Journal: iScience

Article Title: HOXA11 promotes lymphatic metastasis of gastric cancer via transcriptional activation of TGFβ1

doi: 10.1016/j.isci.2023.107346

Figure Lengend Snippet: HOXA11 activates nuclear expression of Smad2 in the TGFβ/Smad signaling pathway through transcriptional regulation of TGFβ-1 (A) ChIP-qPCR assay revealed the potential binding sites of HOXA11 in the TGFβ1 promoter region. (B and C) Dual-luciferase assays demonstrated that HOXA11 activated TGFβ-1 transcription through direct regulation. (D–G) Cellular immunofluorescence assays detected Smad2 entry into the nucleus in HOXA11 overexpressed (D and E) or knockdown (F and G) gastric cancer cells, Scale bar: 10 μm. (H and I) Western blot assays examined the protein expression of Smad-2 and P-smad2 in gastric cancer cells overexpressing (H) or knocking down (I) HOXA11 or after treatment with the TGFβ1 signaling pathway inhibitor. The data are presented as the mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocking with 5% normal donkey serum, cells were incubated overnight at 4°C with primary antibodies specific for E-cadherin (1:200, CST), N-cadherin (1:200, CST), Vimentin (1:100, CST) and Smad2 (1:400, CST), followed by incubation with fluorescence-conjugated secondary antibodies at room temperature for 1 hours.

Techniques: Expressing, ChIP-qPCR, Binding Assay, Luciferase, Immunofluorescence, Knockdown, Western Blot

Journal: iScience

Article Title: HOXA11 promotes lymphatic metastasis of gastric cancer via transcriptional activation of TGFβ1

doi: 10.1016/j.isci.2023.107346

Figure Lengend Snippet: HOXA11 promotes VEGF-C expression and secretion through activation of the TGFβ1/SMAD2 pathway (A–D) Expression of VEGF-C and VEGF-D in gastric cancer cells affected by HOXA11 overexpression (A and C) or knockdown (B and D) was determined by RT-qPCR and Western blot assays. (E and F) ELISA assays determined VEGF-C expression in supernatant cultures of gastric cancer cells with HOXA11 overexpression (E) or knockdown (F). (G) ChIP-qPCR assay revealed the potential binding sites of Smad2 in the VEGF-C promoter region. The data are presented as the mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocking with 5% normal donkey serum, cells were incubated overnight at 4°C with primary antibodies specific for E-cadherin (1:200, CST), N-cadherin (1:200, CST), Vimentin (1:100, CST) and Smad2 (1:400, CST), followed by incubation with fluorescence-conjugated secondary antibodies at room temperature for 1 hours.

Techniques: Expressing, Activation Assay, Over Expression, Knockdown, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Binding Assay

Journal: iScience

Article Title: HOXA11 promotes lymphatic metastasis of gastric cancer via transcriptional activation of TGFβ1

doi: 10.1016/j.isci.2023.107346

Figure Lengend Snippet:

Article Snippet: After blocking with 5% normal donkey serum, cells were incubated overnight at 4°C with primary antibodies specific for E-cadherin (1:200, CST), N-cadherin (1:200, CST), Vimentin (1:100, CST) and Smad2 (1:400, CST), followed by incubation with fluorescence-conjugated secondary antibodies at room temperature for 1 hours.

Techniques: Recombinant, Membrane, Chromatin Immunoprecipitation, Magnetic Beads, Luciferase, Reporter Gene Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Nature communications

Article Title: TGFβ induces the formation of tumour-initiating cells in claudinlow breast cancer.

doi: 10.1038/ncomms2039

Figure Lengend Snippet: Figure 5 | NEDD9 is a TGF/Smad and MRTF-SRF target. (a) Western blot using the indicated antibodies of untreated ( − ) and TGFβ (2.5 ng ml − 1)- treated ( + ) MDA-MB-231 M1 cultures transfected with siRNA pools against SRF, Smad2 (S2), Smad3 (S3) Smad2 and 3 (S2 + 3) and Smad4 (S4). (b) qRT–PCR analysis of NEDD9 in same conditions as in (a). Error bars represent ± s.d. of three experiments. (c) qRT–PCR of the indicated transcripts in HCC1954 (left panel) and MDA-MB-231 (right panel) M1 mammospheres treated with TGFβ (2.5 ng ml − 1), 5 µM CD or 0.5 µM LB as indicated. Expression was normalized against beta-2-microglobulin and expressed as arbitrary units. Error bars represent ± s.d. of at least three independent experiments. (d) ChIP assay on MDA-MB-231 M1 mammospheres, untreated ( − ) or treated with TGFβ (2.5 ng ml − 1) (Tβ) for 1 h to detect binding of endogenous SRF and Smad2/3 to R1, R2 and R3. KRT19SBE region was used as an internal positive control for Smad2/3 binding. Results are expressed as TGFβ-induced fold change in binding from untreated control cultures. Error bars represent ± s.d. of three independent experiments. (e) ChIP assay as described in (d) to detect binding of endogenous MRTF-A and MRTF-B. (f) UCSC Genome Browser display showing regions R1, R2 and R3 that were selected to design primers to detect binding of Smad, SRF, MRTF-A and MRTF-B by ChIP-qPCR assays. Error bars represent ± s.d. of three independent experiments. Also showing ChIP-sequencing enrichment of Smad2/3 binding in M1 mammosphere cultures treated with TGFβ (2.5 ng ml − 1) for 1 h across the NEDD9 genomic region. Black boxes correspond to called binding events obtained using both MACS and SWEMBL algorithms.

Article Snippet: Cell suspensions were treated with TGFβ for 1 h at 37 °C, where indicated, and ChIP was performed following the method outlined in Schmidt et al.55, using SRF rabbit polyclonal antibody (Santa Cruz Biotechnology Inc. sc-335 X) and smad2/3 rabbit polyclonal antibody (Santa Cruz Biotechnology Inc. sc-8332 X) and Protein G Dynabeads (Dynal).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Expressing, Binding Assay, Positive Control, Control, ChIP-qPCR, ChIP-sequencing

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Characterization of pCRMs instructing ZGA. (a) Timeline (hpf, hours post-fertilization, at 25°C) of the maternal-to-zygotic transition and earliest signaling events (nuclear accumulation of Wnt, Nodal and Bmp signal mediators β-catenin, Smad2 and Smad1, respectively) during early X. tropicalis development up to the late gastrula embryo (12 hpf) with ∼40,000 (40K) cells. (b) Signal transduction pathway causing signal mediators to enter the nucleus and engage with pCRMs (e.g. marked by RNAPII). (c) Snapshot of RNAPII recruitment to pCRMs of the zygotic gene locus tbxt from the 32-cell to the late gastrula stage. The underlying DNA sequence of RNAPII + pCRMs are used to discover enriched DNA motifs de novo (illustrated as colored arrows for one RNAPII + pCRM). (d) Spearman correlations (R s ) of RNAPII binding levels across ∼27,000 pCRMs (Supplementary Table 2) between the indicated developmental stages. (e) Temporal enrichment (Z-score) of consensus DNA motifs known to be recognized by indicated TF families among RNAPII + pCRMs. (f) MBT-staged heat map of DNase-probed chromatin accessibility (n=2), RNAPII binding and H3K4me1 (n=2) marking across ∼17,500 pCRMs (Supplementary Table 3) grouped by sequence conservation levels (phastCons) and sorted by the statistical significance of pCRM accessibility. Abbreviations used for the developmental timeline: 32, 128 and 1K, 32-, 128- and 1,024-cell stage; MBT, mid-blastula transition; eG, mG and lG, early, mid- and late gastrula stage.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Transduction, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Search for ZGA-critical proteins based on their significantly enriched DNA recognition motifs at accessible and engaged (RNAPII + /H3K4me1 + ) pCRMs and their high translation frequency before MBT. (a) Maternal protein concentrations in the egg versus ribosome footprint (translation) levels at the 8-cell stage . Most frequently translated representatives of various TF families are labeled. (b) Matching canonical pCRM-enriched DNA motifs (sorted by statistical significance) with frequently translated TFs and signal mediators. (c) WMIHC of Sox3 protein in control and Sox3 loss-of-function (LOF) embryos at the 64-cell and blastula stage. Nuclear accumulation of Sox3 protein was detected in both the animal (An) and vegetal (Vg) hemisphere of control embryos. Scale bar, 0.5 mm. (d) Graphical illustration of protein levels (derived from mass spectrometry data ) and nuclear localizations (mainly derived from WMIHC, see references below) of selected TFs and signal mediators based on our and previously published results: Sox3 (this study and ref. ), mPouV (Pou5f3.2 and Pou5f3.3) and (zygotic) Pou5f3.1 (deduced from transcript data ), mVegT and zVegT , β-catenin , , Smad1 (this study and ref. , ) and Smad2 (this study and ref. , ). Shaded boxes indicate periods of non-nuclear protein localization. Arrows indicate tissue movements of gastrulation. Abbreviations used for the developmental timeline: 8 and 32, 8-cell and 32-cell stage; MBT, mid-blastula transition; and eG, early gastrula stage.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Labeling, Derivative Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Verification of antibodies against frequently translated TFs and signal mediators. ( a ) Line chart shows the level of ribosome footprints of selected TFs and signal mediators across MBT in rpkm (reads per kilobase of transcript per million mapped reads). Abbreviations used for the developmental timeline: 8, 8-cell stage; MBT, mid-blastula transition; lG, late gastrula stage. ( b ) Western blot shows the immunoprecipitation (IP) of Sox3 protein extracted from early gastrula embryos. IgG H and L, detected IgG heavy and light chains of the IP antibody. ( c ) Western blot shows the level of Sox3 protein immunoprecipitated from control (control MO) and Sox3-depleted (Sox3 MO) blastula embryos (Sox3 LOF). α-tubulin, IP input control. ( d , g , i , j , n ) Bar graphs show the ChIP-qPCR results as a percentage of ChIP input (mean+SD; n=2-3) for Sox3, β-catenin, Smad1 and Smad2 at the early gastrula stage and for Tbx6 at the early neurula stage. One-tailed Student’s t-test (comparing to IgG control): *, p ≤0.1 and ≥2-fold enrichment relative to the lowest DNA recovery with the ChIP antibody (dotted line). ( e ) Ribosome footprinting tracks show the post-MBT switch of translation from the maternal (m) to the zygotic (z) VegT transcript. The VegT translation-blocking MO was designed to block translation of the maternal transcript only. ( f ) Western blot shows the level of mVegT protein immunoprecipitated from control (control MO) and mVegT-depleted (mVegT MO) blastula embryos (mVegT LOF). Dotted line indicates the elimination of irrelevant lanes from the western blot. α-tubulin, IP input control. ( h ) WMIHC shows the spatial distribution of nuclear Smad1 and Smad2 protein on bisected early gastrula embryos. Scale bar, 0.5 mm. ( k ) Western blot shows the level of Tbx6 immunoprecipitated from X. tropicalis and X. laevis late gastrula embryos. Antibody #4596 was chosen for subsequent IP and ChIP experiments, and #5061 for Western blotting. ( l ) Western blot (WB) shows the level of Tbx6 protein immunoprecipitated from standard control and Tbx6 morphants at the early tailbud stage. α-tubulin, IP input control. ( m ) Line charts show the relative level of tbx6 transcripts (RT-qPCR) and Tbx6 protein (IP/WB) from the early blastula to the early tailbud stage. α-tubulin, IP input control.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Western Blot, Immunoprecipitation, One-tailed Test, Footprinting, Blocking Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Chromatin engagement of TFs and signal mediators during the maternal-to-zygotic transition. ( a ) Chromatin profiling (ChIP-Seq, n=1-3) of selected TFs and signal mediators from the 32-cell to the early tailbud stage. In all subsequent figure panels, the chromatin factors and developmental stages profiled are consistently color-coded as illustrated here. The excerpt of multiple chromatin tracks shows the binding of maternal TFs (Sox3, Foxh1 and VegT) and signal mediators (β-catenin, Smad2 and Smad1) to the siamois2 ( sia 2 and sia2l ) super-enhancer at the 1,024-cell stage (see for the temporal progression of chromatin engagement to the siamois2 and ventx super-enhancers). ( b ) Bubble plot shows significantly enriched biological processes associated with zygotic super-enhancer + genes (i.e. genes possessing engaged super-enhancers ≤5 kb from their active TSS at indicated developmental stages). ( c ) Meta-plots (mean [solid line] ± SD [dotted line or polygon]) summarize the level of RNAPII engagement across 2,000 pCRMs most frequently occupied by the indicated TFs or signal mediators at the 1,024-cell and early gastrula stage, respectively. The pie chart next to each meta-plot shows the percentage of these TF + or signal mediator + pCRMs bound by RNAPII (ChIP ≥2x input tag density): 98% (1,024-cell stage) and 99% (early gastrula stage) of Sox3 + pCRMs, 100% and 90% of Foxh1 + pCRMs, 93% and 97% of VegT + pCRMs, 65% and 98% of β-catenin + pCRMs, 96% and 98% of Smad2 + pCRMs, and 78% and 99% of Smad1 + pCRMs. ( d ) Biplot of principal component (PC) 1 (accounting for 40% variance) and 2 (28% variance) shows the similarity (or dissimilarity) of TF (Sox3, mVegT, Foxh1 , , Eomes , zVegT , Tbxt and Tbx6) and signal mediator (β-catenin, Smad1, Smad2) binding levels across ∼12,500 highly engaged pCRMs (compiled from the 2,000 pCRMs with the highest DNA occupancy levels detected per protein and developmental stage) over several developmental stages. Note that developmental time (arrow) separates these profiles best. ( e ) Biplot of PC1 (accounting for 60% variance) and PC2 (18% variance) for the temporal progression of RNAPII and TF binding levels across the same set of pCRMs as in ( d ). Abbreviation: r, biological replicates. ( f ) Meta-plots (mean [solid line] ± SD [dotted line or polygon]) summarizes the level of signal mediator engagement across 2,000 pCRMs most frequently occupied by the indicated TF at the 1,024-cell and early gastrula stage, respectively. The pie chart next to each meta-plot shows the percentage of these TF + pCRMs bound by signal mediators (ChIP ≥2x input tag density): β-catenin at Sox3 + pCRMs (10% at the 1,024-cell stage and 100% at the early gastrula stage), Smad2 at Sox3 + pCRMs (96% and 91%), Smad1 at Sox3 + pCRMs (96% and 85%), β-catenin at Foxh1 + pCRMs (12% and 87%), Smad2 at Foxh1 + pCRMs (94% and 82%), Smad1 at Foxh1 + pCRMs (63% and 59%), β-catenin at VegT + pCRMs (11% and 100%), Smad2 at VegT + pCRMs (64% and 92%) and Smad1 at VegT + pCRMs (55% and 68%). ( g ) Heat map shows the statistical significance of finding TF- and signal-specific DNA consensus motifs (y-axis) across 2,000 pCRMs most frequently occupied by the indicated TFs or signal mediators (x-axis). Abbreviations used for the developmental timeline: 32 and 1K, 32-cell and 1,024-cell stage; MBT, mid-blastula transition; eG and lG, early and late gastrula stage; and eTailbud, early tailbud.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Snapshots of TF and signal mediator binding to super-enhancers during early embryogenesis. ( a ) Dynamic chromatin engagement of endogenous Sox3, Foxh1 , VegT, β-catenin, Smad2, Smad1 and RNAPII to putative super-enhancers of the siamois and ventx gene cluster from the 32-cell to the early gastrula stage. Illustrated sagittal sections (dorsal side is right) show the nuclear localization of the selected TFs with arrows pointing to the tissue movements of gastrulation. ( b ) Snapshot of endogenous Sox3, Eomes, zVegT, Tbxt and Tbx6 binding to the putative super-enhancer of the mesp gene cluster at the late gastrula and/or early tailbud stage. Illustrated sagittal sections (dorsal side is right) show the nuclear localization of selected TFs in the late gastrula embryo and at the caudal end of the early tailbud embryo with arrows pointing to the tissue movements during axial elongation . All super-enhancers were formed by stitching together engaged pCRMs that are ≤25 kb apart .

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: The relationship of chromatin recruitment among TFs and signal mediators and its underlying preferences for specific DNA recognition motifs in pCRMs of pluripotent and neuro-mesodermal embryonic cells. ( a ) Heat map shows pairwise Spearman correlations (R s ) of DNA occupancy levels at ∼12,500 pCRMs among TFs (Sox3, mVegT, Foxh1 , , Eomes , zVegT , Tbxt and Tbx6) and signal mediators (β-catenin, Smad1, Smad2) at indicated developmental stages. The pCRM coordinates were generated by collating the strongest 2,000 peaks of each binding profile. Abbreviations used for the developmental timeline: 32 and 1K, 32- and 1,024-cell stage; MBT, mid-blastula transition; eG and lG, early and late gastrula stage; eTailbud, early tailbud stage. ( b ) Bubble plots and heat maps show the coverage and enrichment of DNA recognition motifs, respectively, among the top 2,000 pCRMs in each chromatin profile.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Generated, Binding Assay

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: pCRM binding patterns correlate with the occurrence of specific DNA motifs. Heat map to the left shows the relative DNA occupancy levels across ∼12,500 pCRMs for a selection of TFs (mVegT, Sox3, Foxh1 , Eomes , zVegT , Tbxt and Tbx6) and signal mediators (β-catenin, Smad1, Smad2) from the 1,024-cell to the early tailbud stage. pCRM were hierarchically clustered according to the binding levels of all TFs and signal mediators. The pCRM coordinates were generated by collating the strongest 2,000 peaks of each binding profile. Heat map to the right shows the occurrence of specific DNA motifs ±50 bp from the pCRM centre as well as the flanking 50 bp. The consensus sequences of these DNA motifs are shown in . Abbreviations used for the developmental timeline: 1K, 1,024-cell stage; MBT, mid-blastula transition; eG and lG, early and late gastrula stage; eTailbud, early tailbud stage.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Binding Assay, Selection, Generated

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Maternal Pou5f3/Sox3-initiated chromatin remodeling to prime the first transcriptional response to inductive signals during ZGA. (a,b) Superimposed line tracks show the level of promoter-tied chromatin conformations, chromatin accessibility and various chromatin components (Smad2, H3K4me1 and RNAPII) at the Nodal-responsive mesoderm determinants tbxt (a) and eomes (b) between control (uninjected) and mPouV/Sox3 LOF embryos. Heat maps (pΔ) below each superimposed line track show the statistical significance of changes caused by mPouV/Sox3 LOF. The footer highlights the occurrences of canonical POU/SOX motifs (black filled rectangles) at accessible pCRMs (±50 bp from the accessibility centre) and one strongly affected pCRM with an arrowhead. Asterisks on the pΔ heat map mark significant (FDR ≤10%) reductions to pCRM accessibility. pCRMs are boxed in and their frequency of contacts with the promoter are illustrated with an arc of varying strength. Boxes of affected pCRM and arcs of promoter contacts are colored orange. (c) Stacked bar graphs summarize the correlation of unaffected and significantly reduced (FDR ≤10%) pCRM accessibility with RNAPII-mediated expression of all, signal responsive and non-responsive zygotic genes in mPouV/Sox3 LOF embryos at MBT. These correlations and corresponding numbers (placed on the stacked bars) are shown for pCRM-gene associations and zygotic genes. TSS-centric maps of reduced chromatin accessibility are shown for signal responsive and non-responsive genes in - . (d) Model of chromatin pioneering and opportunistic engagement to predefine first zygotic responses to inductive signals.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Expressing

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Maternal pluripotency TFs mPouV and Sox3 remodel 40% of the accessible chromatin landscape to contribute to one quarter of ZGA. (a) Used approach to reveal the effect of mPouV/Sox3 on chromatin accessibility (DNase-Seq; n=2) and chromatin composition (ChIP-Seq; n=2) at MBT. (b) Double-logarithmic volcano plot shows massive chromatin accessibility (DNase cleavage) reductions caused by mPouV/Sox3 LOF. pCRMs (dots) with significant accessibility changes (n=2, FDR ≤10%) are marked in red. (c) Violin plots show the comparison of chromatin accessibility (DNase cleavages) between uninjected and mPouV/Sox3 LOF embryos at all and affected (FDR ≤10%) pCRMs. Wilcoxon test: *, p <2.2×10 −16 . (d) Normalized meta-plots (top row, mean±SD) and heat maps (bottom row) show the level of chromatin accessibility, chromatin engagement (Sox3, H3K4me1, β-catenin, Smad2 and RNAPII) and RNA (n=3) across accessible pCRMs in uninjected and mPouV/Sox3 LOF embryos. The β-catenin binding profile was generated at late blastula stage rather than the 1,024-cell stage (Sox3) or MBT (all others). RNA was profiled at and beyond MBT as shown in . In the heat map, the pCRMs are sorted and grouped by significantly reduced DNase cleavages under mPouV/Sox3 LOF: red group, affected (FDR ≤10%) and blue group, unaffected (FDR >10%). These groups are represented in the meta-plots. Each heat map under mPouV/Sox3 LOF are followed by a heat map showing the statistical significance of changes (pΔ) caused by mPouV/Sox3 LOF. (e) Heat map shows the occurrence of DNA motifs at accessible pCRMs sorted and grouped as in (d). (f) Pie charts summarize the distribution of distances (kb) to nearest zygotic TSSs of affected (top pie chart) and unaffected (bottom pie chart) pCRMs. (g) Panel compares the effect of mPouV/Sox3 LOF on chromatin accessibility and RNAPII-mediated gene expression. Plot to the left shows the localization of accessible pCRMs (affected, dot colored in orange to red with FDR decreasing from 10%; and unaffected, grey dot) relative to the zygotic TSSs that are active by the MBT and produce enough RNA transcripts to show significant ≥two-fold reductions upon α-amanitin injection . Gene loci are sorted by mPouV/Sox3 LOF-induced transcript fold changes as shown in the log-scaled bar graph to the right.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: ChIP-sequencing, Comparison, Binding Assay, Generated, Expressing, Injection

Journal: bioRxiv

Article Title: The role of maternal pioneer factors in predefining first zygotic responses to inductive signals

doi: 10.1101/306803

Figure Lengend Snippet: Pioneering activity of mPouV/Sox3 initiates extensive chromatin remodeling such as the chromatin looping of distal pCRMs with promoters. (a) Used approach to reveal the effect of mPouV/Sox3 on chromatin conformation between 30 promoters and distal pCRMs (next-generation capture-C; n=3) at MBT. (b) Bar graph shows the number of normalized contacts (mean+SD; n=6) derived from non-redundant capture-reporter FLASH reads for each promoter captured with one or two probes (Supplementary Table 10). (c) Violin plots compare the number of promoter contacts with accessible pCRMs between uninjected and mPouV/Sox3 LOF embryos. The comparison is stratified into pCRMs with stable and lost accessibility upon mPouV/Sox3 LOF and shown for both all and affected (FDR ≤10%) promoter contacts. Wilcoxon tests and effect size estimates: *, p=5×10 −5 and r effect =0.12 (small effect); **, p=5×10 −30 and r effect =0.34 (medium effect); and ***, p=5×10 −10 and r effect =0.78 (large effect). (d) Superimposed line tracks show the level of promoter-tied chromatin conformations, chromatin accessibility and various chromatin components (Smad2, β-catenin, H3K4me1 and RNAPII) at the cdc25b gene locus between control (uninjected) and mPouV/Sox3 LOF embryos. The RNA track is split into a high (0-12) and low (0-0.01) expression window. Note that the low-expression window shows that locally transcribed non-coding super-enhancer RNA depend on mPouV/Sox3 as well as the gene cdc25b . Heat maps (pΔ) below each superimposed line plot show the statistical significance of changes caused by mPouV/Sox3 LOF. The footer highlights the occurrences of canonical POU/SOX motifs (black filled rectangles) at accessible pCRMs (±50 bp from the accessibility centre) and one strongly affected pCRM with an arrowhead. Asterisks on the pΔ heat map mark significant (FDR ≤10%) reductions to pCRM accessibility. pCRMs are boxed in and their frequency of contacts with the cdc25b promoter are illustrated with an arc of varying strength. Boxes of affected pCRM and arcs of promoter contacts are colored orange.

Article Snippet: The embryos were incubated at 4°C for 1-3 days in 50 µl blocking solution containing the primary antibody at the following dilutions: 1:1,000 rabbit polyclonal anti-Sox3 , 1:500 rabbit polyclonal anti phospho-Smad1/5/8 (Cell Signaling, #9511) or 1:500 goat polyclonal Smad2/3 (R&D Systems, #AF3797).

Techniques: Activity Assay, Capture-C, Derivative Assay, Comparison, Expressing

Journal: bioRxiv

Article Title: Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

doi: 10.1101/2024.11.25.625323

Figure Lengend Snippet: A Scheme of mouse breeding and tamoxifen injection. B Isolation of ECs from the inner curvature of the aortic arch (AA) of SMAD2/3 WT (red) or SMAD2/3 iΔEC (green) mice for ATAC-Seq (n=2/3 SMAD2/3 WT and SMAD2/3 iΔEC mice). C Number of SMAD motifs ( GGCGCC ) found in differentially accessible peaks (FDR<0.2) compared to random genomic regions of similar size and number. D Genome browser view showing ATAC-Seq tracks of ECs isolated from the inner curvature of the AA of WT (red) or SMAD2/3 knock-out (green) mice. Gray shading highlights areas of interest. E Motifs enriched in ATAC-Seq peaks that are lost upon SMAD2/3 knock-out identified by HOMER. Found motifs were screened for similarities to motifs of SMADs or known SMAD co-factors.

Article Snippet: Immunoprecipitation was performed using either 3 µg of either SMAD1 (SMAD1 XP, Cell Signaling Technology 6944), SMAD2/3 (SMAD 2/3 XP, Cell Signaling Technology 8685) or isotype control (Rabbit IgG XP, Cell Signaling Technology 3900) antibody overnight followed by incubation with protein A coupled Sepharose beads (VWR).

Techniques: Injection, Isolation, Knock-Out

Journal: bioRxiv

Article Title: Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

doi: 10.1101/2024.11.25.625323

Figure Lengend Snippet: A Images showing western blots from co-immunoprecipitation of total SMAD1 (S1) or total SMAD2/3 (S2) with FOXO1. Total cell lysate represents cell lysate before immunoprecipitation. IP – Immunoprecipitation, IB-Immunoblot. B Scheme of pneumatic pump system used to apply oscillatory (OS) and pulsatile (PS) flow on human aortic endothelial cells (HAoECs). C Monocyte adhesion assay using THP-1 cells labeled in green on HAoEC monolayers exposed to OS or PS (n=3). Scale bars are 100 µm. D Quantification of adhered monocytes from C . Quantification from 10 images of 3 biological donors. Statistics were calculated using a two-tailed Student’s t -test. E Confocal images of SMAD1-FOXO1 interaction assessed by proximity ligation assay (PLA) on HAoECs exposed to OS or PS (n=2). Scale bars are 100 µm. F Quantification of PLA events per cell from E . Quantification from 10 images of 2 biological donors. Statistics were calculated using a two-tailed Student’s t -test.

Article Snippet: Immunoprecipitation was performed using either 3 µg of either SMAD1 (SMAD1 XP, Cell Signaling Technology 6944), SMAD2/3 (SMAD 2/3 XP, Cell Signaling Technology 8685) or isotype control (Rabbit IgG XP, Cell Signaling Technology 3900) antibody overnight followed by incubation with protein A coupled Sepharose beads (VWR).

Techniques: Western Blot, Immunoprecipitation, Cell Adhesion Assay, Labeling, Two Tailed Test, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

doi: 10.1101/2024.11.25.625323

Figure Lengend Snippet: A Epifluorescence microscopy images of FOXO1 immunostainings (red) in human aortic ECs (HAoECs) exposed to oscillatory (OS) or pulsatile (PS) flow. Nuclei are counterstained with DAPI (blue). Representative images of 3 biological replicates with 4 images each. Scale bars are 20 µm. B Confocal images of SMAD2/3-FOXO1 interaction assessed by proximity ligation assay (PLA) on ECs exposed to OS or PS. Scale bars are 100 µm. C Quantification of PLA events per cell from B . Quantification from 10 images of 3 biological donors. Statistics were calculated using a two-tailed Student’s t -test. D Confocal images of PLA control samples which were incubated with only the secondary PLA probes. Scale bars are 40 µm.

Article Snippet: Immunoprecipitation was performed using either 3 µg of either SMAD1 (SMAD1 XP, Cell Signaling Technology 6944), SMAD2/3 (SMAD 2/3 XP, Cell Signaling Technology 8685) or isotype control (Rabbit IgG XP, Cell Signaling Technology 3900) antibody overnight followed by incubation with protein A coupled Sepharose beads (VWR).

Techniques: Epifluorescence Microscopy, Proximity Ligation Assay, Two Tailed Test, Control, Incubation

Journal: bioRxiv

Article Title: Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

doi: 10.1101/2024.11.25.625323

Figure Lengend Snippet: A Venn diagram showing the percentage of differentially accessible ATAC-Seq regions (DARs) (adjusted p-value < 0.05, fold change > |log 2 (0.585)|) positive for SMAD and/ or FOXO motifs in human aortic endothelial cells (HAoECs) (n=3). B SMAD and FOXO-positive regions which are associated with genes that display enhanced expression. BMP target genes are highlighted. C Genome browser view of the EDN1 promoter displaying ATAC-Seq data from HAoECs exposed to oscillatory (OS) or pulsatile (PS) flow. SMAD and FOXO motifs are highlighted below. The dashed line indicates regions used in D . Gray shading highlights areas of interest. D Reporter gene assay in HEK293T cells using a genomic region of the EDN1 promoter region indicated by a dashed line in C . Values are fold inductions of FOXO1 overexpression compared to an empty vector control (n=4). Quantification shows the mean of experiments +/- standard deviation. Statistics were calculated using a two-tailed Student’s t -test. E Reporter gene assay of a synthetic SMAD and FOXO motif containing plasmid in HEK293T cells (n=3). Values are normalized to the unstimulated empty vector control and represent means of 3 independent experiments +/- standard deviation. Statistics were calculated using a two-way ANOVA with Šidák’s post-hoc test. F Genome Browser view of SMAD1 and SMAD2/3 CUT&RUN (C&R) of BMP9 stimulated HAoECs (n=2 each) and FOXO1 ChIP-Seq in human umbilical vein ECs upon overexpression of constitutively active FOXO1 (GSE128635). Gray shading highlights area of interest. G qPCR analysis of BMP target genes in HAoECs exposed to OS upon siRNA-mediated depletion of FOXO1. Values are normalized to the scrambled control and are depicted as mean +/- standard deviation from 4 independent experiments. Statistics were calculated using one sample t -test for siFOXO1.

Article Snippet: Immunoprecipitation was performed using either 3 µg of either SMAD1 (SMAD1 XP, Cell Signaling Technology 6944), SMAD2/3 (SMAD 2/3 XP, Cell Signaling Technology 8685) or isotype control (Rabbit IgG XP, Cell Signaling Technology 3900) antibody overnight followed by incubation with protein A coupled Sepharose beads (VWR).

Techniques: Expressing, Reporter Gene Assay, Over Expression, Plasmid Preparation, Control, Standard Deviation, Two Tailed Test, ChIP-sequencing

Journal: bioRxiv

Article Title: Atheroprone Flow Activates SMAD-FOXO1 to drive Endothelial-to-Mesenchymal Transition and Atherosclerosis

doi: 10.1101/2024.11.25.625323

Figure Lengend Snippet: A Schematic overview of the experimental setup. B Volcano plot of differentially expressed genes (DEGs) (adjusted p-value < 0.05, fold change > |log 2 (0.585)|) from RNA-Seq data of HAoECs exposed to oscillatory flow (OS) in absence or presence of FOXO1 inhibitor AS1842856 (0.5 µM) (n=3). Genes corresponding to EndoMT are highlighted. C Gene ontology enrichment analysis of DEGs corresponding to active FOXO1. D , E Genome browser views of ATAC-Seq data from HAoECs exposed to OS in the presence or absence of FOXO1 inhibitor AS1842856 (0.5 µM) (n=3). Gray shading highlights areas of interest. F Genome browser view of ATAC-Seq data from HAoECs exposed to OS or pulsatile flow (PS), SMAD1; SMAD2/3 CUT&RUN of BMP9 stimulated HAoECs, and ChIP-Seq of FOXO1 and histone 3 lysine 27 acetylation (H3K27Ac) in human umbilical vein ECs (HUVECs) upon overexpression of constitutively active FOXO1 (FOXO1, H3K27Ac) or an empty control vector (H3K27Ac) (GSE128635). Gray shading highlights areas of interest. G Normalized counts from RNA-Seq of HUVECs transduced with control vector or constitutively active FOXO1 (GSE128636). Displayed p-values were calculated using DESeq2 (adjusted p-value) to account for multiple testing and non-normality of RNA-Seq data. Wald test p-values are corrected for multiple testing using the Benjamini and Hochberg method. H RT-qPCR analysis of HAoECs transfected with control or FOXO1 siRNA (n=3). Values are depicted as mean normalized expression +/- standard deviation. Statistics were calculated using a two-tailed Student’s t -test.

Article Snippet: Immunoprecipitation was performed using either 3 µg of either SMAD1 (SMAD1 XP, Cell Signaling Technology 6944), SMAD2/3 (SMAD 2/3 XP, Cell Signaling Technology 8685) or isotype control (Rabbit IgG XP, Cell Signaling Technology 3900) antibody overnight followed by incubation with protein A coupled Sepharose beads (VWR).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Over Expression, Control, Plasmid Preparation, Transduction, Quantitative RT-PCR, Transfection, Expressing, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Identification of regulatory elements important for TGF-β-induced transactivation of the MIXL1 promoter. A, schematic representation of HNF4α-binding motifs in the Smad2/3 and HNF4α binding region of the MIXL1 promoter. Promoter reporters with mutations in their HNF4α-binding motifs are shown in the lower panel. B, activation of the MIXL1 gene promoter by TGF-β and effects of mutations in putative HNF4α-binding motifs. HepG2 cells were transfected with the MIXL1 promoter and its mutants and treated with TGF-β for 24 h. C, conserved Smad-binding elements ( SBEs ) of the MIXL1 promoter. Four Smad-binding elements that were conserved between mouse and human (SBE 1–4) are shown with their relative positions from the transcription start site. Nucleotide sequences of Smad-binding elements and their mutations used in D are also shown. WT , wild-type; mut , mutant. D, effect of mutations in Smad-binding elements on TGF-β-induced transcriptional activity of MIXL1 promoter. Cells were treated as in B , and luciferase activities were determined. *, p < 0.05 compared with WT without TGF-β; **, p < 0.05 compared with SBE4 mutant, without TGF-β; n.s., not significant compared with WT and SBE2 mutant, without TGF-β; error bars, S.D.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay, Activation Assay, Transfection, Mutagenesis, Activity Assay, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Identification of Smad2/3 binding regions in HepG2 cells. A, Smad2/3 binding to the SERPINE1/PAI-1 locus in HepG2 cells. MAT scores were plotted at the SERPINE1 and HPRT1 loci to obtain a graphical representation of Smad2/3 binding in these regions. Significant Smad2/3 binding regions as determined by detection of p values of 10 −4 are shown by black bars. B, percent input values of Smad2/3 binding compared with input genome as determined by ChIP-qPCR. Cells were treated with 120 p m TGF-β for 1.5 h. Cells were cross-linked sequentially with dimethyl adipimidate and formaldehyde. Error bars represent S.D.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Comparison of the Smad2/3 binding regions among different cell lines. A, Venn diagrams showing the overlaps of Smad2/3 binding regions in HaCaT, HepG2, and Hep3B cells. Numbers in the circles indicate percentages of the Smad2/3 binding region of each cell line ( red, HaCaT; black, HepG2; blue, Hep3B). B, identification of a motif conserved in HepG2-specific Smad binding regions. Partial genomic sequences within 250 bp from the peak positions of HepG2-specific Smad2/3 binding regions ( n = 2,955) were analyzed using the CisGenome Gibbs motif sampler. Default parameters were used for the calculation, except for the numbers of motifs to be identified ( n = 10). Matrix datum of the motif calculated by CisGenome was graphically shown using the SegLogo function of the R software. C, HNF4α-binding motif that matched the predicted motif in HepG2-specific Smad2/3 binding regions. The JASPAR CORE data base was used to identify known transcription factor binding motifs similar to the calculated matrix data in B . An HNF4α motif (ID: MA0114.1) was identified as the most similar motif with a comparison score of 21.3, which reached 96.9% of the potential maximal score. D, frequencies of the HNF4α-binding motif in Smad2/3 binding regions. Presence of the HNF4α-binding motif in each Smad2/3 binding region (within 250 bp from the peak signal position) was determined using CisGenome. Frequencies of the motif in either HepG2- or HaCaT-specific Smad2/3 binding regions were then calculated. As a control, matched genomic regions to HaCaT-specific Smad2/3 binding regions were obtained using CisGenome, and the frequency of the HNF4α motif was determined. E, expression of the HNF4α protein and phosphorylation of Smad2/3 in HaCaT and HepG2 cells. Cells were treated with TGF-β for 1.5 h, and the expression of each protein was determined by immunoblotting ( IB ).

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay, Genomic Sequencing, Software, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Identification of HNF4α binding regions in the presence and absence of TGF-β stimulation . A, graphical representation of HNF4α binding to the APOA4/APOC3/APOA1 gene loci. Sequence read numbers of 100-bp sliding window were plotted for HNF4α ChIP-seq samples. Smad2/3 bindings as determined by ChIP-chip analysis were shown in the upper two panels as in A. Black bars represent significant binding regions (FDR, <0.1%). B, Venn diagrams showing overlap between TGF-β-treated and untreated HNF4α binding regions. HNF4α binding regions were determined for each sample (FDR, <0.1%). HNF4α binding regions that have overlapping regions within 500 bp from their positions of maximum read numbers were considered as shared binding regions. C, changes in the HNF4α binding to APOC3 and APOA1 loci were quantitatively determined by ChIP-qPCR analysis. Error bars , S.D. D, frequencies of in vivo HNF4α binding to the Smad2/3 binding regions. Percentages of HNF4α binding within 250 bp from the peak signal position of Smad2/3 binding regions were calculated for the indicated Smad2/3 binding groups. E, frequencies of canonical Smad-binding elements in HNF4α binding regions compared with Smad binding regions in HepG2 cells. A Smad-binding element, M00974.SMAD that was identified as an enriched motif in HepG2-specific Smad2/3 binding regions using CEAS (see text), was selected for calculation. CisGenome was used for mapping of the motif. Presence of the motif for each HNF4α binding region and Smad2/3 binding region was determined using PerlScript.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay, Sequencing, ChIP-sequencing, ChIP-chip, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Effect of knockdown of HNF4α on TGF-β-induced gene expression in HepG2 cells. A, confirmation of HNF4α-knocked down samples for microarray analysis. HepG2 cells were transfected with HNF4α siRNA and treated with 120 p m TGF-β for the indicated times and harvested. Expression of HNF4α was determined by RT-qPCR and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). siNC , negative control siRNA. B, down-regulation of HNF4α protein expression by siRNA. C, phosphorylation levels of Smad2/3 by using HNF4α siRNA. The top two panels show phosphorylation of Smad2 and Smad3. The 3rd panel indicates the expression of total Smad2/3, and the bottom panel is a loading control. IB , immunoblot. D, heat map of the TGF-β-induced expression of target genes of Smad2/3 and HNF4α and effect of HNF4α siRNA. Target genes that have overlapping binding regions of Smad2/3 and HNF4α were sorted by their induction of probe signal values by TGF-β stimulation for 1.5 h and are represented by color bars in the 1st column , using the TM4 microarray software . Relative expression of these genes in HNF4α siRNA samples to the control siRNA is shown in the 2nd column . In addition, a list of genes whose expressions changed more than 1.5-fold is shown in the right panel with their expression changes. E, heat map of target genes of TGF-β with Smad2/3 binding regions common to HNF4α at 24 h after TGF-β stimulation are shown as in D . Genes whose expressions were changed more than 2-fold are shown in the right panel .

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Expressing, Microarray, Transfection, Quantitative RT-PCR, Negative Control, Western Blot, Binding Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: TGF-β-induced changes in gene expression in relation to Smad2/3 binding Expression array data transfected with control siRNA and stimulated with TGF-β were compared with Smad2/3 ChIP-chip data. A total of 8,653 genes that had values of more than 100 at least at one time point for one of their probes ( n = 13,720) was used for the analysis. Up-regulated or down-regulated genes were determined compared with 0-h values. The positions of peak signals of Smad binding regions (SBRs) relative to the nearby RefSeq genes were first determined, and regions within 5 kb upstream from the transcription start site and the first intron were selected. *a indicates number of genes analyzed by microarray. *b indicates number of genes which have Smad2/3 binding regions.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Expressing, Binding Assay, Transfection, Microarray

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: TGF-β-induced genes with Smad2/3 and HNF4α binding at 1.5 h Target genes of TGF-β in HepG2 cells that were induced more than 2-fold at 1.5 h and that have common binding regions for Smad2/3 and HNF4α were sorted by their expression changes in the presence or absence of HNF4α siRNA. Presence of Smad2/3 binding regions in HaCaT cells is also shown in the 2nd column.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Smad2/3 and HNF4α bindings in the MIXL1 locus. A, Smad2/3- and HNF4α-enriched regions in the MIXL1 locus are shown as in A. B, HepG2 cells were treated with 120 p m TGF-β for 1.5 h, fixed in formaldehyde, and harvested. Smad2/3 binding to the MIXL1 locus was verified by ChIP-qPCR. HPRT1 served as a negative control. C, HepG2 cells were treated with or without 120 p m TGF-β for 1.5 h, and ChIP-qPCR analysis of the MIXL1 locus using anti-HNF4α was performed as in B. n.s ., not significant. D, effects of knockdown of HNF4α on TGF-β-induced expression changes of MIXL1 . HepG2 cells were transfected with control siRNA ( siNC ) or siHNF4α, treated with 3 ng/ml TGF-β for the indicated times, and harvested. HNF4α expression was quantified by RT-qPCR. *, p < 0.05; error bars , S.D.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay, Negative Control, Expressing, Transfection, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells * ^♦

doi: 10.1074/jbc.M110.217745

Figure Lengend Snippet: Roles of HNF4α on Smad2/3 binding and transcriptional activity of MIXL1 promoter. A, effects of HNF4α knockdown on transactivation of the MIXL1 promoter. HepG2 cells were transfected with siRNAs 1 day before transfection with the reporter constructs. siNC , negative control siRNA. B, effects of exogenous HNF4α on transactivation of the MIXL1 promoter. HNF4α was exogenously expressed in HaCaT cells, and transcriptional activity of MIXL1 reporter was determined. The lower panel shows the protein expression of HNF4α. IB , immunoblot. C, HNF4α (variant 2, RefSeq ID: NM_000457) or its C115R ( CR ) mutant, which does not bind to DNA, was overexpressed in HepG2 cells. The lower panel shows the protein expression of HNF4α and its mutant. D, effect of HNF4α siRNA on Smad2/3 binding to the MIXL1 locus. HepG2 cells were transfected with siRNAs 24 h before TGF-β stimulation. Cells were fixed 1.5 h after treatment, and ChIP-qPCR was performed as in B. Error bars, S.D.; *, p < 0.05; n.s. , not significant.

Article Snippet: Sample preparation for ChIP-chip analysis was performed as described previously, using anti-Smad2/3 (BD Biosciences) ( , , ).

Techniques: Binding Assay, Activity Assay, Transfection, Construct, Negative Control, Expressing, Western Blot, Variant Assay, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: mTOR inhibition rescues osteopenia in mice with systemic sclerosis

doi: 10.1084/jem.20140643

Figure Lengend Snippet: TGF-β and STAT6 synergistically enhance the expression of IL4Rα in Fbn +/− BMMSCs. (A) ELISA assay showed the level of TGF-β in Fbn1 +/− mouse serum and Fbn1 +/− BMMSCs cultured medium compared with WT group. (B) Western blotting showed the expression levels of p-SMAD3 and SP-1 in WT and Fbn1 +/− BMMSCs. (C and D) Western blotting showed that TGF-β treatment elevated IL4Rα expression in BMMSCs, which could be blocked by TGF-β neutralizing antibody (NAb). (E and F) Western blot showed that Fbn1 knockdown by siRNA in BMMSCs elevated IL4Rα expression, which could be blocked by TGF-β NAb. (G) Western blot showed the expression of IL4Rα after Smad2/3 siRNA treatment compared with vehicle-treated group. (H) Il4rα promoter luciferase fusions were examined in WT, TGF-β–treated, and Fbn1 +/− BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of cotransfected Renilla luciferase. (I) ChIP-qPCR assay showed enrichment of direct association of SP1 and STAT6 on Il4rα promoter in TGF-β–treated and Fbn1 +/− BMMSCs. (J) Western blot showed the expression of p-JAK1 and p-STAT6 in Fbn1 +/− BMMSCs. (K and L) Western blotting showed the expression of IL4Rα and p-mTOR after TGF-β, IL4, or combinatorial treatment with TGF-β and IL4. All experimental data verified in at least three independent experiments. Error bars represent the SD from the mean values. ***, P < 0.005.

Article Snippet: Antibodies to mouse ALP, LPL, PPARγ2, IL4Rα, PI3K-p110, phosphor-SMAD2/3, and SP1 were purchased from Santa Cruz Biotechnology, Inc. Antibodies to human RUNX2, ALP, and OCN were purchased from Santa Cruz Biotechnology, Inc. Antibodies to phosphor-mTOR (Ser2448), mTOR, phospho-Akt (Ser473), Akt, phosphor-P70S6K (T389; S371), phosphor-Rictor, phosphor-Raptor, and SMAD2/3 were obtained from Cell Signaling Technology.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Knockdown, Luciferase, Activity Assay, ChIP-qPCR