3361. Representative data are shown below the summary graph (B). (D) p53-dependent HDAC activity at SBE/p53RE. Immobilized SBE/p53RE DNA oligomers were incubated in 100 g of HepG2 extract, either tryptic soy agar treated (10 M; black bars) or untreated (gray bars), in the presence or absence of recombinant, activated p53. Protein complexes bound to the oligomers were washed and incubated with in vivo-labeled, 3H-acetylated histones, which were isolated and purified. HDAC enzymatic activity was measured by scintillation counting of released [3H]acetyl groups. The graph

Article Snippet: The primary antibodies used are as follows: p53 (Oncogene Ab1 and Santa Cruz SC-240), SnoN (SC-9141; Santa Cruz), Phospho-Smad2 (07–392; Upstate), Smad4 (06–693; Upstate) (SC-7966; Santa Cruz), AFP (SC C-19; Santa Cruz), and actin (A5316; Sigma).

Techniques: In Vitro, Recombinant, Incubation, Functional Assay, Southern Blot, Labeling, Activity Assay, In Vivo, Isolation

Journal: bioRxiv

Article Title: Modulation of ERK1/MAPK3 potentiates ERK nuclear signalling, facilitates neuronal cell survival and improves memory in mouse models of neurodegenerative disorders

doi: 10.1101/496141

Figure Lengend Snippet: a Dose-response curve of RB5 peptide for ERK activation. Different doses of RB5 were used on fresh striatal slices from adult mice. The level of activation is expressed on the Y-axis as arbitrary units (AU). Doses are reported in a logarithmic scale (Log10) on the X-axis. RB5 was effective in enhancing pERK with an EC50 of 3.4 μM. b-c (Upper panels) Representative merged images showing histone AcH3 phosphorylation (b) and ribosomal S6 protein phosphorylation (c) on ex vivo acute slices pre-treated for 1h with Scramble (SCR) or RB5 peptide (50μM), and stimulated for 10 min with glutamate (GLU, 100μM) or Vehicle (Veh). (Lower panel b) RB5 enhanced pAcH3 in response to glutamate (Two-way ANOVA, peptide x treatment interaction F 1,59 =73.314 P<0.0001, simple main effect of glutamate F 1,59 =119.285 P<0.0001, Bonferroni’s post-hoc, SCR GLU (n=13) vs RB5 GLU (n=19) P<0.0001). (Lower panel c) RB5 prevented glutamate mediated pS6 increase (Two-way ANOVA, peptide x treatment interaction F 1,45 =40.970 P<0.0001, simple main effect of glutamate F 1,45 =61.369 P<0.0001, Bonferroni’s post-hoc, SCR Veh (n=9) vs SCR GLU (n=12) P<0.0001, RB5 Veh (n=14) vs RB5 GLU (n=14) P=1.000). d-e (Upper panels) Representative merged images showing pAcH3 (d), and pS6 (e) on slices from ERK1-/-mice pre-treated for 1h with SCR or RB5 peptide (50μM) and stimulated with glutamate or vehicle for 10 min. (Lower panel d) ERK1-/- slices showed a greater enhancement of pAcH3 in response to glutamate (Two-way ANOVA, genotype x treatment interaction F 1,41 =5.448 P<0.05, simple main effect of glutamate F 1,41 =9.617 P<0.01, Bonferroni’s post-hoc, ERK1-/- GLU (n=11) vs ERK1+/+ GLU (n=11) P<0.01). (Lower panel e) Absence of ERK1 prevented glutamate mediated pS6 enhancement (Two-way ANOVA, genotype x treatment interaction F 1,43 =9.900 P<0.01, simple main effect of glutamate F 1,43 =11.315 P<0.01, Bonferroni’s post-hoc, ERK1+/+ GLU (n=15) vs ERK1-/- GLU (n=8) P<0.01, ERK1-/- Veh (n=9) vs ERK1-/- GLU P=0.701). f PERK positive cells in dorsal striatum induced by a single dose of RB5 (20 mg/kg, i.p.) at different time-points (n=5 mice/group). Scramble-injected mice (20 mg/kg, i.p.) were perfused after 1h. RB5-mediated enhancement of ERK phosphorylation lasted up to 6h post-injection (One-way ANOVA, F5,2 4 =10.942 p<0.0001, Bonferroni’s post-hoc, SCR vs RB5 1h and 6h P<0.0001, SCR vs RB5 3h P<0.001). g RB5 brain levels measured with mass spectrometry at different hours after a single administration (20 mg/kg i.p.). High levels of RB5 were detected up to 6h (Oneway ANOVA, F 4,18 =17.469 P<0.0001, Bonferroni’s post-hoc, SCR vs RB5 1h P<0.01, SCR vs RB5 3h P<0.0001, SCR vs RB5 6h P<0.05). h PERK positive cells in dorsal striatum induced by increasing doses of RB5 (1.5, 2.5, 5, 10, and 20 mg/kg, i.p.) at 1h after administration (n=5 mice/group). SCR peptide was injected at 20 mg/kg, i.p. All mice were perfused after 1h. RB5 caused a significant increase of ERK phosphorylation at both 10 and 20 mg (One-way ANOVA, F 5,24 =14.292 P<0.0001, Bonferroni’s post-hoc, SCR vs RB5 10, and vs RB5 20 P<0.0001). i-t (Upper panels) Representative images showing p-ERK, nuclear ERK-dependent markers and cytoplasmic markers in striata of mice injected either with RB5 or SCR peptide (20 mg/kg, i.p.) and perfused 1h later. (Lower panels) RB5 increased ERK phosphorylation ( i , Independent sample t-test t 28 = −5.340 P<0.0001; SCR (n=15) RB5 (n=15)) and promoted a selective activation of nuclear ERK-dependent markers as pMSK-1 ( j , Independent sample t-test t 29 = −2.808 P<0.01; SCR (n=16) RB5 (n=15)), pAcH3 ( k , Independent sample t-test t 28 = −5.404 P<0.0001; SCR (n=16) RB5 (n=14)), pELK ( l , Independent sample t-test t 29 = −12.106 P<0.0001; SCR (n=16) RB5 (n=15)) and c-Fos ( m , Independent sample t-test t 29 = −12.743 P<0.0001; SCR (n=16) RB5 (n=15)). Differently, in the cytoplasm RB5 did not affect the phosphorylation of pMEK-1 ( n , Independent sample t-test t 28 = 0.609 P=0.547; SCR (n=15) RB5 (n=15)) or the phosphorylation of Kv4.2 ( o , Independent sample t-test t 28 = 0.618 P=0.541; SCR (n=16) RB5 (n=14)). In addition, RB5 slightly reduced ERK dependent phosphorylation of S6 (Thr235/236) ( p , Independent sample t-test t 29 = 2.191 p<0.05; SCR (n=16) RB5 (n=15)) while had no effect on mTOR/TORC1 dependent phosphorylation of S6 (Thr240/244) ( q , Independent sample t-test t 28 = 1.878 P=0.071; SCR (n=16) RB5 (n=14)). r-t (Upper panels) Representative images of phosphorylation of ERK, AcH3 and S6 in striata of ERK1-/- mice after a single RB5 injection. r (Lower panel) RB5 increased ERK phosphorylation in WT but not in KO mice (Two-way ANOVA, genotype x peptide interaction F 1,36 =23.301 P<0.0001, simple main effect of peptide F 1,36 =37.707 p<0.0001, Bonferroni’s post-hoc, ERK1+/+ SCR (n=10) vs ERK1+/+ RB5 (n=8) P<0.0001; ERK1 -/- SCR (n=12) ERK1 -/- RB5 (n=10)). s (Lower panel) RB5 increased pAcH3 in WT mice (Two-way ANOVA, genotype x peptide interaction F 1,30 =17.494 P<0.0001, simple main effect of peptide F 1,30 =37.505 P<0.0001, Bonferroni’s post-hoc, ERK1+/+ SCR (n=8) vs ERK1+/+ RB5 (n=8) P<0.0001; ERK1 -/- SCR (n=9) ERK1 -/- RB5 (n=9)). t (Lower panel) RB5 treatment did not change the phosphorylation of S6 (Thr235/236) in both genotypes (Two-way ANOVA, genotype x peptide interaction F 1,31 =2.601 P=0.117, main effect of peptide F 1,31 =3.266 P=0.080, main effect of genotype F 1,31 =1.185 P=0.285; ERK1+/+ SCR (n=9) ERK1+/+ RB5 (n=10) ERK1-/- SCR (n=8) ERK1-/- RB5 (n=8)). Results show mean±s.e.m. White star P<0.0001, ★★★P<0.001, ★★ P<0.01, ★ P<0.05.

Article Snippet: Briefly, 1h after blocking in 5% normal goat serum, slices were incubated overnight at 4°C with the following primary antibodies: anti-phospho (Ser10)-acetylated (Lys14) histone H3 (1:1000, Millipore), anti-phospho-S6 ribosomal protein (Ser235/236) (1:200, Cell Signalling Technology) anti-phospho-S6 ribosomal protein (Ser240/244) (1:200, Cell Signalling) or Cleaved Caspase 3 (1:200, Cell Signalling Technology) and NeuN (1:1000, Millipore).

Techniques: Activation Assay, Ex Vivo, Injection, Mass Spectrometry