Structured Review

Cell Signaling Technology Inc histone h3
TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice. After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated <t>histone</t> H3 and anti-acetylated histone H4. ( A ) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. ( B ) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. ( C ) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ** P
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1) Product Images from "Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα"

Article Title: Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

Journal: PLoS ONE

doi: 10.1371/journal.pone.0153330

TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice. After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 and anti-acetylated histone H4. ( A ) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. ( B ) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. ( C ) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ** P
Figure Legend Snippet: TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice. After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 and anti-acetylated histone H4. ( A ) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. ( B ) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. ( C ) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ** P

Techniques Used: Mouse Assay, Chromatin Immunoprecipitation

TSA or MCP30 restores the HQ-induced increased HDAC activity, decreased Topo IIα expression, and the resulting apoptosis in human bone marrow mononuclear cells. ( A—C ) Mononuclear cells were isolated from bone marrow aspirates from four healthy donors and subsequently treated with or without 100 μM HQ, in the presence or absence of TSA (0.5 μM) or MCP30 (1 μg/ml). After 24 h, the activity of HDAC ( A ), the expression of Topo IIα ( B ), and apoptosis ( C ) were determined using HDAC activity assay kit, western blot, and Annexin V/PI double staining, respectively. NS stands for normal saline. Histone H3 was used as a loading control for nuclear protein in western blot analysis. (D) Human bone marrow mononuclear cells were treated with or without a Topo IIα inhibitor daunorubicin (200 nM) for 48 h and apoptosis was measured using Annexin V staining. Statistical data and representatives of four independent experiments from different healthy donors were shown. * P
Figure Legend Snippet: TSA or MCP30 restores the HQ-induced increased HDAC activity, decreased Topo IIα expression, and the resulting apoptosis in human bone marrow mononuclear cells. ( A—C ) Mononuclear cells were isolated from bone marrow aspirates from four healthy donors and subsequently treated with or without 100 μM HQ, in the presence or absence of TSA (0.5 μM) or MCP30 (1 μg/ml). After 24 h, the activity of HDAC ( A ), the expression of Topo IIα ( B ), and apoptosis ( C ) were determined using HDAC activity assay kit, western blot, and Annexin V/PI double staining, respectively. NS stands for normal saline. Histone H3 was used as a loading control for nuclear protein in western blot analysis. (D) Human bone marrow mononuclear cells were treated with or without a Topo IIα inhibitor daunorubicin (200 nM) for 48 h and apoptosis was measured using Annexin V staining. Statistical data and representatives of four independent experiments from different healthy donors were shown. * P

Techniques Used: Activity Assay, Expressing, Isolation, HDAC Activity Assay, Western Blot, Double Staining, Staining

2) Product Images from "Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation"

Article Title: Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation

Journal: bioRxiv

doi: 10.1101/2020.05.07.081216

Proximity biotin ligation assay of KIF2A isoforms in Neuro2A cells. A . Immunofluorescence staining of myc (red), Streptavidin (green) and DNA (blue) in Neuro2A cells transfected with myc BirA* , myc BirA*-Kif2a.1, mycBirA*-Kif2a.2, mycBirA*-Kif2a.3  or  mycBirA*-Kif2a H321D  separately. Transfected cells were identified based on myc immunostaining. Scale bar, 10 µm.  B . Top panel: Immunoblotting with anti-myc showing expression of mycBirA*-KIF2A in Neuro2A cells. Middle panel: Immunoblotting with anti-biotin confirming biotinylation. Bottom panel: Anti-Histone H3 as loading control.  C . Pull-down with streptavidin beads followed by anti-biotin immunoblotting of Neuro2A cell lysates. Biotin supplementation to cell media is indicated.
Figure Legend Snippet: Proximity biotin ligation assay of KIF2A isoforms in Neuro2A cells. A . Immunofluorescence staining of myc (red), Streptavidin (green) and DNA (blue) in Neuro2A cells transfected with myc BirA* , myc BirA*-Kif2a.1, mycBirA*-Kif2a.2, mycBirA*-Kif2a.3 or mycBirA*-Kif2a H321D separately. Transfected cells were identified based on myc immunostaining. Scale bar, 10 µm. B . Top panel: Immunoblotting with anti-myc showing expression of mycBirA*-KIF2A in Neuro2A cells. Middle panel: Immunoblotting with anti-biotin confirming biotinylation. Bottom panel: Anti-Histone H3 as loading control. C . Pull-down with streptavidin beads followed by anti-biotin immunoblotting of Neuro2A cell lysates. Biotin supplementation to cell media is indicated.

Techniques Used: Ligation, Immunofluorescence, Staining, Transfection, Immunostaining, Expressing

3) Product Images from "A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination"

Article Title: A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.1769

Premature formation of hyperphosphorylated RPA2 impedes recruitment of RPA and RAD51 to chromatinized DNA damage–induced foci. ( a ) RPA2 focus formation after CPT or mock (represented as Un) treatment of U2OS cells expressing RPA2 WT or RPA2 D4. Cells were stained for RPA2 and DAPI, and images were captured by fluorescence microscopy. The RPA2 focus–positive cells ( > 30 foci) were quantified manually by comparison with DAPI images (~300 cells total). Representative images are shown on the left. ( b ) Interaction of Myc-tagged RPA2 WT, RPA2 D2 or RPA2 D4 with RAD51 after CPT treatment. Cells were subjected to immunoprecipitation after CPT treatment using an anti-Myc and probed for RAD51. ( c ) Reduced nuclear staining of RAD51 in damaged cells lacking PP4R2 or replaced with RPA2 D4. Following replacement of endogenous RPA2 with phosphomimetic RPA2 mutants or depletion of PP4R2, U2OS cells were mock- or CPT-treated. Cells were then extracted to remove soluble RAD51, stained with DAPI and anti-RAD51, and imaged by epifluorescence microscopy using identical exposure times. RAD51 nuclear staining was quantified and plotted. ( d ) Nuclear localization of RAD51 is altered by hyperphosphorylated RPA2. RAD51 localization after CPT treatment of U2OS cells, either where endogenous RPA2 was replaced by RPA2 WT or RPA2 D4 (left) or where PP4R2 was silenced (right). Nuclei were biochemically fractionated, and nuclear soluble (NS) and chromatin-bound (Chr) fractions were probed for RAD51. Topoisomerase II (TOPII) and histone H3 (H3) was probed for loading and fractionation controls, respectively. The relative amounts of RAD51 and RPA1 are shown in parentheses.
Figure Legend Snippet: Premature formation of hyperphosphorylated RPA2 impedes recruitment of RPA and RAD51 to chromatinized DNA damage–induced foci. ( a ) RPA2 focus formation after CPT or mock (represented as Un) treatment of U2OS cells expressing RPA2 WT or RPA2 D4. Cells were stained for RPA2 and DAPI, and images were captured by fluorescence microscopy. The RPA2 focus–positive cells ( > 30 foci) were quantified manually by comparison with DAPI images (~300 cells total). Representative images are shown on the left. ( b ) Interaction of Myc-tagged RPA2 WT, RPA2 D2 or RPA2 D4 with RAD51 after CPT treatment. Cells were subjected to immunoprecipitation after CPT treatment using an anti-Myc and probed for RAD51. ( c ) Reduced nuclear staining of RAD51 in damaged cells lacking PP4R2 or replaced with RPA2 D4. Following replacement of endogenous RPA2 with phosphomimetic RPA2 mutants or depletion of PP4R2, U2OS cells were mock- or CPT-treated. Cells were then extracted to remove soluble RAD51, stained with DAPI and anti-RAD51, and imaged by epifluorescence microscopy using identical exposure times. RAD51 nuclear staining was quantified and plotted. ( d ) Nuclear localization of RAD51 is altered by hyperphosphorylated RPA2. RAD51 localization after CPT treatment of U2OS cells, either where endogenous RPA2 was replaced by RPA2 WT or RPA2 D4 (left) or where PP4R2 was silenced (right). Nuclei were biochemically fractionated, and nuclear soluble (NS) and chromatin-bound (Chr) fractions were probed for RAD51. Topoisomerase II (TOPII) and histone H3 (H3) was probed for loading and fractionation controls, respectively. The relative amounts of RAD51 and RPA1 are shown in parentheses.

Techniques Used: Recombinase Polymerase Amplification, Cycling Probe Technology, Expressing, Staining, Fluorescence, Microscopy, Immunoprecipitation, Epifluorescence Microscopy, Fractionation

4) Product Images from "Treatment of human pre-B acute lymphoblastic leukemia with the Aurora kinase inhibitor PHA-739358 (Danusertib)"

Article Title: Treatment of human pre-B acute lymphoblastic leukemia with the Aurora kinase inhibitor PHA-739358 (Danusertib)

Journal: Molecular Cancer

doi: 10.1186/1476-4598-11-42

Monitoring of PHA-739358 treatment effect in vivo on molecular targets. Fully leukemic NSG mice transplanted with Pt2 Ph-positive ALL were treated with PHA-739358 (45 mg/kg, iv). Samples of one non-treated (−) and two drug-treated (+) mice are shown. Two hours after injection, spleen and bone marrow were collected. The expression of p-Tyr, p-histone H3 (Ser 10), p-Crkl, histone H3 and Crkl was assessed by Western blot. Bcr and Gapdh serve as a loading control.
Figure Legend Snippet: Monitoring of PHA-739358 treatment effect in vivo on molecular targets. Fully leukemic NSG mice transplanted with Pt2 Ph-positive ALL were treated with PHA-739358 (45 mg/kg, iv). Samples of one non-treated (−) and two drug-treated (+) mice are shown. Two hours after injection, spleen and bone marrow were collected. The expression of p-Tyr, p-histone H3 (Ser 10), p-Crkl, histone H3 and Crkl was assessed by Western blot. Bcr and Gapdh serve as a loading control.

Techniques Used: In Vivo, Mouse Assay, Injection, Expressing, Western Blot

PHA-739358 eliminates activity of both Bcr/Abl and Aurora kinase. (A) . BLQ1 (Ph-positive T315I mutation) and UCSF02 (Ph-positive no mutation) cells were incubated with the indicated concentrations of PHA-739358 with or without dasatinib (100 nM) for 24 hours. Phosphorylation status of Bcr/Abl, Stat5, Crkl and Src was assessed by Western blot. Blots were stripped and reprobed with Bcr, Src and Gapdh as loading controls.  (B) . BLQ1 and US6 cells were exposed to 1 μM PHA-739358 for 24 and 48 hours and the percentage of phospho-histone H3 (Ser10) positive cells was defined by flow cytometry.
Figure Legend Snippet: PHA-739358 eliminates activity of both Bcr/Abl and Aurora kinase. (A) . BLQ1 (Ph-positive T315I mutation) and UCSF02 (Ph-positive no mutation) cells were incubated with the indicated concentrations of PHA-739358 with or without dasatinib (100 nM) for 24 hours. Phosphorylation status of Bcr/Abl, Stat5, Crkl and Src was assessed by Western blot. Blots were stripped and reprobed with Bcr, Src and Gapdh as loading controls. (B) . BLQ1 and US6 cells were exposed to 1 μM PHA-739358 for 24 and 48 hours and the percentage of phospho-histone H3 (Ser10) positive cells was defined by flow cytometry.

Techniques Used: Activity Assay, Mutagenesis, Incubation, Western Blot, Flow Cytometry, Cytometry

5) Product Images from "Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors ▿Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors ▿ †"

Article Title: Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors ▿Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01839-06

Recruitment of Eos, MITF, and PU.1 to the  Acp5  and  Ctsk  promoters during osteoclast differentiation. (A) (Left) Graphic representation of regions analyzed for  Ctsk  and  Acp5  genes in ChIP assays. (Right) Representative gel pictures for Eos ChIPs on  Ctsk  gene, following 40 cycles of PCR. Input indicates the total DNA in each assay before antibody was added. Negative controls included no antibody (No Ab) and 3′ exon/intron region (Exon). Anti-histone H3 was used as a positive control. d, days. (B) ChIP assays to study the association of Eos, MITF, and PU.1 with  Ctsk  and  Acp5  promoters in cells treated with CSF-1 alone (day 0) or subsequently with CSF-1 and RANKL for 0.5, 3, and 5 days. rel enrichment, relative enrichment. (C) Analysis of enrichment of MITF, PU.1, and Eos at  Bcl2  and c- Fms  promoters in committed osteoclast precursors. (D) Analysis of enrichment of MITF, PU.1, and Eos at  Acp5  promoter in cells treated with CSF-1 alone derived from hypomorphic  MITF vga/vga  mice (left) or Pu.1 conditional knockout cells (right). Results from three independent experiments are represented as means ± standard errors of the means (error bars) for each experiment in panels B to D.
Figure Legend Snippet: Recruitment of Eos, MITF, and PU.1 to the Acp5 and Ctsk promoters during osteoclast differentiation. (A) (Left) Graphic representation of regions analyzed for Ctsk and Acp5 genes in ChIP assays. (Right) Representative gel pictures for Eos ChIPs on Ctsk gene, following 40 cycles of PCR. Input indicates the total DNA in each assay before antibody was added. Negative controls included no antibody (No Ab) and 3′ exon/intron region (Exon). Anti-histone H3 was used as a positive control. d, days. (B) ChIP assays to study the association of Eos, MITF, and PU.1 with Ctsk and Acp5 promoters in cells treated with CSF-1 alone (day 0) or subsequently with CSF-1 and RANKL for 0.5, 3, and 5 days. rel enrichment, relative enrichment. (C) Analysis of enrichment of MITF, PU.1, and Eos at Bcl2 and c- Fms promoters in committed osteoclast precursors. (D) Analysis of enrichment of MITF, PU.1, and Eos at Acp5 promoter in cells treated with CSF-1 alone derived from hypomorphic MITF vga/vga mice (left) or Pu.1 conditional knockout cells (right). Results from three independent experiments are represented as means ± standard errors of the means (error bars) for each experiment in panels B to D.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Derivative Assay, Mouse Assay, Knock-Out

Eos expression is downregulated during osteoclast differentiation. (A) Relative (rel) expression of Eos , Acp5 , and Ctsk mRNA was measured by qRT-PCR at the indicated times (in days [d]) and cytokine treatments. Results from three independent experiments are presented as means plus standard errors of the means (error bars). (B) Nuclear extracts from osteoclasts harvested at the indicated times (in days [d]) were analyzed by Western blotting using anti-Eos antibody. Histone H3 was used as a loading control.
Figure Legend Snippet: Eos expression is downregulated during osteoclast differentiation. (A) Relative (rel) expression of Eos , Acp5 , and Ctsk mRNA was measured by qRT-PCR at the indicated times (in days [d]) and cytokine treatments. Results from three independent experiments are presented as means plus standard errors of the means (error bars). (B) Nuclear extracts from osteoclasts harvested at the indicated times (in days [d]) were analyzed by Western blotting using anti-Eos antibody. Histone H3 was used as a loading control.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

6) Product Images from "Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein"

Article Title: Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1308673110

T-cell stimulation by Tat enhances histone acetylation and viral infection. ( A ) Tat stimulation of T cells for 24 h increases acetylated histone H3 in nuclear extracts. Data are mean ± SD from three independent experiments compared by two-way paired T test, * P
Figure Legend Snippet: T-cell stimulation by Tat enhances histone acetylation and viral infection. ( A ) Tat stimulation of T cells for 24 h increases acetylated histone H3 in nuclear extracts. Data are mean ± SD from three independent experiments compared by two-way paired T test, * P

Techniques Used: Cell Stimulation, Infection

7) Product Images from "Coxiella burnetii Subverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages"

Article Title: Coxiella burnetii Subverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages

Journal: Infection and Immunity

doi: 10.1128/IAI.00608-17

Nrf2 translocates to the nucleus during infection. THP-1 cells were infected with NMII for 72 h and then processed for immunofluorescence confocal microscopy to detect C. burnetii and Nrf2 (A) and GFP-Nrf2 expression in HeLa cells (B) or immunoblot analysis to detect Nrf2 and histone H3 (nuclear fraction control) in the nuclear fraction of infected THP-1 cells (C). The results presented are representative of those from three individual experiments. *, P
Figure Legend Snippet: Nrf2 translocates to the nucleus during infection. THP-1 cells were infected with NMII for 72 h and then processed for immunofluorescence confocal microscopy to detect C. burnetii and Nrf2 (A) and GFP-Nrf2 expression in HeLa cells (B) or immunoblot analysis to detect Nrf2 and histone H3 (nuclear fraction control) in the nuclear fraction of infected THP-1 cells (C). The results presented are representative of those from three individual experiments. *, P

Techniques Used: Infection, Immunofluorescence, Confocal Microscopy, Expressing

8) Product Images from "SETDB1 accelerates tumorigenesis by regulating WNT signaling pathway"

Article Title: SETDB1 accelerates tumorigenesis by regulating WNT signaling pathway

Journal: The Journal of pathology

doi: 10.1002/path.4482

Protein levels of β-catenin in the NSCLC cells with either stably overexpressed or silenced SETDB1 A. Western analysis of β-catenin accumulation in NSCLC cell lines with either silenced or overexpressed SETDB1. Ctrl, sh-Scramble; sh1,2,3, three shRNA targeting to SETDB1. B. Relative TOP/FOP activities when overexpressing SETDB1 in H1299 cells. The stably forced expression of either SETDB1 or GFP containing cells were transfected with either pGL-TOP or pGL-FOP luciferase-reporter constructs. Luciferase activities were measured 72 hours after the transfection and normalized to the corresponding co-transfected Renilla luciferase activity. Data are shown as the ratio between TOP/FOP and Renilla. Error bars represent SD of three independent experiments. C. Silencing of IGFBP4, LRP8 or FZD1 reduced the WNT activity in H1299 NSCLC cells. Cells stably infected with either IGFBP4, LRP8 or FZD1 were transfected with either TOP or FOP, as well as Renilla control vector, and the levels of WNT pathway activity were determined by TOP/FOP assay. D. Cytoplasmic and nuclear fraction of H1299 cells stably overexpressing either GFP or SETDB1. Cells were grown on 10 cm dishes to 50% ~ 70% confluence, and the cytoplasmic and nuclear proteins were prepared as described in Materials and Methods. Cytoplasmic α-tubulin and nuclear histone H3 was used as controls of protein fractionation. E. Western analysis of alternations of downstream genes (c-MYC, Cyclin D1) of the WNT pathway in SETDB1 stably overexpressing H1299 NSCLC cells. F. Effect of stable silencing of β-catenin on clonogenic growth of H1299 cells overexpressing either GFP (control) or SETDB1. Values are expressed as % of GFP control cells, mean ± SD (three independent experiments). The silencing efficiency of β-catenin was determined by Western blot (right side). G. Clonogenic growth of either GFP or SETDB1 forced expressing H1299 cells after stable silencing of FZD1, LRP8 or IGFBP4. Values are expressed as % of GFP control cells. Experiments were performed in triplicate, and results are presented as mean ± SD. H. Schematic illustration of the proposed signaling interaction of SETDB1 and WNT pathway.
Figure Legend Snippet: Protein levels of β-catenin in the NSCLC cells with either stably overexpressed or silenced SETDB1 A. Western analysis of β-catenin accumulation in NSCLC cell lines with either silenced or overexpressed SETDB1. Ctrl, sh-Scramble; sh1,2,3, three shRNA targeting to SETDB1. B. Relative TOP/FOP activities when overexpressing SETDB1 in H1299 cells. The stably forced expression of either SETDB1 or GFP containing cells were transfected with either pGL-TOP or pGL-FOP luciferase-reporter constructs. Luciferase activities were measured 72 hours after the transfection and normalized to the corresponding co-transfected Renilla luciferase activity. Data are shown as the ratio between TOP/FOP and Renilla. Error bars represent SD of three independent experiments. C. Silencing of IGFBP4, LRP8 or FZD1 reduced the WNT activity in H1299 NSCLC cells. Cells stably infected with either IGFBP4, LRP8 or FZD1 were transfected with either TOP or FOP, as well as Renilla control vector, and the levels of WNT pathway activity were determined by TOP/FOP assay. D. Cytoplasmic and nuclear fraction of H1299 cells stably overexpressing either GFP or SETDB1. Cells were grown on 10 cm dishes to 50% ~ 70% confluence, and the cytoplasmic and nuclear proteins were prepared as described in Materials and Methods. Cytoplasmic α-tubulin and nuclear histone H3 was used as controls of protein fractionation. E. Western analysis of alternations of downstream genes (c-MYC, Cyclin D1) of the WNT pathway in SETDB1 stably overexpressing H1299 NSCLC cells. F. Effect of stable silencing of β-catenin on clonogenic growth of H1299 cells overexpressing either GFP (control) or SETDB1. Values are expressed as % of GFP control cells, mean ± SD (three independent experiments). The silencing efficiency of β-catenin was determined by Western blot (right side). G. Clonogenic growth of either GFP or SETDB1 forced expressing H1299 cells after stable silencing of FZD1, LRP8 or IGFBP4. Values are expressed as % of GFP control cells. Experiments were performed in triplicate, and results are presented as mean ± SD. H. Schematic illustration of the proposed signaling interaction of SETDB1 and WNT pathway.

Techniques Used: Stable Transfection, Western Blot, shRNA, Expressing, Transfection, Luciferase, Construct, Activity Assay, Infection, Plasmid Preparation, Fractionation

Aberrant expression of SETDB1 in NSCLC cells affects their proliferation in liquid culture, clonogenic growth in semi-soft cultures and tumor size in nude mice A. Effect of stably silencing SETDB1 on NSCLC cell growth in liquid culture. MTT assay was performed in 96 wells plate. Mean ± SD of 6 wells. B. Effect of silencing of SETDB1 on colony formation of NSCLC cells. Cells were seeded into soft agar in triplicate, and colonies were counted after 21~28 days of culturing. Mean ± SD (3 wells) are expressed as percent variation relative to scramble shRNA infected cells (control). C. Effect of forced expression of SETDB1 on colony formation of PC14 and H1299 NSCLC cells. Cells were seeded into soft agar in triplicate dishes; and colonies were counted on day 28 of culture. Values are expressed as fold variation relative to GFP overexpressing cells (control). Results are mean ± SD of 3 experiments. Right panel, representative pictures of colonies of H1299 overexpressing SETDB1. D. Histone H3K9 methylation status of NSCLC cells with stable overexpression of SETDB1 (H1299 and A549) was examined by western blot using antibodies specific for H3K9me1, H3K9me2, and H3K9me3 (mono-, di- and tri- methylation of H3K9, respectively). Expression levels of histone H3 protein were used as an internal control. E. SETDB1 stimulates NSCLC tumor growth in mice. Photograph of tumors dissected from nude mice which were injected with H1299 cells overexpressing either GFP (upper row) or SETDB1 (lower row). Tumor cells (3 × 10 6 ) were suspended in a 1:1 mixture of fetal bovine serum and Matrigel (BD Labware) and injected into both flanks of five week-old nude mice. Tumors were removed on day 21 from initiation of the experiment. F. Weights of tumors are shown in Fig. 3 E. The bars show differences in average weight of tumors in two groups (mean ± S.D., n = 8). Difference of mean weights between control (GFP) and SETDB1 overexpressing tumors was statistically significant (p-value of 0.0003). G. Expression level of SETDB1 in the tumors with forced expression of either GFP or SETDB1. Proteins and mRNA were isolated from xenografts. Tumor mRNA values were measured by RT-PCR and normalized with β-actin. Values are expressed as fold variation of SETDB1 overexpressing tumors relative to GFP control tumors. SETDB1 protein was measured by western blot (inner picture).
Figure Legend Snippet: Aberrant expression of SETDB1 in NSCLC cells affects their proliferation in liquid culture, clonogenic growth in semi-soft cultures and tumor size in nude mice A. Effect of stably silencing SETDB1 on NSCLC cell growth in liquid culture. MTT assay was performed in 96 wells plate. Mean ± SD of 6 wells. B. Effect of silencing of SETDB1 on colony formation of NSCLC cells. Cells were seeded into soft agar in triplicate, and colonies were counted after 21~28 days of culturing. Mean ± SD (3 wells) are expressed as percent variation relative to scramble shRNA infected cells (control). C. Effect of forced expression of SETDB1 on colony formation of PC14 and H1299 NSCLC cells. Cells were seeded into soft agar in triplicate dishes; and colonies were counted on day 28 of culture. Values are expressed as fold variation relative to GFP overexpressing cells (control). Results are mean ± SD of 3 experiments. Right panel, representative pictures of colonies of H1299 overexpressing SETDB1. D. Histone H3K9 methylation status of NSCLC cells with stable overexpression of SETDB1 (H1299 and A549) was examined by western blot using antibodies specific for H3K9me1, H3K9me2, and H3K9me3 (mono-, di- and tri- methylation of H3K9, respectively). Expression levels of histone H3 protein were used as an internal control. E. SETDB1 stimulates NSCLC tumor growth in mice. Photograph of tumors dissected from nude mice which were injected with H1299 cells overexpressing either GFP (upper row) or SETDB1 (lower row). Tumor cells (3 × 10 6 ) were suspended in a 1:1 mixture of fetal bovine serum and Matrigel (BD Labware) and injected into both flanks of five week-old nude mice. Tumors were removed on day 21 from initiation of the experiment. F. Weights of tumors are shown in Fig. 3 E. The bars show differences in average weight of tumors in two groups (mean ± S.D., n = 8). Difference of mean weights between control (GFP) and SETDB1 overexpressing tumors was statistically significant (p-value of 0.0003). G. Expression level of SETDB1 in the tumors with forced expression of either GFP or SETDB1. Proteins and mRNA were isolated from xenografts. Tumor mRNA values were measured by RT-PCR and normalized with β-actin. Values are expressed as fold variation of SETDB1 overexpressing tumors relative to GFP control tumors. SETDB1 protein was measured by western blot (inner picture).

Techniques Used: Expressing, Mouse Assay, Stable Transfection, MTT Assay, shRNA, Infection, Methylation, Over Expression, Western Blot, Injection, Significance Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

9) Product Images from "Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair"

Article Title: Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn014

Loss of p21 results in a reduced HAT activity after UV irradiation. ( A ) Western blot analysis of acetylated forms (K9-K14) and total content of histone H3 performed in p21 +/+ (lanes 1–4) and p21 −/− (lanes 5–8) cell extracts, before (lanes 1 and 5) and 1 h (lanes 2 and 6), 3 h (lanes 3 and 7) or 6 h (lanes 4 and 8) after UV irradiation. In lane 9, extract from cells treated with trichostatin A (TSA) to enhance H3 acetylated forms, is shown as positive control of acetylation. ( B ) Densitometric analysis of the ratio acetylated form (K9–K14)/total levels of histone H3 in p21 +/+ (empty bars) and p21 −/− (solid bars) fibroblasts collected 3 or 24 h after UV irradiation. Normalized mean values ± SD of three independent experiments are reported (3 h, P = 0.002; 24 h, P = 0.01). ( C ) Western blot analysis of acetylated form (K9–K14) and of the total levels of histone H3, and p21. LF1 fibroblasts not transfected, or transfected with p21-specific or control (GFP) siRNA pools were collected 24 h after UV irradiation. ( D ) Densitometric analysis of the ratio acetylated form (K9–K14)/total levels of histone H3, in UV-irradiated LF1 fibroblasts not transfected, or transfected with p21-specific or control (GFP) siRNA pools. Normalized mean values ± SD of three independent experiments, are reported ( P = 0.03, p21-siRNA versus untreated control; P = 0.04, p21-siRNA versus GFP-siRNA).
Figure Legend Snippet: Loss of p21 results in a reduced HAT activity after UV irradiation. ( A ) Western blot analysis of acetylated forms (K9-K14) and total content of histone H3 performed in p21 +/+ (lanes 1–4) and p21 −/− (lanes 5–8) cell extracts, before (lanes 1 and 5) and 1 h (lanes 2 and 6), 3 h (lanes 3 and 7) or 6 h (lanes 4 and 8) after UV irradiation. In lane 9, extract from cells treated with trichostatin A (TSA) to enhance H3 acetylated forms, is shown as positive control of acetylation. ( B ) Densitometric analysis of the ratio acetylated form (K9–K14)/total levels of histone H3 in p21 +/+ (empty bars) and p21 −/− (solid bars) fibroblasts collected 3 or 24 h after UV irradiation. Normalized mean values ± SD of three independent experiments are reported (3 h, P = 0.002; 24 h, P = 0.01). ( C ) Western blot analysis of acetylated form (K9–K14) and of the total levels of histone H3, and p21. LF1 fibroblasts not transfected, or transfected with p21-specific or control (GFP) siRNA pools were collected 24 h after UV irradiation. ( D ) Densitometric analysis of the ratio acetylated form (K9–K14)/total levels of histone H3, in UV-irradiated LF1 fibroblasts not transfected, or transfected with p21-specific or control (GFP) siRNA pools. Normalized mean values ± SD of three independent experiments, are reported ( P = 0.03, p21-siRNA versus untreated control; P = 0.04, p21-siRNA versus GFP-siRNA).

Techniques Used: HAT Assay, Activity Assay, Irradiation, Western Blot, Positive Control, Transfection

10) Product Images from "HDAC inhibitors, MS-275 and salermide, potentiates the anticancer effect of EF24 in human pancreatic cancer cells"

Article Title: HDAC inhibitors, MS-275 and salermide, potentiates the anticancer effect of EF24 in human pancreatic cancer cells

Journal: EXCLI Journal

doi: 10.17179/excli2016-186

Acetylated histone H3 and H4 levels in BxPC-3 cells treated with MS (a) and SAL (b) alone or in combination with EF for 24 h. MS: MS-275; SAL: Salermide; EF: EF24 *p
Figure Legend Snippet: Acetylated histone H3 and H4 levels in BxPC-3 cells treated with MS (a) and SAL (b) alone or in combination with EF for 24 h. MS: MS-275; SAL: Salermide; EF: EF24 *p

Techniques Used: Mass Spectrometry

11) Product Images from "PPAR?-Sirt1 complex mediates cardiac hypertrophy and failure through suppression of the ERR transcriptional pathway"

Article Title: PPAR?-Sirt1 complex mediates cardiac hypertrophy and failure through suppression of the ERR transcriptional pathway

Journal: Cell metabolism

doi: 10.1016/j.cmet.2011.10.001

Involvement of PPARα/Sirt1-mediated ERRE suppression in pressure overload(PO)-induced cardiac hypertrophy and fasting. (A–B) After 4 weeks of TAC, ChIP assays were performed with anti-Sirt1, anti-PPARα and anti-histone H3 acetyllysine
Figure Legend Snippet: Involvement of PPARα/Sirt1-mediated ERRE suppression in pressure overload(PO)-induced cardiac hypertrophy and fasting. (A–B) After 4 weeks of TAC, ChIP assays were performed with anti-Sirt1, anti-PPARα and anti-histone H3 acetyllysine

Techniques Used: Chromatin Immunoprecipitation

12) Product Images from "Muscle hypertrophy in hypoxia with inflammation is controlled by bromodomain and extra-terminal domain proteins"

Article Title: Muscle hypertrophy in hypoxia with inflammation is controlled by bromodomain and extra-terminal domain proteins

Journal: Scientific Reports

doi: 10.1038/s41598-017-12112-0

Effects of pulmonary inflammation and hypoxia exposure on histone H3 acetylation levels in soleus muscle subjected to an overload surgery. Panel a shows blots of the acetylated and the total form of histone 3. Numbers represent different animals. Western blots with antibodies against acetyl-lysine recognizing preferentially acetylated histone H3 and total histone H3 were quantified and normalized (panel b) and related to the soleus muscle weight (panel c). (n = 6-7; Mean ± SEM); NC: Normoxia Control; NS: Normoxia + Surgery; NSI: Normoxia + Surgery + Inflammation; HS: Hypoxia + Surgery; HSI: Hypoxia + Surgery + Inflammation. *Global effect of inflammation (NSI + HSI) vs . (NS + HS), p
Figure Legend Snippet: Effects of pulmonary inflammation and hypoxia exposure on histone H3 acetylation levels in soleus muscle subjected to an overload surgery. Panel a shows blots of the acetylated and the total form of histone 3. Numbers represent different animals. Western blots with antibodies against acetyl-lysine recognizing preferentially acetylated histone H3 and total histone H3 were quantified and normalized (panel b) and related to the soleus muscle weight (panel c). (n = 6-7; Mean ± SEM); NC: Normoxia Control; NS: Normoxia + Surgery; NSI: Normoxia + Surgery + Inflammation; HS: Hypoxia + Surgery; HSI: Hypoxia + Surgery + Inflammation. *Global effect of inflammation (NSI + HSI) vs . (NS + HS), p

Techniques Used: Western Blot

13) Product Images from "Down-regulation of G9a triggers DNA damage response and inhibits colorectal cancer cells proliferation"

Article Title: Down-regulation of G9a triggers DNA damage response and inhibits colorectal cancer cells proliferation

Journal: Oncotarget

doi:

G9a depletion synergizes with TOPO I inhibitors in CRC cells (A)  SW620 shG9a or shCon cells were treated with different concentrations of SN38 or CPT, followed by subsequent proliferation inhibition assessment using the SRB assay. CRC cells were treated with different concentrations of UNC0638 and/or SN38, and  (B)  cell proliferation was measured with the SRB assay and CIs were calculated, ( C)  colony formation was measured.  (D)  Protein levels of γH2AX were analyzed by microscopy (scale bar 10 μM), and  (E)  protein levels of histone H3, H3K9me2, p-Chk1, p-Chk2, γH2AX and GAPDH were determined by western blot.  (F)  Protein levels of GFP, G9a, p-Chk1(Ser 317), p-Chk2 (Thr 68), γH2AX, and β-actin (loading control) from 293T cells stably transfected with pLEX-mock, pLEX-hG9a, or pLEX-ΔSET-hG9a and treated with the indicated concentrations of CPT. Data are means ± SD,  n  ≥ 3. ** P
Figure Legend Snippet: G9a depletion synergizes with TOPO I inhibitors in CRC cells (A) SW620 shG9a or shCon cells were treated with different concentrations of SN38 or CPT, followed by subsequent proliferation inhibition assessment using the SRB assay. CRC cells were treated with different concentrations of UNC0638 and/or SN38, and (B) cell proliferation was measured with the SRB assay and CIs were calculated, ( C) colony formation was measured. (D) Protein levels of γH2AX were analyzed by microscopy (scale bar 10 μM), and (E) protein levels of histone H3, H3K9me2, p-Chk1, p-Chk2, γH2AX and GAPDH were determined by western blot. (F) Protein levels of GFP, G9a, p-Chk1(Ser 317), p-Chk2 (Thr 68), γH2AX, and β-actin (loading control) from 293T cells stably transfected with pLEX-mock, pLEX-hG9a, or pLEX-ΔSET-hG9a and treated with the indicated concentrations of CPT. Data are means ± SD, n ≥ 3. ** P

Techniques Used: Cycling Probe Technology, Inhibition, Sulforhodamine B Assay, Microscopy, Western Blot, Stable Transfection, Transfection

14) Product Images from "Cell Type-Specific Activation of the Cytomegalovirus Promoter by Dimethylsulfoxide and 5-Aza-2?-deoxycytidine"

Article Title: Cell Type-Specific Activation of the Cytomegalovirus Promoter by Dimethylsulfoxide and 5-Aza-2?-deoxycytidine

Journal: The international journal of biochemistry & cell biology

doi: 10.1016/j.biocel.2008.02.014

Chromatin Immunoprecipitation (ChIP)-PCR assay for CMV promoter sequence from CHO-LacZ. Chromatin DNA was immunoprecipitated with antibodies specific for acetylated histone H3 (acetyl-H3). DNA fragments were amplified with primers specific for CMV promoter and control GAPDH. ChIP-PCR products for CMV promoter (IP) (161 bp): Lane 1, 1 kb+ ladder; Lane 2, No DNA control; Lane 3, Negative control; Lane 4, No treatment control; Lane 5, 2% DMSO; Lane 6, 4% DMSO; Lane 7, 5-Aza CdR; Lane 8, 2% DMSO + 5-Aza CdR. ChIP-PCR products obtained for CMV promoter inputs under various conditions as described above are designated as (Input) (161 bp). ChIP-PCR products obtained for GAPDH control under various conditions as described above are designated as (GAPDH) (162 bp). The fold changes in signal intensity were measured by the ratio of IP/inputs to control GAPDH genes as described under materials and methods. Increased signal intensity observed in CHO-LacZ cells treated with various agents was: 2% DMSO, 10%; 4% DMSO, 20%; 5-Aza CdR, 23%; and 5-Aza CdR + 2% DMSO, 30% when compared with that of the untreated control cells.
Figure Legend Snippet: Chromatin Immunoprecipitation (ChIP)-PCR assay for CMV promoter sequence from CHO-LacZ. Chromatin DNA was immunoprecipitated with antibodies specific for acetylated histone H3 (acetyl-H3). DNA fragments were amplified with primers specific for CMV promoter and control GAPDH. ChIP-PCR products for CMV promoter (IP) (161 bp): Lane 1, 1 kb+ ladder; Lane 2, No DNA control; Lane 3, Negative control; Lane 4, No treatment control; Lane 5, 2% DMSO; Lane 6, 4% DMSO; Lane 7, 5-Aza CdR; Lane 8, 2% DMSO + 5-Aza CdR. ChIP-PCR products obtained for CMV promoter inputs under various conditions as described above are designated as (Input) (161 bp). ChIP-PCR products obtained for GAPDH control under various conditions as described above are designated as (GAPDH) (162 bp). The fold changes in signal intensity were measured by the ratio of IP/inputs to control GAPDH genes as described under materials and methods. Increased signal intensity observed in CHO-LacZ cells treated with various agents was: 2% DMSO, 10%; 4% DMSO, 20%; 5-Aza CdR, 23%; and 5-Aza CdR + 2% DMSO, 30% when compared with that of the untreated control cells.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Sequencing, Immunoprecipitation, Amplification, Negative Control

15) Product Images from "Protective effects of sulforaphane on di-n-butylphthalate-induced testicular oxidative stress injury in male mice offsprings via activating Nrf2/ARE pathway"

Article Title: Protective effects of sulforaphane on di-n-butylphthalate-induced testicular oxidative stress injury in male mice offsprings via activating Nrf2/ARE pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.19981

SFN pretreatment enhances Nrf2 nuclear translocation and increases HO-1 and NQO-1 protein expression in Nrf2 +/+ mice (A) Protein expression levels of nuc NF-κB and p-I-kB in different groups. Histone H3 or β-actin was used as a protein Control to normalize volume of protein expression. (B-C) Protein levels were determined by densitometric analysis and normalized to the Histone H3 signal or the β-actin signal. (D) Protein expression levels of Nrf2, HO-1 and NQO-1 in different groups. Histone H3 or β-actin was used as a protein Control to normalize volume of protein expression. (E-G) Protein levels were determined by densitometric analysis and normalized to Histone H3 or the β-actin signal. Data are expressed as mean ± SD. *significant difference vs. Control group (P
Figure Legend Snippet: SFN pretreatment enhances Nrf2 nuclear translocation and increases HO-1 and NQO-1 protein expression in Nrf2 +/+ mice (A) Protein expression levels of nuc NF-κB and p-I-kB in different groups. Histone H3 or β-actin was used as a protein Control to normalize volume of protein expression. (B-C) Protein levels were determined by densitometric analysis and normalized to the Histone H3 signal or the β-actin signal. (D) Protein expression levels of Nrf2, HO-1 and NQO-1 in different groups. Histone H3 or β-actin was used as a protein Control to normalize volume of protein expression. (E-G) Protein levels were determined by densitometric analysis and normalized to Histone H3 or the β-actin signal. Data are expressed as mean ± SD. *significant difference vs. Control group (P

Techniques Used: Translocation Assay, Expressing, Mouse Assay

16) Product Images from "Use of a genome-wide haploid genetic screen to identify treatment predicting factors: a proof-of-principle study in pancreatic cancer"

Article Title: Use of a genome-wide haploid genetic screen to identify treatment predicting factors: a proof-of-principle study in pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.18879

Effects of entinostat on histone H3 acetylation PANC-1 and SUIT2 Clone 1 cells were incubated for 72 hours in the presence of variable concentrations of entinostat (0-50μM). Cells were then lysed and subjected to Western blotting using an antibody directed at acetylated histone H3 or total H3. The membrane was then probed for β-actin to confirm equal loading of lanes.
Figure Legend Snippet: Effects of entinostat on histone H3 acetylation PANC-1 and SUIT2 Clone 1 cells were incubated for 72 hours in the presence of variable concentrations of entinostat (0-50μM). Cells were then lysed and subjected to Western blotting using an antibody directed at acetylated histone H3 or total H3. The membrane was then probed for β-actin to confirm equal loading of lanes.

Techniques Used: Incubation, Western Blot

17) Product Images from "Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma"

Article Title: Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma

Journal: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology

doi: 10.1016/j.radonc.2010.06.010

Src family kinase mediates radiation-induced EGFR translocation to the nucleus SCC1, SCC6, SCC1483 cells were pretreated with or without dasatinib (25 nM) for 24 hours before irradiation with 4Gy. 30 minutes after radiation, cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.
Figure Legend Snippet: Src family kinase mediates radiation-induced EGFR translocation to the nucleus SCC1, SCC6, SCC1483 cells were pretreated with or without dasatinib (25 nM) for 24 hours before irradiation with 4Gy. 30 minutes after radiation, cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.

Techniques Used: Translocation Assay, Irradiation, SDS Page

Radiation induces EGFR translocation to the nucleus A) SCC1, SCC6, SCC1483 cells were irradiated with 4Gy. At the indicated times, nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. B) Comparison of time courses of EGFR nuclear translocation respectively induced by EGF, cetuximab and radiation. SCC1, CC6, SCC1483 cells were treated with or without EGF (200ng/ml), cetuximab (400nM) and radiation (4Gy) for indicated times. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for EGFR. Bands were quantitated using ImageJ and presented graphically as fold increase relative to untreated control. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.
Figure Legend Snippet: Radiation induces EGFR translocation to the nucleus A) SCC1, SCC6, SCC1483 cells were irradiated with 4Gy. At the indicated times, nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. B) Comparison of time courses of EGFR nuclear translocation respectively induced by EGF, cetuximab and radiation. SCC1, CC6, SCC1483 cells were treated with or without EGF (200ng/ml), cetuximab (400nM) and radiation (4Gy) for indicated times. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for EGFR. Bands were quantitated using ImageJ and presented graphically as fold increase relative to untreated control. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.

Techniques Used: Translocation Assay, Irradiation, SDS Page

Cetuximab treatment increases phosphorylation of the EGFR in HNSCC tumor cells A) SCC1, SCC6, SCC1483 cells were treated with or without lOOng/ml EGF for 30 minutes. The EGFR was immunoprecipitated from whole cell lysate and blotted for the indicated proteins. B) SCC1, SCC6, SCC1483 cells were treated with or without cetuximab (400nM) for 24 hours. The EGFR was immunoprecipitated from whole cell lysate and blotted for the indicated proteins. C and D) SCC1, SCC6, SCC1483 cells were treated with or without cetuximab (400nM) for 24 hours. Cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.
Figure Legend Snippet: Cetuximab treatment increases phosphorylation of the EGFR in HNSCC tumor cells A) SCC1, SCC6, SCC1483 cells were treated with or without lOOng/ml EGF for 30 minutes. The EGFR was immunoprecipitated from whole cell lysate and blotted for the indicated proteins. B) SCC1, SCC6, SCC1483 cells were treated with or without cetuximab (400nM) for 24 hours. The EGFR was immunoprecipitated from whole cell lysate and blotted for the indicated proteins. C and D) SCC1, SCC6, SCC1483 cells were treated with or without cetuximab (400nM) for 24 hours. Cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.

Techniques Used: Immunoprecipitation, SDS Page

Src family kinases mediate cetuximab-induced EGFR translocation to the nucleus SCC1, SCC6, SCC1483 cells were pretreated with dasatinib (25nM) for 24 hours followed by cetuximab (400nM) treatment for 24 hours. Cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and iblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.
Figure Legend Snippet: Src family kinases mediate cetuximab-induced EGFR translocation to the nucleus SCC1, SCC6, SCC1483 cells were pretreated with dasatinib (25nM) for 24 hours followed by cetuximab (400nM) treatment for 24 hours. Cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and iblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.

Techniques Used: Translocation Assay, SDS Page

Dasatinib blocks cetuximab and radiation-induced EGFR nuclear translocation A) SCC1, SCC6, SCC1483 cells were pretreated with or without cetuximab (400 nM) for 1 hour prior to irradiation with 4Gy and harvested after 24 hours. Protein was extracted and immunoblotted for the indicated proteins. B) SCC1, SCC6, SCC1483 cells were pretreated with or without cetuximab (400 nM) and dasatinib (25nM) for 1 hours prior to irradiation with 4Gy and harvested after 24 hours. Protein was extracted and immunoblotted for the indicated proteins. Cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.
Figure Legend Snippet: Dasatinib blocks cetuximab and radiation-induced EGFR nuclear translocation A) SCC1, SCC6, SCC1483 cells were pretreated with or without cetuximab (400 nM) for 1 hour prior to irradiation with 4Gy and harvested after 24 hours. Protein was extracted and immunoblotted for the indicated proteins. B) SCC1, SCC6, SCC1483 cells were pretreated with or without cetuximab (400 nM) and dasatinib (25nM) for 1 hours prior to irradiation with 4Gy and harvested after 24 hours. Protein was extracted and immunoblotted for the indicated proteins. Cytoplasmic and nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.

Techniques Used: Translocation Assay, Irradiation, SDS Page

Cetuximab induces EGFR translocation to the nucleus A) Lung (H226, A549, Calu-3), Colon (HT29, SW480, SK-CO-1), and HNSCC (SCC1, SCC6, SCC1483) tumor cells were treated with or without 400 nM of cetuximab for 24 hours. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. B) SCC1, SCC6, SCC1483 cells were treated with increasing concentrations of cetuximab for 24 hours. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. C) SCC1, SCC6, SCC1483 cells were treated with cetuximab (400 nM) for the indicated times. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. D) Time course of cetuximab-induced EGFR nuclear translocation. SCC1, SCC6, SCC1483 cells were treated with cetuximab (400 nM) for the indicated times. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for EGFR. Bands were quantitated using ImageJ and presented graphically as fold increase relative to untreated control. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.
Figure Legend Snippet: Cetuximab induces EGFR translocation to the nucleus A) Lung (H226, A549, Calu-3), Colon (HT29, SW480, SK-CO-1), and HNSCC (SCC1, SCC6, SCC1483) tumor cells were treated with or without 400 nM of cetuximab for 24 hours. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. B) SCC1, SCC6, SCC1483 cells were treated with increasing concentrations of cetuximab for 24 hours. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. C) SCC1, SCC6, SCC1483 cells were treated with cetuximab (400 nM) for the indicated times. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. D) Time course of cetuximab-induced EGFR nuclear translocation. SCC1, SCC6, SCC1483 cells were treated with cetuximab (400 nM) for the indicated times. Nuclear fractions were obtained, fractionated by SDS-PAGE and immunoblotted for EGFR. Bands were quantitated using ImageJ and presented graphically as fold increase relative to untreated control. Histone H3 and α-Tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively.

Techniques Used: Translocation Assay, SDS Page

18) Product Images from "Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity"

Article Title: Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity

Journal: Scientific Reports

doi: 10.1038/s41598-017-03148-3

MCM7 keep binding with chromatin in G2/M of HepG2 and UMUC-3 Cells. Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. The chromatin binding (chromatin) and non-chromatin binding (supernatant) MCMs in HepG2 cells ( A ) and UMUC-3 cells ( B ) were examined by Chromatin-binding assays. Histone H3 and GAPDH were used as loading control for chromatin-binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments. N: asynchronized cells.
Figure Legend Snippet: MCM7 keep binding with chromatin in G2/M of HepG2 and UMUC-3 Cells. Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. The chromatin binding (chromatin) and non-chromatin binding (supernatant) MCMs in HepG2 cells ( A ) and UMUC-3 cells ( B ) were examined by Chromatin-binding assays. Histone H3 and GAPDH were used as loading control for chromatin-binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments. N: asynchronized cells.

Techniques Used: Binding Assay, Incubation

MCM7 constantly bind chromatin throughout cell cycle in SPC-A1 Cells. SPC-A1 Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. ( A ) SPC-A1 cell cycle was analyzed by FACS. ( B ) P-Histone H3 was detected by western-blot. ( C ) Chromatin binding and non-chromatin binding MCM4, MCM5, MCM6 and MCM7 were analyzed by chromatin binding assay. Histone H3 and GAPDH were used as loading control for chromatin-binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments.
Figure Legend Snippet: MCM7 constantly bind chromatin throughout cell cycle in SPC-A1 Cells. SPC-A1 Cells were treated with double thymine and then incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. ( A ) SPC-A1 cell cycle was analyzed by FACS. ( B ) P-Histone H3 was detected by western-blot. ( C ) Chromatin binding and non-chromatin binding MCM4, MCM5, MCM6 and MCM7 were analyzed by chromatin binding assay. Histone H3 and GAPDH were used as loading control for chromatin-binding proteins and non-chromatin binding proteins respectively. Data are presented as optical density fold difference related to loading control from three independent experiments.

Techniques Used: Incubation, FACS, Western Blot, Binding Assay

MCMs’ mRNA express in HepG2 and UMUC-3 Cells. ( A ) Cells were synchronized with double thymine and incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. Cell cycle was analyzed by FACS. Data are shown as mean ± SD of three independent experiments. N: asynchronized cells. ( B ) P-Histone H3 was detected by western-blot. ( C ) MCMs’ mRNA levels were examined by real-time RCR. Data are normalized to the beta-actin level in each sample. These experiments were performed in triplicate, and the values are expressed as mean ± SD.
Figure Legend Snippet: MCMs’ mRNA express in HepG2 and UMUC-3 Cells. ( A ) Cells were synchronized with double thymine and incubated in fresh medium for 0 h, 3 h, 6 h, 9 h, 12 h and 15 h. Cell cycle was analyzed by FACS. Data are shown as mean ± SD of three independent experiments. N: asynchronized cells. ( B ) P-Histone H3 was detected by western-blot. ( C ) MCMs’ mRNA levels were examined by real-time RCR. Data are normalized to the beta-actin level in each sample. These experiments were performed in triplicate, and the values are expressed as mean ± SD.

Techniques Used: Incubation, FACS, Western Blot

19) Product Images from "A selective inhibitor of histone deacetylase 3 prevents cognitive deficits and suppresses striatal CAG repeat expansions in Huntington’s disease mice"

Article Title: A selective inhibitor of histone deacetylase 3 prevents cognitive deficits and suppresses striatal CAG repeat expansions in Huntington’s disease mice

Journal: Scientific Reports

doi: 10.1038/s41598-017-05125-2

RGFP966 treatment induces Arc but not Egr1 or c-Fos protein levels in primary hippocampal cultures. (a ) Representative immunoblots showing histone H3 acetylation levels at position lysine 9 (AcH3K9) with actin as loading control and (b) Arc, Egr1 and c-Fos protein levels normalized to actin levels in DMSO and RGFP966 treated primary hippocampal cultures obtained from Hdh Q7/Q7 (WT) and Hdh Q7/Q111 (KI) embryos. The blots in (a and b) are cropped; full-length images are provided in Supplementary Figure 3 . ** p
Figure Legend Snippet: RGFP966 treatment induces Arc but not Egr1 or c-Fos protein levels in primary hippocampal cultures. (a ) Representative immunoblots showing histone H3 acetylation levels at position lysine 9 (AcH3K9) with actin as loading control and (b) Arc, Egr1 and c-Fos protein levels normalized to actin levels in DMSO and RGFP966 treated primary hippocampal cultures obtained from Hdh Q7/Q7 (WT) and Hdh Q7/Q111 (KI) embryos. The blots in (a and b) are cropped; full-length images are provided in Supplementary Figure 3 . ** p

Techniques Used: Western Blot

Systemic RGFP966 treatment efficiently inhibits hippocampal and striatal HDAC activity. (a) Schematic of chronic treatment and experimental plan. ARTP, accelerating rotarod task procedure; OLT, object location task; NORT, novel object recognition task. ( b) Body weight measurements. Data represent the mean ± SEM (n = 6–10). ( c) Representative immunoblots showing histone H3 acetylation levels at position lysine 9 (AcH3K9) normalized to total histone H3 and (d) HDAC3 levels normalized to α-tubulin in striatal and hippocampal extracts from vehicle and RGFP966 chronically treated wild type Hdh Q7/Q7 (WT) and mutant Hdh Q7/Q111 (KI) mice. The blots in (c) and (d) are cropped; full-length images are provided in Supplementary Figure 2 . * p
Figure Legend Snippet: Systemic RGFP966 treatment efficiently inhibits hippocampal and striatal HDAC activity. (a) Schematic of chronic treatment and experimental plan. ARTP, accelerating rotarod task procedure; OLT, object location task; NORT, novel object recognition task. ( b) Body weight measurements. Data represent the mean ± SEM (n = 6–10). ( c) Representative immunoblots showing histone H3 acetylation levels at position lysine 9 (AcH3K9) normalized to total histone H3 and (d) HDAC3 levels normalized to α-tubulin in striatal and hippocampal extracts from vehicle and RGFP966 chronically treated wild type Hdh Q7/Q7 (WT) and mutant Hdh Q7/Q111 (KI) mice. The blots in (c) and (d) are cropped; full-length images are provided in Supplementary Figure 2 . * p

Techniques Used: Activity Assay, Western Blot, Mutagenesis, Mouse Assay

20) Product Images from "Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein"

Article Title: Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1308673110

T-cell stimulation by Tat enhances histone acetylation and viral infection. ( A ) Tat stimulation of T cells for 24 h increases acetylated histone H3 in nuclear extracts. Data are mean ± SD from three independent experiments compared by two-way paired T test, * P
Figure Legend Snippet: T-cell stimulation by Tat enhances histone acetylation and viral infection. ( A ) Tat stimulation of T cells for 24 h increases acetylated histone H3 in nuclear extracts. Data are mean ± SD from three independent experiments compared by two-way paired T test, * P

Techniques Used: Cell Stimulation, Infection

21) Product Images from "Epigenetic Repression of Matrix Metalloproteinases in Myofibroblastic Hepatic Stellate Cells through Histone Deacetylases 4"

Article Title: Epigenetic Repression of Matrix Metalloproteinases in Myofibroblastic Hepatic Stellate Cells through Histone Deacetylases 4

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2010.100011

Reduced histone acetylation during HSC transdifferentiation. A: Quiescent (day two), intermediate (day five), and myofibroblastic (day nine) HSCs were examined for H3 and H4 histone acetylation and methylation by Western blot analysis. Transdifferentiation was monitored by increased expression of α-SMA. B: Quantification of acetyl histone H4 and α-SMA by Western blot was measured by densitometry, representing three independent experiments. C: Cells were fractionated into cytoplasmic and nuclear/membrane fractions. Decreased acetylation of histone H3 and H4 during transdifferentiation is obvious. D: In quiescent (day two, qHSC) and myofibroblastic (day nine, mHSC) HSCs, basal levels of acetyl histone H4 in the transcription start site of MMP13 gene were measured by ChIP assay and qRT-PCR. Data represent the output/input ratios and are the averages of three independent experiments. Statistical significance, * P
Figure Legend Snippet: Reduced histone acetylation during HSC transdifferentiation. A: Quiescent (day two), intermediate (day five), and myofibroblastic (day nine) HSCs were examined for H3 and H4 histone acetylation and methylation by Western blot analysis. Transdifferentiation was monitored by increased expression of α-SMA. B: Quantification of acetyl histone H4 and α-SMA by Western blot was measured by densitometry, representing three independent experiments. C: Cells were fractionated into cytoplasmic and nuclear/membrane fractions. Decreased acetylation of histone H3 and H4 during transdifferentiation is obvious. D: In quiescent (day two, qHSC) and myofibroblastic (day nine, mHSC) HSCs, basal levels of acetyl histone H4 in the transcription start site of MMP13 gene were measured by ChIP assay and qRT-PCR. Data represent the output/input ratios and are the averages of three independent experiments. Statistical significance, * P

Techniques Used: Methylation, Western Blot, Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR

Impaired histone acetylation and failed recruitment of RNA Pol II to the 5′-promoters of MMP genes in myofibroblastic HSCs. Quiescent and myofibroblastic HSCs were stimulated by IL-1 and harvested at the time points indicated. Binding of Ser-5-p-Pol II and acetylation of histone H4 in MMP9 ( A ) and MMP 13 ( B ) promoters were measured by ChIP assay followed by qPCR analysis. Data were calculated as output/input ratios and further converted to fold changes over basal levels. Results are averages of three experiments with standard deviations.
Figure Legend Snippet: Impaired histone acetylation and failed recruitment of RNA Pol II to the 5′-promoters of MMP genes in myofibroblastic HSCs. Quiescent and myofibroblastic HSCs were stimulated by IL-1 and harvested at the time points indicated. Binding of Ser-5-p-Pol II and acetylation of histone H4 in MMP9 ( A ) and MMP 13 ( B ) promoters were measured by ChIP assay followed by qPCR analysis. Data were calculated as output/input ratios and further converted to fold changes over basal levels. Results are averages of three experiments with standard deviations.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

22) Product Images from "The p38 Mitogen-Activated Protein Kinase Signaling Pathway Is Coupled to Toll-Like Receptor 5 To Mediate Gene Regulation in Response to Pseudomonas aeruginosa Infection in Human Airway Epithelial Cells ▿"

Article Title: The p38 Mitogen-Activated Protein Kinase Signaling Pathway Is Coupled to Toll-Like Receptor 5 To Mediate Gene Regulation in Response to Pseudomonas aeruginosa Infection in Human Airway Epithelial Cells ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.00678-07

p38 MAPK is activated by P. aeruginosa and remains in the cytoplasm of HAECs. (A) Time-response curve for p38 MAPK phosphorylation stimulated by PAO1. HAECs were seeded in 24-well plates and stimulated with medium or heat-killed PAO1 at an MOI of 100 at 37°C. The cell lysates were analyzed by Western blotting using phosphorylated p38 (P-p38) MAPK antibody (upper panel). Variations in sample loading were examined by detection of total p38 MAPK (lower panel). (B) Dose-response curve for p38 MAPK with PAO1 stimulation. HAECs were cultured in 24-well plates and were incubated with heat-killed PAO1 at the MOI indicated for 30 min. Phosphorylated and total p38 MAPK were analyzed by Western blotting. (C) Intracellular localization of P-p38 MAPK in HAECs. HAECs grown on glass coverslips placed in 24-well plates were challenged with heat-killed PAO1 (MOI of 100). Immunocytochemical analysis using P-p38 MAPK antibody was carried out as described in Materials and Methods. The numbers indicate time (in minutes). (D) Subcellular fractionation of P-p38 MAPK in HAECs. HAECs were grown on 10-cm culture plates and incubated with PAO1 (MOI of 100) alone or with medium for 30 min. Cytoplasmic and nuclear extracts were prepared as described in Materials and Methods and subjected to blotting with antibodies against either P-p38 MAPK (upper panel) or histone H3 subunit (lower panel). All images are representative of at least two independent experiments.
Figure Legend Snippet: p38 MAPK is activated by P. aeruginosa and remains in the cytoplasm of HAECs. (A) Time-response curve for p38 MAPK phosphorylation stimulated by PAO1. HAECs were seeded in 24-well plates and stimulated with medium or heat-killed PAO1 at an MOI of 100 at 37°C. The cell lysates were analyzed by Western blotting using phosphorylated p38 (P-p38) MAPK antibody (upper panel). Variations in sample loading were examined by detection of total p38 MAPK (lower panel). (B) Dose-response curve for p38 MAPK with PAO1 stimulation. HAECs were cultured in 24-well plates and were incubated with heat-killed PAO1 at the MOI indicated for 30 min. Phosphorylated and total p38 MAPK were analyzed by Western blotting. (C) Intracellular localization of P-p38 MAPK in HAECs. HAECs grown on glass coverslips placed in 24-well plates were challenged with heat-killed PAO1 (MOI of 100). Immunocytochemical analysis using P-p38 MAPK antibody was carried out as described in Materials and Methods. The numbers indicate time (in minutes). (D) Subcellular fractionation of P-p38 MAPK in HAECs. HAECs were grown on 10-cm culture plates and incubated with PAO1 (MOI of 100) alone or with medium for 30 min. Cytoplasmic and nuclear extracts were prepared as described in Materials and Methods and subjected to blotting with antibodies against either P-p38 MAPK (upper panel) or histone H3 subunit (lower panel). All images are representative of at least two independent experiments.

Techniques Used: Western Blot, Cell Culture, Incubation, Fractionation

23) Product Images from "A grape seed procyanidin extract inhibits HDAC activity leading to increased Pparα phosphorylation and target gene expression"

Article Title: A grape seed procyanidin extract inhibits HDAC activity leading to increased Pparα phosphorylation and target gene expression

Journal: Molecular nutrition & food research

doi: 10.1002/mnfr.201600347

GSPE increases histone acetylation and blocks Class I HDAC activity in vivo (A) Global profile of protein acetylation and histone deacetylase (HDAC)1, 2 and 3 expression in the mouse liver treated with GSPE (250 mg/kg) or vehicle (water). (B) Relative protein expression of acetylated (Ac)-H3K9, Ac-Lys and Ac-Tub. (C) Class I, IIa and IIb HDAC activities following GSPE treatment. *p
Figure Legend Snippet: GSPE increases histone acetylation and blocks Class I HDAC activity in vivo (A) Global profile of protein acetylation and histone deacetylase (HDAC)1, 2 and 3 expression in the mouse liver treated with GSPE (250 mg/kg) or vehicle (water). (B) Relative protein expression of acetylated (Ac)-H3K9, Ac-Lys and Ac-Tub. (C) Class I, IIa and IIb HDAC activities following GSPE treatment. *p

Techniques Used: Activity Assay, In Vivo, Histone Deacetylase Assay, Expressing

24) Product Images from "Kaempferol targets RSK2 and MSK1 to suppress ultraviolet radiation-induced skin cancer"

Article Title: Kaempferol targets RSK2 and MSK1 to suppress ultraviolet radiation-induced skin cancer

Journal: Cancer prevention research (Philadelphia, Pa.)

doi: 10.1158/1940-6207.CAPR-14-0126

Kaempferol attenuates SUV-induced phosphorylation of CREB and histone H3 and activation of AP-1 and NF-κB in mouse skin cells
Figure Legend Snippet: Kaempferol attenuates SUV-induced phosphorylation of CREB and histone H3 and activation of AP-1 and NF-κB in mouse skin cells

Techniques Used: Activation Assay

25) Product Images from "Insulin detemir enhances proglucagon gene expression in the intestinal L cells via stimulating ?-catenin and CREB activities"

Article Title: Insulin detemir enhances proglucagon gene expression in the intestinal L cells via stimulating ?-catenin and CREB activities

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00328.2011

d-INS stimulates cAMP response element-binding protein (CREB) phosphorylation, which is phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK)/ERK dependent in GLUTag cells. Cells were serum starved overnight and treated with d-INS at indicated concentrations for 10 min ( A ) or 100 nM d-INS for indicated times ( B ). Cell lysates were subjected to Western blotting. C : serum-starved GLUTag cells were treated with 100 nM d-INS for 10 min in the presence of an indicated inhibitor (PD, 50 μM; LY, 50 μM), and nuclear fractions were subjected to Western blotting using Histone H3 as loading controls. Bar graphs show the densitometry analysis. Data are means ± SE. * P
Figure Legend Snippet: d-INS stimulates cAMP response element-binding protein (CREB) phosphorylation, which is phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK)/ERK dependent in GLUTag cells. Cells were serum starved overnight and treated with d-INS at indicated concentrations for 10 min ( A ) or 100 nM d-INS for indicated times ( B ). Cell lysates were subjected to Western blotting. C : serum-starved GLUTag cells were treated with 100 nM d-INS for 10 min in the presence of an indicated inhibitor (PD, 50 μM; LY, 50 μM), and nuclear fractions were subjected to Western blotting using Histone H3 as loading controls. Bar graphs show the densitometry analysis. Data are means ± SE. * P

Techniques Used: Binding Assay, Western Blot

26) Product Images from "MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM"

Article Title: MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1819085116

MM-131 inhibits HGF-dependent and HGF-independent Met signaling by antagonizing HGF binding to Met and inducing down-regulation of Met. ( A ) MM-131 blocks HGF binding to Met. Biolayer interferometry (ForteBio) was used to measure the HGF-blocking properties of anti-Met antibodies MM-131, huOA-5D5, LY2875358, and ABT-700. Antibodies were loaded onto anti-human IgG Fc capture biosensors and incubated with 200 nM human Met. After 2 min, 200 nM human HGF was added, followed by addition of buffer at 4 min. ( B ) MM-131 inhibits HGF-induced Met signaling in a dose-dependent manner. HT-29 cells were incubated without HGF, with 1 nM HGF, or with 1 nM HGF + 0.1–1,000 nM MM-131 for 10 min and analyzed by immunoblotting with the indicated antibodies. ( C ) MM-131 down-regulates total Met levels. NCI-H2170 cells were incubated with 100 nM indicated antibodies or PBS for 24 h and analyzed by immunoblotting. Histone H3 served as a loading control. Bands were quantified using the LI-COR Odyssey imaging system. MM-131 EpCAMmut is a version of MM-131 with a mutation in the EpCAM scFv, preventing EpCAM binding. MM-131 Metmut is a version of MM-131 that cannot bind Met. Data are the mean of two independent samples; error bars indicate SD.
Figure Legend Snippet: MM-131 inhibits HGF-dependent and HGF-independent Met signaling by antagonizing HGF binding to Met and inducing down-regulation of Met. ( A ) MM-131 blocks HGF binding to Met. Biolayer interferometry (ForteBio) was used to measure the HGF-blocking properties of anti-Met antibodies MM-131, huOA-5D5, LY2875358, and ABT-700. Antibodies were loaded onto anti-human IgG Fc capture biosensors and incubated with 200 nM human Met. After 2 min, 200 nM human HGF was added, followed by addition of buffer at 4 min. ( B ) MM-131 inhibits HGF-induced Met signaling in a dose-dependent manner. HT-29 cells were incubated without HGF, with 1 nM HGF, or with 1 nM HGF + 0.1–1,000 nM MM-131 for 10 min and analyzed by immunoblotting with the indicated antibodies. ( C ) MM-131 down-regulates total Met levels. NCI-H2170 cells were incubated with 100 nM indicated antibodies or PBS for 24 h and analyzed by immunoblotting. Histone H3 served as a loading control. Bands were quantified using the LI-COR Odyssey imaging system. MM-131 EpCAMmut is a version of MM-131 with a mutation in the EpCAM scFv, preventing EpCAM binding. MM-131 Metmut is a version of MM-131 that cannot bind Met. Data are the mean of two independent samples; error bars indicate SD.

Techniques Used: Binding Assay, Blocking Assay, Incubation, Imaging, Mutagenesis

27) Product Images from "SIRT6 Acts as a Negative Regulator in Dengue Virus-Induced Inflammatory Response by Targeting the DNA Binding Domain of NF-κB p65"

Article Title: SIRT6 Acts as a Negative Regulator in Dengue Virus-Induced Inflammatory Response by Targeting the DNA Binding Domain of NF-κB p65

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00113

NF-κB p65 is the target of SIRT6. (A) SIRT6 inhibits RLR and TLR3-activated NF-κB signaling pathway. Quantification of NF-κB promoter activity of HEK293T cells expressing increasing amounts of SIRT6 together with TRAF6, TAK1/TAB1, IKKα, IKKβ, or p65, respectively. (B) SIRT6 interacts with NF-κB p65. Coimmunoprecipitation (CoIP) of SIRT6 and p65 from HEK293T cells expressing Flag-SIRT6 and HA-p65 using α-Flag or α-HA antibody pull-down, followed by immunoblotting. (C) CoIP of endogenous SIRT6 and p65 from Raw264.7 cells infected with DENV (MOI = 1) for 12 h using α-SIRT6 or α-p65 antibody pull-down, followed by immunoblotting. (D) Immunoblot analysis of SIRT6, p65, Caspase 3 and Histone H3 in cytoplasm and nucleus of Raw264.7 cells after DENV infection (MOI = 1–5) for 12 h. (E) Immunofluorescence microscopy of HEK293T-TLR3 cells transfected with plasmids of Flag-SIRT6 (green) with HA-p65 (red) stimulated by HMW Poly I:C. White Bar: 50 um. Data shown are the mean ± SEM; * p
Figure Legend Snippet: NF-κB p65 is the target of SIRT6. (A) SIRT6 inhibits RLR and TLR3-activated NF-κB signaling pathway. Quantification of NF-κB promoter activity of HEK293T cells expressing increasing amounts of SIRT6 together with TRAF6, TAK1/TAB1, IKKα, IKKβ, or p65, respectively. (B) SIRT6 interacts with NF-κB p65. Coimmunoprecipitation (CoIP) of SIRT6 and p65 from HEK293T cells expressing Flag-SIRT6 and HA-p65 using α-Flag or α-HA antibody pull-down, followed by immunoblotting. (C) CoIP of endogenous SIRT6 and p65 from Raw264.7 cells infected with DENV (MOI = 1) for 12 h using α-SIRT6 or α-p65 antibody pull-down, followed by immunoblotting. (D) Immunoblot analysis of SIRT6, p65, Caspase 3 and Histone H3 in cytoplasm and nucleus of Raw264.7 cells after DENV infection (MOI = 1–5) for 12 h. (E) Immunofluorescence microscopy of HEK293T-TLR3 cells transfected with plasmids of Flag-SIRT6 (green) with HA-p65 (red) stimulated by HMW Poly I:C. White Bar: 50 um. Data shown are the mean ± SEM; * p

Techniques Used: Activity Assay, Expressing, Co-Immunoprecipitation Assay, Infection, Immunofluorescence, Microscopy, Transfection

28) Product Images from "Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo"

Article Title: Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo

Journal: Nature medicine

doi: 10.1038/nm.3826

Nicotine treatment results in greater pMKP1-Ser334 levels and subsequent degradation through AMPK. ( a ) MKP1 expression (top) and its serine (Ser) /threonine (Thr) phosphorylation (bottom) in WAT from Veh- or Nic-treated mice. Gapdh was detected as a loading control; n = 8 each. ( b ) AMPKα2-MKP1binding in MEF-derived adipocytes; n = 5 each. ( c ) AMPK activation and MKP1 serine phosphorylation in nicotine-treated isolated adipocytes; n = 4 each. ( d ) MKP1 phosphorylation in HEK293 cells transfected with vector, GFP-tagged WT MKP1 (WT) or the indicated site-directed mutants of MKP1; n = 4 each. ( e ) The indicated peptides (the SAMS peptide and its mutant, SAMSmu) or peptides spanning the indicated amino acid residues of MKP1; n = 3 each. ( f ) MKP1 ubiquitination in HEK293 cells transfected with WT MKP1 (WT) or MKP1-S334A; n = 4 each. ( g ) MKP1 protein expression in MG132-treated isolated adipocytes; n = 5 each. ( h ) The subcellular localization of AMPKα1/2, p38 and MKP1 in adipocytes treated with or without nicotine. LDH (lactate dehydrogenase), cytoplasmic marker; H3 (histone 3), nuclear marker; n = 5 each. ( i ) Insulin signaling (western blot, top) and lipolysis (bottom) in Nic- or Veh-treated MEF-derived adipocytes transfected with adenovirus GFP (Ad-GFP) or MKP1 (Ad-MKP1).; n = 5 each. ( j ) Detection of pMKP1-S334 in WAT from the indicated strains in Nic- or Veh-treated mice (top) and its quantitation (bottom). Beta-actin was detected as a loading control; n = 6 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test ( a,i,j ) and * P
Figure Legend Snippet: Nicotine treatment results in greater pMKP1-Ser334 levels and subsequent degradation through AMPK. ( a ) MKP1 expression (top) and its serine (Ser) /threonine (Thr) phosphorylation (bottom) in WAT from Veh- or Nic-treated mice. Gapdh was detected as a loading control; n = 8 each. ( b ) AMPKα2-MKP1binding in MEF-derived adipocytes; n = 5 each. ( c ) AMPK activation and MKP1 serine phosphorylation in nicotine-treated isolated adipocytes; n = 4 each. ( d ) MKP1 phosphorylation in HEK293 cells transfected with vector, GFP-tagged WT MKP1 (WT) or the indicated site-directed mutants of MKP1; n = 4 each. ( e ) The indicated peptides (the SAMS peptide and its mutant, SAMSmu) or peptides spanning the indicated amino acid residues of MKP1; n = 3 each. ( f ) MKP1 ubiquitination in HEK293 cells transfected with WT MKP1 (WT) or MKP1-S334A; n = 4 each. ( g ) MKP1 protein expression in MG132-treated isolated adipocytes; n = 5 each. ( h ) The subcellular localization of AMPKα1/2, p38 and MKP1 in adipocytes treated with or without nicotine. LDH (lactate dehydrogenase), cytoplasmic marker; H3 (histone 3), nuclear marker; n = 5 each. ( i ) Insulin signaling (western blot, top) and lipolysis (bottom) in Nic- or Veh-treated MEF-derived adipocytes transfected with adenovirus GFP (Ad-GFP) or MKP1 (Ad-MKP1).; n = 5 each. ( j ) Detection of pMKP1-S334 in WAT from the indicated strains in Nic- or Veh-treated mice (top) and its quantitation (bottom). Beta-actin was detected as a loading control; n = 6 each. Significance determined by one-way ANOVA with Bonferroni’s post-hoc test ( a,i,j ) and * P

Techniques Used: Expressing, Mouse Assay, Derivative Assay, Activation Assay, Isolation, Transfection, Plasmid Preparation, Mutagenesis, Marker, Western Blot, Quantitation Assay

29) Product Images from "Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource"

Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx900

Epigenetic profiles associated with expressed single- versus multi-exon genes in K562 and primary CD4+ human T cells. ( A ) Read coverage of transcription and splicing-associated histone post-translational modifications (PTMs) across the gene bodies of expressed genes in K562 cells. Genes were segregated into sets with different length restrictions; single- and multi-exon genes are shown in blue and red, respectively. ( B ) Heatmaps showing ChIP-seq read coverage for histone PTMs, pol II, and TATA binding protein (TBP) centered around transcription start sites (TSS) of single- and multi-exon genes; rows are sorted by decreasing pol II density. ( C ) Read coverage for 5-methylcytosine (5mC) MeDIP-seq in CD4 + T-cells over gene bodies of expressed genes. Read coverage is presented for all genes or segregated based on gene length. RPKM, reads per kilobase per million mapped (log scale); MeDIP, methylated DNA immunoprecipitation.
Figure Legend Snippet: Epigenetic profiles associated with expressed single- versus multi-exon genes in K562 and primary CD4+ human T cells. ( A ) Read coverage of transcription and splicing-associated histone post-translational modifications (PTMs) across the gene bodies of expressed genes in K562 cells. Genes were segregated into sets with different length restrictions; single- and multi-exon genes are shown in blue and red, respectively. ( B ) Heatmaps showing ChIP-seq read coverage for histone PTMs, pol II, and TATA binding protein (TBP) centered around transcription start sites (TSS) of single- and multi-exon genes; rows are sorted by decreasing pol II density. ( C ) Read coverage for 5-methylcytosine (5mC) MeDIP-seq in CD4 + T-cells over gene bodies of expressed genes. Read coverage is presented for all genes or segregated based on gene length. RPKM, reads per kilobase per million mapped (log scale); MeDIP, methylated DNA immunoprecipitation.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Methylated DNA Immunoprecipitation, Methylation, Immunoprecipitation

30) Product Images from "P53 Is Involved in a Three-Dimensional Architecture-Mediated Decrease in Chemosensitivity in Colon Cancer"

Article Title: P53 Is Involved in a Three-Dimensional Architecture-Mediated Decrease in Chemosensitivity in Colon Cancer

Journal: Journal of Cancer

doi: 10.7150/jca.14506

Knockdown of p53 decreases sensitivity to platinum in HCT116. (A) There was no detectable difference in the p53 protein level between Parental and shcontrol cells. Relative quantities of p53 were normalized to GAPDH and diagrammed (right). (western blot) (B) The protein p53 was knocked down by lentiviral delivery of shRNA (shp53), and the efficiency was assayed by western blot. Relative quantities of p53 were normalized to β-actin and diagrammed (right). (C) Immunohistochemical staining of p53 in HCT116 3D cultures. (D) After treatment of CDDP for 24 hours, p53, p-p53 and cleaved caspase 3 in 3D cultures were assayed by western blot. Relative quantities were normalized to their loading control and diagrammed, respectively (right). (E) After treatment of CDDP for 48 hours, p53 and cleaved caspase 3 in 3D cultures were assayed by western blot. Relative quantities were normalized to Histone H3 and diagrammed (right). (F) After treatment of CDDP for 24 hours, p53 and cleaved caspase 3 in 2D cultures were assayed by western blot. Relative quantities were normalized to β-actin and diagrammed (right). (G) After treatment of CDDP for 24 hours, apoptosis was detected with TUNEL stain. (H) Cell viabilities of HCT116 3D cultures were assayed by WST. *: p
Figure Legend Snippet: Knockdown of p53 decreases sensitivity to platinum in HCT116. (A) There was no detectable difference in the p53 protein level between Parental and shcontrol cells. Relative quantities of p53 were normalized to GAPDH and diagrammed (right). (western blot) (B) The protein p53 was knocked down by lentiviral delivery of shRNA (shp53), and the efficiency was assayed by western blot. Relative quantities of p53 were normalized to β-actin and diagrammed (right). (C) Immunohistochemical staining of p53 in HCT116 3D cultures. (D) After treatment of CDDP for 24 hours, p53, p-p53 and cleaved caspase 3 in 3D cultures were assayed by western blot. Relative quantities were normalized to their loading control and diagrammed, respectively (right). (E) After treatment of CDDP for 48 hours, p53 and cleaved caspase 3 in 3D cultures were assayed by western blot. Relative quantities were normalized to Histone H3 and diagrammed (right). (F) After treatment of CDDP for 24 hours, p53 and cleaved caspase 3 in 2D cultures were assayed by western blot. Relative quantities were normalized to β-actin and diagrammed (right). (G) After treatment of CDDP for 24 hours, apoptosis was detected with TUNEL stain. (H) Cell viabilities of HCT116 3D cultures were assayed by WST. *: p

Techniques Used: Western Blot, shRNA, Immunohistochemistry, Staining, TUNEL Assay

31) Product Images from "Inhibition of Histone Deacetylase Activity Promotes Invasion of Human Cancer Cells through Activation of Urokinase Plasminogen Activator (uPA)"

Article Title: Inhibition of Histone Deacetylase Activity Promotes Invasion of Human Cancer Cells through Activation of Urokinase Plasminogen Activator (uPA)

Journal:

doi: 10.1074/jbc.M705867200

TSA induces accumulation of acetylated histones H3 and H4 in chromatin associated with the uPA gene
Figure Legend Snippet: TSA induces accumulation of acetylated histones H3 and H4 in chromatin associated with the uPA gene

Techniques Used:

Inhibition of HDAC activity induces the acetylation of histones and concomitant uPA expression in human cancer cells
Figure Legend Snippet: Inhibition of HDAC activity induces the acetylation of histones and concomitant uPA expression in human cancer cells

Techniques Used: Inhibition, Activity Assay, Expressing

32) Product Images from "Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells"

Article Title: Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-98

Nucleus distribution of NF-κB family members in human esophageal squamous cell carcinoma cell lines. Nuclear extracts of TE-1, Eca109, KYSE150, KYSE510 and HaCaT were prepared as described in Methods and analyzed with antibodies against NF-κB subunits p50, p52, p65, c-Rel and RelB, respectively. Histone H3 was used as a nuclear protein loading control.
Figure Legend Snippet: Nucleus distribution of NF-κB family members in human esophageal squamous cell carcinoma cell lines. Nuclear extracts of TE-1, Eca109, KYSE150, KYSE510 and HaCaT were prepared as described in Methods and analyzed with antibodies against NF-κB subunits p50, p52, p65, c-Rel and RelB, respectively. Histone H3 was used as a nuclear protein loading control.

Techniques Used:

33) Product Images from "Subchronic Treatment of Donepezil Rescues Impaired Social, Hyperactive, and Stereotypic Behavior in Valproic Acid-Induced Animal Model of Autism"

Article Title: Subchronic Treatment of Donepezil Rescues Impaired Social, Hyperactive, and Stereotypic Behavior in Valproic Acid-Induced Animal Model of Autism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104927

Acetylation of histone H3 bound to promoter region of Ache was increased by VPA treatment. To confirm the effect of HDACIs on the upregulation of AChE, ChIP was performed using prefrontal cortex from VPA rat and VPA treated cortical NPCs from SD rat. (A) ChIP results using prefrontal cortex of rat prenatally exposed to VPA or PBS (con) (B) ChIP results from cortical neural progenitor cells of rat. Gapdh was used as a positive control for this analysis. (AcH:acetylated Histone H3, Ache:acetylcholinesterase).
Figure Legend Snippet: Acetylation of histone H3 bound to promoter region of Ache was increased by VPA treatment. To confirm the effect of HDACIs on the upregulation of AChE, ChIP was performed using prefrontal cortex from VPA rat and VPA treated cortical NPCs from SD rat. (A) ChIP results using prefrontal cortex of rat prenatally exposed to VPA or PBS (con) (B) ChIP results from cortical neural progenitor cells of rat. Gapdh was used as a positive control for this analysis. (AcH:acetylated Histone H3, Ache:acetylcholinesterase).

Techniques Used: Chromatin Immunoprecipitation, Positive Control

34) Product Images from "Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus"

Article Title: Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus

Journal: Biochemistry Research International

doi: 10.1155/2015/731595

Nuclear regulation of p53 during late torpor. (a) Western blot is shown for nuclear levels of p53 and histone H3 during euthermic control and late torpor. (b) p53 DNA binding activity towards double stranded probe containing p53 consensus binding sequence (5′-GGACATGCCCGGGCATGTCC-3′) in control and late torpor nuclear lysates. Data show mean ± SEM, N = 4 independent trials on tissues from different animals. ∗ denotes significant statistical difference from EC values, p
Figure Legend Snippet: Nuclear regulation of p53 during late torpor. (a) Western blot is shown for nuclear levels of p53 and histone H3 during euthermic control and late torpor. (b) p53 DNA binding activity towards double stranded probe containing p53 consensus binding sequence (5′-GGACATGCCCGGGCATGTCC-3′) in control and late torpor nuclear lysates. Data show mean ± SEM, N = 4 independent trials on tissues from different animals. ∗ denotes significant statistical difference from EC values, p

Techniques Used: Western Blot, Binding Assay, Activity Assay, Sequencing

35) Product Images from "SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin"

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

Journal: Nature Communications

doi: 10.1038/ncomms11996

SARI inhibits VEGF expression by directly binding to Cp. ( a ) Co-immunoprecipitation products after adding anti-SARI antibody were separated by SDS–PAGE, and the differential band was analysed by MS. ( b ) Co-immunoprecipitation analysis of SW480-control and SW480-SARI cell lysates using anti-SARI and anti-Cp antibody. ( c ) Staining for Cp (green) and SARI (red) in SW480-control and SW480-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. ( d ) Immunoblots of SARI and Cp expression in SW480 and HCT116-blank, control and SARI cells. GAPDH was used as a loading control. ( e ) Western blotting detection of Cp expression of collected nuclear and cytoplasmic proteins. Histone H3 was used as the nucleus loading control and β-actin as the cytoplasm loading control. ( f ) Immunoblots of Cp and VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. GAPDH was used as the loading control. ( g ) ELISA detection of VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. ( n =4; ** P
Figure Legend Snippet: SARI inhibits VEGF expression by directly binding to Cp. ( a ) Co-immunoprecipitation products after adding anti-SARI antibody were separated by SDS–PAGE, and the differential band was analysed by MS. ( b ) Co-immunoprecipitation analysis of SW480-control and SW480-SARI cell lysates using anti-SARI and anti-Cp antibody. ( c ) Staining for Cp (green) and SARI (red) in SW480-control and SW480-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. ( d ) Immunoblots of SARI and Cp expression in SW480 and HCT116-blank, control and SARI cells. GAPDH was used as a loading control. ( e ) Western blotting detection of Cp expression of collected nuclear and cytoplasmic proteins. Histone H3 was used as the nucleus loading control and β-actin as the cytoplasm loading control. ( f ) Immunoblots of Cp and VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. GAPDH was used as the loading control. ( g ) ELISA detection of VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. ( n =4; ** P

Techniques Used: Expressing, Binding Assay, Immunoprecipitation, SDS Page, Mass Spectrometry, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

36) Product Images from "The characterization of an intestine-like genomic signature maintained during Barrett’s-associated adenocarcinogenesis reveals an NR5A2-mediated promotion of cancer cell survival"

Article Title: The characterization of an intestine-like genomic signature maintained during Barrett’s-associated adenocarcinogenesis reveals an NR5A2-mediated promotion of cancer cell survival

Journal: Scientific Reports

doi: 10.1038/srep32638

Induction of NR5A2 and GATA6 expression by pulsed gastric acid in oesophageal cells. SKGT4 cells were exposed to simulated reflux events, 10 min in duration, using acidified cell culture media at pH4.5 or non-acidified media at pH7.4 (control) followed by incubation at pH7.4 for 2 hs prior to repeating the reflux event. ( A ) pH levels monitored throughout experiment and 2 hs prior to initial insult demonstrates only minor fluctuations in intended pH. ( B ) FOSb (positive control), ( C ) NR5A2 and ( D ) GATA6 mRNA levels in SKGT4 cells exposed to differing acidic environments (continuous (cont) or pulsatile (pulse) (10 min) at pH4.5 or pH7.4) examined by real time RT-PCR at t-2, t0, t+2, t+4 and t+24 hs. ( E ) Cell viability following acidic treatments in SKGT4 cells measured by MTT assay following 2 repeated exposures. ( F ) Induction of GATA6 protein expression following exposure to pulsatile low pH4.5 following 2 repeated exposures measured over 8 hs using histone H3 blotting as loading control (image details live zoomed cropped exposure of GATA6 and loading control bands as capture by CCD imager). Significance was achieved by two-tailed non-parametric Mann-Whitney testing for RT-PCR data and students t-test in viability and Western blotting experiments, p
Figure Legend Snippet: Induction of NR5A2 and GATA6 expression by pulsed gastric acid in oesophageal cells. SKGT4 cells were exposed to simulated reflux events, 10 min in duration, using acidified cell culture media at pH4.5 or non-acidified media at pH7.4 (control) followed by incubation at pH7.4 for 2 hs prior to repeating the reflux event. ( A ) pH levels monitored throughout experiment and 2 hs prior to initial insult demonstrates only minor fluctuations in intended pH. ( B ) FOSb (positive control), ( C ) NR5A2 and ( D ) GATA6 mRNA levels in SKGT4 cells exposed to differing acidic environments (continuous (cont) or pulsatile (pulse) (10 min) at pH4.5 or pH7.4) examined by real time RT-PCR at t-2, t0, t+2, t+4 and t+24 hs. ( E ) Cell viability following acidic treatments in SKGT4 cells measured by MTT assay following 2 repeated exposures. ( F ) Induction of GATA6 protein expression following exposure to pulsatile low pH4.5 following 2 repeated exposures measured over 8 hs using histone H3 blotting as loading control (image details live zoomed cropped exposure of GATA6 and loading control bands as capture by CCD imager). Significance was achieved by two-tailed non-parametric Mann-Whitney testing for RT-PCR data and students t-test in viability and Western blotting experiments, p

Techniques Used: Expressing, Reflux, Cell Culture, Incubation, Positive Control, Quantitative RT-PCR, MTT Assay, Two Tailed Test, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Western Blot

37) Product Images from "Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion"

Article Title: Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion

Journal: Cell Cycle

doi: 10.1080/15384101.2016.1261765

Regulation of DNA synthesis and expression of histone genes by CASP8AP2, NPAT and HINFP. (A) Flow cytometric analysis of DNA content (x-axis) and DNA replication (EdU incorporation; y-axis) shows partial or complete DNA synthesis progression 3 d after knockdown of CASP8AP2, NPAT and HINFP in tumor (U2OS) and normal (hTERT-RPE1) cells. Note that in both cell lines, CASP8AP2 RNAi results in formation of a population of S-phase cells with low EdU incorporation (red arrowheads). (B) Analysis of expression of histone genes following knockdown of the indicated genes in U2OS and hTERT-RPE1 cells. Replication-independent histone genes are marked with an asterisk. (C) Location-analysis of transcriptional regulators at histone gene cluster on chromosome 6p22. Cell lines and antibodies used in ChIP-Seq are indicated on the left, and signal intensity as number of reads is shown in parentheses above each track. Note that CASP8AP2 and NPAT co-bind to transcription start sites of replication-dependent histone genes (indicated in bottom) in this cluster.
Figure Legend Snippet: Regulation of DNA synthesis and expression of histone genes by CASP8AP2, NPAT and HINFP. (A) Flow cytometric analysis of DNA content (x-axis) and DNA replication (EdU incorporation; y-axis) shows partial or complete DNA synthesis progression 3 d after knockdown of CASP8AP2, NPAT and HINFP in tumor (U2OS) and normal (hTERT-RPE1) cells. Note that in both cell lines, CASP8AP2 RNAi results in formation of a population of S-phase cells with low EdU incorporation (red arrowheads). (B) Analysis of expression of histone genes following knockdown of the indicated genes in U2OS and hTERT-RPE1 cells. Replication-independent histone genes are marked with an asterisk. (C) Location-analysis of transcriptional regulators at histone gene cluster on chromosome 6p22. Cell lines and antibodies used in ChIP-Seq are indicated on the left, and signal intensity as number of reads is shown in parentheses above each track. Note that CASP8AP2 and NPAT co-bind to transcription start sites of replication-dependent histone genes (indicated in bottom) in this cluster.

Techniques Used: DNA Synthesis, Expressing, Flow Cytometry, Chromatin Immunoprecipitation

CASP8AP2 siRNA triggers activation of a p53 dependent S-phase checkpoint in normal but not in tumor cells. (A) Analysis of p53 target gene expression 3 d after knockdown of the indicated transcriptional regulators of histone genes. (B, C) Flow cytometry analysis of CASP8AP2 siRNA treated hTERT-RPE1 (B) and U2OS (C) cells indicates that non-replicated cells arrested in S-phase express a marker for DNA damage signaling (γ-H2AX; low right hand corner). (D, E) hTERT-RPE1 (D) but not U2OS (E) cells with activated DNA damage signaling have activated p53, based on expression of the p53 target-gene p21 (upper right hand corner).
Figure Legend Snippet: CASP8AP2 siRNA triggers activation of a p53 dependent S-phase checkpoint in normal but not in tumor cells. (A) Analysis of p53 target gene expression 3 d after knockdown of the indicated transcriptional regulators of histone genes. (B, C) Flow cytometry analysis of CASP8AP2 siRNA treated hTERT-RPE1 (B) and U2OS (C) cells indicates that non-replicated cells arrested in S-phase express a marker for DNA damage signaling (γ-H2AX; low right hand corner). (D, E) hTERT-RPE1 (D) but not U2OS (E) cells with activated DNA damage signaling have activated p53, based on expression of the p53 target-gene p21 (upper right hand corner).

Techniques Used: Activation Assay, Expressing, Flow Cytometry, Cytometry, Marker

Tumor cells continue to progress in S-phase despite low nucleosome levels induced by CASP8AP2 knockdown. (A, B) Flow cytometry analysis of DNA content, DNA replication and histone H3 levels in siRNA treated cells. Note that tumor cells continue to replicate their DNA slowly (A) for multiple days, despite low amount of histone H3 (red arrowheads) (B). (C, D) Flow cytometry analysis of DNA content, DNA replication (EdU staining) and histone H3 levels in siRNA treated hTERT-RPE1 cells. Note that normal cells arrest in S-phase (C), and show decrease in histone H3 levels in early S-phase (red arrowheads) (D).
Figure Legend Snippet: Tumor cells continue to progress in S-phase despite low nucleosome levels induced by CASP8AP2 knockdown. (A, B) Flow cytometry analysis of DNA content, DNA replication and histone H3 levels in siRNA treated cells. Note that tumor cells continue to replicate their DNA slowly (A) for multiple days, despite low amount of histone H3 (red arrowheads) (B). (C, D) Flow cytometry analysis of DNA content, DNA replication (EdU staining) and histone H3 levels in siRNA treated hTERT-RPE1 cells. Note that normal cells arrest in S-phase (C), and show decrease in histone H3 levels in early S-phase (red arrowheads) (D).

Techniques Used: Flow Cytometry, Cytometry, Staining

38) Product Images from "CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma"

Article Title: CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

Journal: Oncotarget

doi: 10.18632/oncotarget.12434

Subcellular localization of CCND1 mutants ( A ) Cytosolic and nuclear extracts were prepared as described in Materials and Methods from UPN-1 cells that stably expressed WT or Y44D CCND1. The extracts (30 μg per lane) were immunoblotted with indicated antibodies. β-ACTIN or GAPDH and histone H3 were used to confirm cytosolic and nuclear fractions, respectively. Numbers below the blots are relative densitometric values of corresponding bands after normalization to β-ACTIN (for WCL and Cytosolic) or histone H3 (for Nuclear) loading controls. WCL, whole cell lysates; Endo., endogenous. ( B ) Subcellular distribution of WT and mutant CCND1-HA proteins, which were transiently expressed in HEK-293T cells, was evaluated by immunofluorescence. Shown are representative confocal immunofluorescence images of WT and mutant CCND1-expressing cells stained with anti-HA antibody (red) followed by nuclear staining with DAPI (blue). Scale bars, 20 μm. Bar graphs show the percentages of HA positive cells that have HA staining in the nucleus from two separate experiments. Error bars, SEM; *** *P
Figure Legend Snippet: Subcellular localization of CCND1 mutants ( A ) Cytosolic and nuclear extracts were prepared as described in Materials and Methods from UPN-1 cells that stably expressed WT or Y44D CCND1. The extracts (30 μg per lane) were immunoblotted with indicated antibodies. β-ACTIN or GAPDH and histone H3 were used to confirm cytosolic and nuclear fractions, respectively. Numbers below the blots are relative densitometric values of corresponding bands after normalization to β-ACTIN (for WCL and Cytosolic) or histone H3 (for Nuclear) loading controls. WCL, whole cell lysates; Endo., endogenous. ( B ) Subcellular distribution of WT and mutant CCND1-HA proteins, which were transiently expressed in HEK-293T cells, was evaluated by immunofluorescence. Shown are representative confocal immunofluorescence images of WT and mutant CCND1-expressing cells stained with anti-HA antibody (red) followed by nuclear staining with DAPI (blue). Scale bars, 20 μm. Bar graphs show the percentages of HA positive cells that have HA staining in the nucleus from two separate experiments. Error bars, SEM; *** *P

Techniques Used: Stable Transfection, Mutagenesis, Immunofluorescence, Expressing, Staining

39) Product Images from "Inhibition of histone deacetylase 1 ameliorates renal tubulointerstitial fibrosis via modulation of inflammation and extracellular matrix gene transcription in mice"

Article Title: Inhibition of histone deacetylase 1 ameliorates renal tubulointerstitial fibrosis via modulation of inflammation and extracellular matrix gene transcription in mice

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2017.3218

Effect of VPA on UUO-induced renal tubular injury and fibrosis. (A) Representative western blot analysis of H3Ac at lysine 9 and 14 from sham- and UUO-operated mice treated with Veh or VPA. Data from densitometric analyses are presented as the relative ratio of each protein to histone H3. The relative ratio measured in the kidneys from sham-operated mice treated with Veh is arbitrarily presented as 1. Data are expressed as the mean ± SD of 3 independent experiments.  * P
Figure Legend Snippet: Effect of VPA on UUO-induced renal tubular injury and fibrosis. (A) Representative western blot analysis of H3Ac at lysine 9 and 14 from sham- and UUO-operated mice treated with Veh or VPA. Data from densitometric analyses are presented as the relative ratio of each protein to histone H3. The relative ratio measured in the kidneys from sham-operated mice treated with Veh is arbitrarily presented as 1. Data are expressed as the mean ± SD of 3 independent experiments. * P

Techniques Used: Western Blot, Mouse Assay

40) Product Images from "Transcription Factor EB Activation Rescues Advanced αB‐Crystallin Mutation‐Induced Cardiomyopathy by Normalizing Desmin Localization"

Article Title: Transcription Factor EB Activation Rescues Advanced αB‐Crystallin Mutation‐Induced Cardiomyopathy by Normalizing Desmin Localization

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.118.010866

Intermittent fasting (IF) activates transcription factor EB ( TFEB) and restores autophagic flux in αB‐crystallin R120G mutant transgenic mice. A , Schematic depicting experimental intervention with IF in Myh6 ‐Cry ABR 120G transgenic mice. B , Survival curves in IF and ad‐lib (AL) fed Myh6 ‐Cry ABR 120G transgenic mice and AL fed controls over the 6‐week experimental duration. P value depicted is by log‐rank test. C and D , Representative immunoblots ( C ) demonstrating expression of TFEB with quantitation ( D ) in the cytosolic and nuclear fractions from hearts of mice treated as in A (at 46 weeks) and subjected to biochemical fractionation. Expression of GAPDH and histone H3 is used to detect enrichment of cytosolic and nuclear proteins, respectively. N=4/group. E , Assessment of autophagic flux in mice Myh6 ‐Cry ABR 120G transgenic mice subjected to IF or provided access to food AL as in A , and injected with chloroquine (40 mg/kg for 4 hours) or diluent with immunoblotting for LC 3 and p62. F , Quantitation of LC 3‐ II and p62 in cardiac tissue from mice treated as in E . N=4/group. G and H , Representative immunoblot ( G ) with quantitation ( H ) of lysosome proteins LAMP 1 and LAMP 2 in total cardiac protein extracts from Myh6 ‐Cry ABR 120G transgenic mice subjected to IF or AL feeding, or littermate wild type (WT) as in A . N=4/group. I and J , Representative immunoblots ( I ) demonstrating expression of mammalian target of rapamycin ( mTOR ) pathway proteins with quantitation ( J ) of phosphorylated mTOR (p‐ mTOR) , phosphorylated 70S6K (p‐S6K), and phosphorylated 4EBP1 (p‐4 EBP 1) in Myh6 –αB‐crystallin R120G transgenic mice subjected to IF or AL feeding, and WT controls as in A . N=4/group. qOD (every other day). * P value by post hoc test after 1‐way ANOVA.
Figure Legend Snippet: Intermittent fasting (IF) activates transcription factor EB ( TFEB) and restores autophagic flux in αB‐crystallin R120G mutant transgenic mice. A , Schematic depicting experimental intervention with IF in Myh6 ‐Cry ABR 120G transgenic mice. B , Survival curves in IF and ad‐lib (AL) fed Myh6 ‐Cry ABR 120G transgenic mice and AL fed controls over the 6‐week experimental duration. P value depicted is by log‐rank test. C and D , Representative immunoblots ( C ) demonstrating expression of TFEB with quantitation ( D ) in the cytosolic and nuclear fractions from hearts of mice treated as in A (at 46 weeks) and subjected to biochemical fractionation. Expression of GAPDH and histone H3 is used to detect enrichment of cytosolic and nuclear proteins, respectively. N=4/group. E , Assessment of autophagic flux in mice Myh6 ‐Cry ABR 120G transgenic mice subjected to IF or provided access to food AL as in A , and injected with chloroquine (40 mg/kg for 4 hours) or diluent with immunoblotting for LC 3 and p62. F , Quantitation of LC 3‐ II and p62 in cardiac tissue from mice treated as in E . N=4/group. G and H , Representative immunoblot ( G ) with quantitation ( H ) of lysosome proteins LAMP 1 and LAMP 2 in total cardiac protein extracts from Myh6 ‐Cry ABR 120G transgenic mice subjected to IF or AL feeding, or littermate wild type (WT) as in A . N=4/group. I and J , Representative immunoblots ( I ) demonstrating expression of mammalian target of rapamycin ( mTOR ) pathway proteins with quantitation ( J ) of phosphorylated mTOR (p‐ mTOR) , phosphorylated 70S6K (p‐S6K), and phosphorylated 4EBP1 (p‐4 EBP 1) in Myh6 –αB‐crystallin R120G transgenic mice subjected to IF or AL feeding, and WT controls as in A . N=4/group. qOD (every other day). * P value by post hoc test after 1‐way ANOVA.

Techniques Used: Mutagenesis, Transgenic Assay, Mouse Assay, Western Blot, Expressing, Quantitation Assay, Fractionation, Injection

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Western Blot:

Article Title: Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate
Article Snippet: .. For Western blot analysis, a monoclonal antibody specific to acetyl-histone H3 (Lys9) was obtained from Cell Signaling Technology (Beverly, MA, USA). .. For chromatin immunoprecipitation (ChIP) assay, a polyclonal antibody against acetyl-histone H3 was purchased from Millipore (Billerica, MA, USA).

Incubation:

Article Title: SIRT1 inhibits apoptosis of degenerative human disc nucleus pulposus cells through activation of Akt pathway
Article Snippet: .. The membranes were blocked in phosphate buffered saline (PBS) containing 0.05 % Tween-20 and 5 % bovine serum albumin (BSA) for 1 h, washed, and incubated with primary antibodies against SIRT1 (Abcam), COL2A1 (Santa Cruz biotechnology), aggrecan (Santa Cruz biotechnology), Akt (Cell Signaling Technology), phospho-Akt (Ser473; Cell Signaling Technology), active caspase-3 (Epitomics), Bcl-2 (Epitomics), acetyl-histone H3 (lys9; Cell Signaling Technology), and GAPDH (Santa Cruz biotechnology) in the same blocking buffer overnight at 4 °C. .. After the membranes were rinsed three times for 5 min in PBS-Tween-20 (PBS-T), they were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody.

Article Title: Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer
Article Snippet: .. Membranes were incubated with Vimentin (clone V9, 1 500, Dako, Carpinteria, CA, USA), BMI-1 (1 500, Millipore, Billerica, MA, USA), Acetyl-Histone H3 Lys9 (1 1500, Cell Signaling, Danvers, MA, USA) or Acetyl-Histone H4 Lys 5, 8, 12 and 16 (1 2000, EMD Millipore, Billerica, MA, USA) primary antibodies at 4°C overnight. .. Membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Sta.

Blocking Assay:

Article Title: SIRT1 inhibits apoptosis of degenerative human disc nucleus pulposus cells through activation of Akt pathway
Article Snippet: .. The membranes were blocked in phosphate buffered saline (PBS) containing 0.05 % Tween-20 and 5 % bovine serum albumin (BSA) for 1 h, washed, and incubated with primary antibodies against SIRT1 (Abcam), COL2A1 (Santa Cruz biotechnology), aggrecan (Santa Cruz biotechnology), Akt (Cell Signaling Technology), phospho-Akt (Ser473; Cell Signaling Technology), active caspase-3 (Epitomics), Bcl-2 (Epitomics), acetyl-histone H3 (lys9; Cell Signaling Technology), and GAPDH (Santa Cruz biotechnology) in the same blocking buffer overnight at 4 °C. .. After the membranes were rinsed three times for 5 min in PBS-Tween-20 (PBS-T), they were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody.

Immunoprecipitation:

Article Title: Spindle Assembly Checkpoint Protein Cdc20 Transcriptionally Activates Expression of Ubiquitin Carrier Protein UbcH10
Article Snippet: .. Immunoprecipitation was carried out with 10 μg each of antibodies specific for RNA pol II, Cdc20, CBP, p300, Cdc27, and Mad2 (Santa Cruz Biotechnology), acetyl-histone H3 (Lys-9/Lys-14) (Cell Signaling Technology), and normal IgG control (Sigma). .. PCR amplification of immunoprecipitated chromatin was done using primers indicated in the Table .

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    Interaction of NMNAT2 with <t>SIRT3</t> promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P
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    Expression changes of Wee1 inhibition signature in xenograft WiDr tumors and their correlation to classical PD marker, pCDC2 . Human WiDr colorectal cancer cells were injected subcutaneously into nude rats. After allowing tumors to establish for 8 days, gencitabine and Wee1 inhibitor were administered in the nude rats as described in the legend of Figure 3. At 8 hr post Wee1 inhibitor administration, mRNA from each WiDr xenograft tumor was applied to quantitative RT-PCR analysis. To correlate the <t>phosphorylated-CDC2</t> and Wee1 gene expression signature, the level of the pCDC2 normalized to total CDC2 is shown. The correlation coefficient (R) of pCDC2 expression and each mRNA expression is shown in each graph. Data represent mean ± SD. **, P
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    PARP-1 inhibitor induces site-specific phosphorylation of <t>H2AX</t> and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing <t>P-Ser139</t> H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.
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    Loss of <t>SIRT1</t> catalytic activity does not affect metastasis. A ) Mouse lungs displaying metastatic nodules (arrows) (H E, left, scale bar 5 mm, right, scale bar 100 μm) and metastatic nodules stained via immunohistochemistry for polyoma middle T antigen and ERα (scale bar 100 μm) B ) The number of individual metastatic nodules in whole lung H E sections assessed at endpoint. Eight 10µm sections spaced 50µm apart were evaluated in each mouse. N=10 mice per genotype. Points represent individual animals and bars represent the mean number of metastatic nodules. C ) The average number of individual metastatic nodules in whole lung H E sections assessed at endpoint correlated with overall survival time in days.
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    Interaction of NMNAT2 with SIRT3 promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    Interaction of NMNAT2 with SIRT3 increases NAD levels of A549. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in the A549 cells alone. Downregulation of SIRT3, decreased significantly (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 increases NAD levels of A549. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in the A549 cells alone. Downregulation of SIRT3, decreased significantly (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    Map of the NMNAT2 and SIRT3 interaction regions. (A) Mapping of SIRT3 interaction region in NMNAT2. (B) Co-immunoprecipitation of NMNAT2 and SIRT3. Map of the NMNAT2 SIRT3-interacting domains. Myc-SIRT3 and NMNAT2-Flag and its derivatives were overexpressed in A549 cells. NMNAT2-Flag protein was pulled down by Protein G Plus/Protein A Agarose Suspension beads. The presence of SIRT3 was detected by Myc immunoblotting.

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Map of the NMNAT2 and SIRT3 interaction regions. (A) Mapping of SIRT3 interaction region in NMNAT2. (B) Co-immunoprecipitation of NMNAT2 and SIRT3. Map of the NMNAT2 SIRT3-interacting domains. Myc-SIRT3 and NMNAT2-Flag and its derivatives were overexpressed in A549 cells. NMNAT2-Flag protein was pulled down by Protein G Plus/Protein A Agarose Suspension beads. The presence of SIRT3 was detected by Myc immunoblotting.

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Immunoprecipitation

    Interaction of NMNAT2 with SIRT3 promotes mitotic entry of A549. (A) Interaction of NMNAT2 with SIRT3 did not affect DNA synthesis. A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse-labeled with BrdU (50 μ M) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of the three experiments. (B) Interaction of NMNAT2 with SIRT3 promoted mitotic entry. Cell cycle progression of > 1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells. Values are means of three experiments. All data are presented as the mean ± SEM.

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 promotes mitotic entry of A549. (A) Interaction of NMNAT2 with SIRT3 did not affect DNA synthesis. A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse-labeled with BrdU (50 μ M) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of the three experiments. (B) Interaction of NMNAT2 with SIRT3 promoted mitotic entry. Cell cycle progression of > 1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells. Values are means of three experiments. All data are presented as the mean ± SEM.

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: DNA Synthesis, Transfection, Labeling, Blocking Assay, Immunostaining

    NMNAT2 interacts with SIRT3 in yeast, in mammalian cells and in vitro . (A) Identification of NMNAT2 as a SIRT3-interacting protein by the yeast two-hybrid assay. Yeast AH109 cells were transformed with different plasmids and grown on SD/-Trp-Leu-His-Ade. +, grown within 96 h; −, no growth within 96 h. Positive colonies were tested for β-galactosidase activity. +, turned blue within 2 h; −, did not turn blue within 2 h. (B) Interaction of NMNAT2 with SIRT3 in mammalian cells. A549 cells, cultured in regular medium, were transfected with expression plasmids as indicated. Immunoprecipitation (IP) and immunoblotting (IB) were performed using anti-FLAG or anti-Myc monoclonal antibody respectively. (C) To test NMNAT2 interacting with SIRT3 in vivo by Mammalian Two-Hybrid system. A549 cells were transfected with expression plasmids as indicated and cultured in regular medium. At 48 h after transfection, cells were evaluated by firely luciferase assays. Significance was determined on the mean of three different experiments. The luciferase levels of pACT-NMNAT2 and pBIND-SIRT3 were ≥2.64 times higher than negative control (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: NMNAT2 interacts with SIRT3 in yeast, in mammalian cells and in vitro . (A) Identification of NMNAT2 as a SIRT3-interacting protein by the yeast two-hybrid assay. Yeast AH109 cells were transformed with different plasmids and grown on SD/-Trp-Leu-His-Ade. +, grown within 96 h; −, no growth within 96 h. Positive colonies were tested for β-galactosidase activity. +, turned blue within 2 h; −, did not turn blue within 2 h. (B) Interaction of NMNAT2 with SIRT3 in mammalian cells. A549 cells, cultured in regular medium, were transfected with expression plasmids as indicated. Immunoprecipitation (IP) and immunoblotting (IB) were performed using anti-FLAG or anti-Myc monoclonal antibody respectively. (C) To test NMNAT2 interacting with SIRT3 in vivo by Mammalian Two-Hybrid system. A549 cells were transfected with expression plasmids as indicated and cultured in regular medium. At 48 h after transfection, cells were evaluated by firely luciferase assays. Significance was determined on the mean of three different experiments. The luciferase levels of pACT-NMNAT2 and pBIND-SIRT3 were ≥2.64 times higher than negative control (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: In Vitro, Y2H Assay, Transformation Assay, Activity Assay, Cell Culture, Transfection, Expressing, Immunoprecipitation, In Vivo, Luciferase, Negative Control

    Interaction of NMNAT2 with SIRT3 inhibited apoptosis of A549 cells. The result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than in A549 cells (P > 0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 inhibited apoptosis of A549 cells. The result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than in A549 cells (P > 0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    SIRT3 deacetylates NMNAT2 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag-NMNAT2 was incubated with PCAF and acetylation of protein was determined by western blotting with antiacetyllysine antibody. (B) Deacetylation of NMNAT2 by SIRT3 in vitro . Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. (C) Quantification of NMNAT2 deacetylation by SIRT3. (D) In vivo deacetylation of NMNAT2 by SIRT3. Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μ M for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by immunoblotting with antiacetyllysine antibody. (E) Quantification of NMNAT2 deacetylation by SIRT3 in vivo . Values are means of three experiments. All data are presented as the mean ± SEM.

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: SIRT3 deacetylates NMNAT2 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag-NMNAT2 was incubated with PCAF and acetylation of protein was determined by western blotting with antiacetyllysine antibody. (B) Deacetylation of NMNAT2 by SIRT3 in vitro . Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. (C) Quantification of NMNAT2 deacetylation by SIRT3. (D) In vivo deacetylation of NMNAT2 by SIRT3. Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μ M for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by immunoblotting with antiacetyllysine antibody. (E) Quantification of NMNAT2 deacetylation by SIRT3 in vivo . Values are means of three experiments. All data are presented as the mean ± SEM.

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: In Vitro, In Vivo, Incubation, Western Blot, Immunoprecipitation, Expressing

    Interaction of NMNAT2 with SIRT3 boosted strongly mitochondrial functions of A549. Intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compare to A549 cells (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 boosted strongly mitochondrial functions of A549. Intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compare to A549 cells (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    Expression changes of Wee1 inhibition signature in xenograft WiDr tumors and their correlation to classical PD marker, pCDC2 . Human WiDr colorectal cancer cells were injected subcutaneously into nude rats. After allowing tumors to establish for 8 days, gencitabine and Wee1 inhibitor were administered in the nude rats as described in the legend of Figure 3. At 8 hr post Wee1 inhibitor administration, mRNA from each WiDr xenograft tumor was applied to quantitative RT-PCR analysis. To correlate the phosphorylated-CDC2 and Wee1 gene expression signature, the level of the pCDC2 normalized to total CDC2 is shown. The correlation coefficient (R) of pCDC2 expression and each mRNA expression is shown in each graph. Data represent mean ± SD. **, P

    Journal: Molecular Cancer

    Article Title: Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor

    doi: 10.1186/1476-4598-8-34

    Figure Lengend Snippet: Expression changes of Wee1 inhibition signature in xenograft WiDr tumors and their correlation to classical PD marker, pCDC2 . Human WiDr colorectal cancer cells were injected subcutaneously into nude rats. After allowing tumors to establish for 8 days, gencitabine and Wee1 inhibitor were administered in the nude rats as described in the legend of Figure 3. At 8 hr post Wee1 inhibitor administration, mRNA from each WiDr xenograft tumor was applied to quantitative RT-PCR analysis. To correlate the phosphorylated-CDC2 and Wee1 gene expression signature, the level of the pCDC2 normalized to total CDC2 is shown. The correlation coefficient (R) of pCDC2 expression and each mRNA expression is shown in each graph. Data represent mean ± SD. **, P

    Article Snippet: CDC2 protein was solubilized by homogenizing cells in buffer containing 1% NP40, 0.1% Triton X-100, and was detected by Western blotting with an anti-p-CDC2Y15 specific antibody (Cell Signaling #9111).

    Techniques: Expressing, Inhibition, Marker, Injection, Quantitative RT-PCR

    PARP-1 inhibitor induces site-specific phosphorylation of H2AX and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing P-Ser139 H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.

    Journal: Biochemical Pharmacology

    Article Title: PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells

    doi: 10.1016/j.bcp.2012.07.024

    Figure Lengend Snippet: PARP-1 inhibitor induces site-specific phosphorylation of H2AX and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing P-Ser139 H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.

    Article Snippet: 2.3 Antibodies The following specific primary antibodies were used to detect the relevant proteins: mouse monoclonal anti-p53 antibody DO-1 and rabbit polyclonal anti-H2AX antibody (from BioLegend, San Diego, CA), anti-BRCA-1 rabbit polyclonal antibody (from Upstate Cell Signaling Solutions, Lake Placid, NY), anti-ER-α rabbit polyclonal antibody (from Sigma–Aldrich, St. Louis, MO), anti-phospho-histone H2AX (Ser139) rabbit polyclonal antibody (from Cell Signaling Technology Inc., Danvers, MA), anti-DBC-1 mouse monoclonal antibody (from Abcam plc, Cambridge, UK), and mouse monoclonal anti-actin antibody (clone C4, ICN Biochemicals, Aurora, OH).

    Techniques: Expressing

    Loss of SIRT1 catalytic activity does not affect metastasis. A ) Mouse lungs displaying metastatic nodules (arrows) (H E, left, scale bar 5 mm, right, scale bar 100 μm) and metastatic nodules stained via immunohistochemistry for polyoma middle T antigen and ERα (scale bar 100 μm) B ) The number of individual metastatic nodules in whole lung H E sections assessed at endpoint. Eight 10µm sections spaced 50µm apart were evaluated in each mouse. N=10 mice per genotype. Points represent individual animals and bars represent the mean number of metastatic nodules. C ) The average number of individual metastatic nodules in whole lung H E sections assessed at endpoint correlated with overall survival time in days.

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Loss of SIRT1 catalytic activity does not affect metastasis. A ) Mouse lungs displaying metastatic nodules (arrows) (H E, left, scale bar 5 mm, right, scale bar 100 μm) and metastatic nodules stained via immunohistochemistry for polyoma middle T antigen and ERα (scale bar 100 μm) B ) The number of individual metastatic nodules in whole lung H E sections assessed at endpoint. Eight 10µm sections spaced 50µm apart were evaluated in each mouse. N=10 mice per genotype. Points represent individual animals and bars represent the mean number of metastatic nodules. C ) The average number of individual metastatic nodules in whole lung H E sections assessed at endpoint correlated with overall survival time in days.

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Staining, Immunohistochemistry, Mouse Assay

    Abolition of SIRT1 enzymatic activity results in blunted ductal morphogenesis in the mammary gland. Right panel, representative photographs of whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age (scale bar, 5mm). Left panel, a higher magnification view of the ductal network in representative mammary gland whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age. 400X magnification (scale bar, 0.7 mm).

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Abolition of SIRT1 enzymatic activity results in blunted ductal morphogenesis in the mammary gland. Right panel, representative photographs of whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age (scale bar, 5mm). Left panel, a higher magnification view of the ductal network in representative mammary gland whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age. 400X magnification (scale bar, 0.7 mm).

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Mouse Assay

    Loss of SIRT1 catalytic activity is associated with increase tumor latency A) Kaplan Meir plot measuring the percentage of mice without any palpable mammary gland mass at the given age. N= 10 mice per genotype. There was a significant delay in the time at which the Sirt1 Y/Y developed their first detectable mass as compared to the Sirt1 +/+ and the Sirt1 Y/+ mice (P

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Loss of SIRT1 catalytic activity is associated with increase tumor latency A) Kaplan Meir plot measuring the percentage of mice without any palpable mammary gland mass at the given age. N= 10 mice per genotype. There was a significant delay in the time at which the Sirt1 Y/Y developed their first detectable mass as compared to the Sirt1 +/+ and the Sirt1 Y/+ mice (P

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Mouse Assay

    Expression of SIRT1, Middle T Antigen, and ERα protein in mammary tumors. Representative immunohistochemical staining for SIRT1 (A-C), Middle T Antigen (D-F), and ERα (G-I) in mammary tumors collected at humane endpoint from Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at 200X magnification (scale bars, 100 μm).

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Expression of SIRT1, Middle T Antigen, and ERα protein in mammary tumors. Representative immunohistochemical staining for SIRT1 (A-C), Middle T Antigen (D-F), and ERα (G-I) in mammary tumors collected at humane endpoint from Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at 200X magnification (scale bars, 100 μm).

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Expressing, Immunohistochemistry, Staining, Mouse Assay

    Loss of SIRT1 catalytic activity does not affect expression of the PyMT transgene. Representative immunohistochemical staining for Middle T Antigen in mammary glands of PyMT + / Sirt1 +/+ and PyMT + / Sirt1 Y/Y mice collected at 6 weeks of age (scale bars equal to 100 μm). Arrows indicate areas of mammary intraepithelial neoplasia.

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Loss of SIRT1 catalytic activity does not affect expression of the PyMT transgene. Representative immunohistochemical staining for Middle T Antigen in mammary glands of PyMT + / Sirt1 +/+ and PyMT + / Sirt1 Y/Y mice collected at 6 weeks of age (scale bars equal to 100 μm). Arrows indicate areas of mammary intraepithelial neoplasia.

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Expressing, Immunohistochemistry, Staining, Mouse Assay

    Abrogation of SIRT1 catalytic activity does not prevent mammary tumor formation in the MMTV-PyMT mouse model of breast cancer. A ) Kaplan Meir plot showing the percentage of surviving animals over time. N= 10 mice per genotype. Sirt1 Y/Y animals had a significantly longer overall survival time than the Sirt1 +/+ and the Sirt1 Y/+ mice (p

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Abrogation of SIRT1 catalytic activity does not prevent mammary tumor formation in the MMTV-PyMT mouse model of breast cancer. A ) Kaplan Meir plot showing the percentage of surviving animals over time. N= 10 mice per genotype. Sirt1 Y/Y animals had a significantly longer overall survival time than the Sirt1 +/+ and the Sirt1 Y/+ mice (p

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Mouse Assay