Structured Review

Merck KGaA histone h3
( A ) Two parallel cell replicates (denoted 1 and 2) were exposed to 6 × 2 Gy fractions of gamma radiation, 2 days per week, aiming to establish gamma radiation-exposed (RE) MDA-MB-231 cells. Analysis was performed at early (2 replicates) and late time points (from replicate 1, three independent experiments). ( B ) Cell growth was assayed and presented as number of doublings during the first 9 days, with the 2 Gy fractions indicated as arrows. The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at 24 h after 2 Gy or in total 6 Gy, i.e., in samples collected on day 2 and 9 ( C ) or in samples 4–6 weeks after 12 Gy ( D ). Trimethylated lysine 9 of <t>histone</t> H3 (H3K9me3) levels, normalised to GAPDH, were assayed using Western blot ( E ), and clonogenic survival was analysed in RE cells versus control cells in response to gamma and alpha radiation in samples 4–6 weeks after 12 Gy ( F ). * p
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Images

1) Product Images from "Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells"

Article Title: Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells

Journal: Cells

doi: 10.3390/cells9051165

( A ) Two parallel cell replicates (denoted 1 and 2) were exposed to 6 × 2 Gy fractions of gamma radiation, 2 days per week, aiming to establish gamma radiation-exposed (RE) MDA-MB-231 cells. Analysis was performed at early (2 replicates) and late time points (from replicate 1, three independent experiments). ( B ) Cell growth was assayed and presented as number of doublings during the first 9 days, with the 2 Gy fractions indicated as arrows. The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at 24 h after 2 Gy or in total 6 Gy, i.e., in samples collected on day 2 and 9 ( C ) or in samples 4–6 weeks after 12 Gy ( D ). Trimethylated lysine 9 of histone H3 (H3K9me3) levels, normalised to GAPDH, were assayed using Western blot ( E ), and clonogenic survival was analysed in RE cells versus control cells in response to gamma and alpha radiation in samples 4–6 weeks after 12 Gy ( F ). * p
Figure Legend Snippet: ( A ) Two parallel cell replicates (denoted 1 and 2) were exposed to 6 × 2 Gy fractions of gamma radiation, 2 days per week, aiming to establish gamma radiation-exposed (RE) MDA-MB-231 cells. Analysis was performed at early (2 replicates) and late time points (from replicate 1, three independent experiments). ( B ) Cell growth was assayed and presented as number of doublings during the first 9 days, with the 2 Gy fractions indicated as arrows. The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at 24 h after 2 Gy or in total 6 Gy, i.e., in samples collected on day 2 and 9 ( C ) or in samples 4–6 weeks after 12 Gy ( D ). Trimethylated lysine 9 of histone H3 (H3K9me3) levels, normalised to GAPDH, were assayed using Western blot ( E ), and clonogenic survival was analysed in RE cells versus control cells in response to gamma and alpha radiation in samples 4–6 weeks after 12 Gy ( F ). * p

Techniques Used: Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot

( A ) Levels of acetylated lysine 8 of histone H4 (H4K8ac) were analysed in MDA-MB-231 cells using Western blot after treatment with 0.25–1 µM trichostatin A (TSA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) Effects of TSA alone on clonogenic survival using 0.5 and 1 µM TSA relative to untreated control cells. * p
Figure Legend Snippet: ( A ) Levels of acetylated lysine 8 of histone H4 (H4K8ac) were analysed in MDA-MB-231 cells using Western blot after treatment with 0.25–1 µM trichostatin A (TSA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) Effects of TSA alone on clonogenic survival using 0.5 and 1 µM TSA relative to untreated control cells. * p

Techniques Used: Multiple Displacement Amplification, Western Blot

( A ) Basal levels of H3K9me3 and total protein levels of histone H3 were analysed in MDA-MB-231 and MCF7 cells using Western blot. GAPDH was used as a loading control. ( B ) γH2AX foci numbers are presented at 30 min and 24 h postexposure to 6 Gy of gamma or 2 Gy of alpha radiation in MDA-MB-231 and MCF7 cells. * p
Figure Legend Snippet: ( A ) Basal levels of H3K9me3 and total protein levels of histone H3 were analysed in MDA-MB-231 and MCF7 cells using Western blot. GAPDH was used as a loading control. ( B ) γH2AX foci numbers are presented at 30 min and 24 h postexposure to 6 Gy of gamma or 2 Gy of alpha radiation in MDA-MB-231 and MCF7 cells. * p

Techniques Used: Multiple Displacement Amplification, Western Blot

2) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

3) Product Images from "Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase"

Article Title: Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02858

ACLY inhibitors suppress IL-4-induced gene expression in ACLY knockout THP-1 cells. (A,B) Western blot analysis of ACLY protein expression (A) and growth curves (B) of control and ACLY knockout (ACLY KO) THP-1 cells. (C) Western blot analysis of histone H3 acetylation at K9, K23, K27, and K14 in control or ACLY KO THP-1 macrophages. (D,E) Q-PCR analysis of IL-4—induced mRNA expression of CCL13 and F13A1 in control or ACLY KO THP-1 macrophages. (F) Western blot analysis of STAT6 phosphorylation in control or ACLY KO THP-1 macrophages. (G,H) Q-PCR analysis of mRNA expression of CCL13 and F13A1 in ACLY KO THP-1 macrophages stimulated with IL-4 in the presence of BMS 303141, SB 204990, MEDICA16 or HC. * p
Figure Legend Snippet: ACLY inhibitors suppress IL-4-induced gene expression in ACLY knockout THP-1 cells. (A,B) Western blot analysis of ACLY protein expression (A) and growth curves (B) of control and ACLY knockout (ACLY KO) THP-1 cells. (C) Western blot analysis of histone H3 acetylation at K9, K23, K27, and K14 in control or ACLY KO THP-1 macrophages. (D,E) Q-PCR analysis of IL-4—induced mRNA expression of CCL13 and F13A1 in control or ACLY KO THP-1 macrophages. (F) Western blot analysis of STAT6 phosphorylation in control or ACLY KO THP-1 macrophages. (G,H) Q-PCR analysis of mRNA expression of CCL13 and F13A1 in ACLY KO THP-1 macrophages stimulated with IL-4 in the presence of BMS 303141, SB 204990, MEDICA16 or HC. * p

Techniques Used: Expressing, Knock-Out, Western Blot, Polymerase Chain Reaction

An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of histone H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p
Figure Legend Snippet: An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of histone H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p

Techniques Used: Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Activity Assay

Effects of ACLY inhibitors on acetyl-CoA levels, histone acetylation, and IL-4-induced STAT6 and STAT3 activation in human MDMs. (A) LC-MS analysis of acetyl-CoA levels in MDMs treated with 5 μM BMS 303141 and 10 μM SB 204990 for 24 h. (B) Western blot analysis of histone H3 acetylation at K14 and K27 in MDMs treated with indicated concentrations of BMS 303141 and SB 204990 for 24 h. (C–F) Q-PCR analysis of mRNA expression of ALOX15 and CCL17 in MDMs pre-treated with acetate, octanoate and TOFA for 1 h prior to treatments with SB 204490, hydroxycitrate, and IL-4 for 24 h. (G,H) Q-PCR analysis of mRNA expression of ALOX15 in MDMs pre-treated with indicated concentrations of BTA (G) or CTPi (H) for 1 h prior to treatment with IL-4 for 24 h. (I) Western blot analysis of STAT3 and STAT6 tyrosine phosphorylation in MDMs pre-treated with ACLY inhibitors 1 h prior to 0.5 h-treatment with IL-4. * p
Figure Legend Snippet: Effects of ACLY inhibitors on acetyl-CoA levels, histone acetylation, and IL-4-induced STAT6 and STAT3 activation in human MDMs. (A) LC-MS analysis of acetyl-CoA levels in MDMs treated with 5 μM BMS 303141 and 10 μM SB 204990 for 24 h. (B) Western blot analysis of histone H3 acetylation at K14 and K27 in MDMs treated with indicated concentrations of BMS 303141 and SB 204990 for 24 h. (C–F) Q-PCR analysis of mRNA expression of ALOX15 and CCL17 in MDMs pre-treated with acetate, octanoate and TOFA for 1 h prior to treatments with SB 204490, hydroxycitrate, and IL-4 for 24 h. (G,H) Q-PCR analysis of mRNA expression of ALOX15 in MDMs pre-treated with indicated concentrations of BTA (G) or CTPi (H) for 1 h prior to treatment with IL-4 for 24 h. (I) Western blot analysis of STAT3 and STAT6 tyrosine phosphorylation in MDMs pre-treated with ACLY inhibitors 1 h prior to 0.5 h-treatment with IL-4. * p

Techniques Used: Activation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Polymerase Chain Reaction, Expressing

IL-4 does not induce ACLY phosphorylation in human MDMs. (A–C) Western blot analysis of ACLY and Akt phosphorylation (A) , nuclear ACLY levels (B) , and histone H3 acetylation at K27 and K14 (C) in MDMs treated with 20 ng/mL IL-4 for indicated times. Data represent mean values ± SE of 3–4 independent experiments. * p
Figure Legend Snippet: IL-4 does not induce ACLY phosphorylation in human MDMs. (A–C) Western blot analysis of ACLY and Akt phosphorylation (A) , nuclear ACLY levels (B) , and histone H3 acetylation at K27 and K14 (C) in MDMs treated with 20 ng/mL IL-4 for indicated times. Data represent mean values ± SE of 3–4 independent experiments. * p

Techniques Used: Western Blot

4) Product Images from "Enhanced Solubility, Permeability and Anticancer Activity of Vorinostat Using Tailored Mesoporous Silica Nanoparticles"

Article Title: Enhanced Solubility, Permeability and Anticancer Activity of Vorinostat Using Tailored Mesoporous Silica Nanoparticles

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics10040283

HDAC inhibition and anti-tumour activity of vorinostat dissolved in media, DMSO or encapsulated within nanoparticles. ( a ) Western blot of acetylated histone H3 and total histone H3 following treatment of HCT116 CRC cells with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) for 1 h. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. ( b ) mRNA expression of ATF3 and JUN determined by qRT-PCR following 24 h treatment with escalating doses of vorinostat dissolved in DMSO or encapsulated within nanoparticles (MCM-41-NH 2 -VOR). ( c ) Apoptosis induction of HCT116 cells following 24 h treatment with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) was determined by propidium-iodide staining and FACS analysis. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. Values shown are mean ± SEM of a representative experiment performed in triplicate. *, p
Figure Legend Snippet: HDAC inhibition and anti-tumour activity of vorinostat dissolved in media, DMSO or encapsulated within nanoparticles. ( a ) Western blot of acetylated histone H3 and total histone H3 following treatment of HCT116 CRC cells with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) for 1 h. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. ( b ) mRNA expression of ATF3 and JUN determined by qRT-PCR following 24 h treatment with escalating doses of vorinostat dissolved in DMSO or encapsulated within nanoparticles (MCM-41-NH 2 -VOR). ( c ) Apoptosis induction of HCT116 cells following 24 h treatment with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) was determined by propidium-iodide staining and FACS analysis. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. Values shown are mean ± SEM of a representative experiment performed in triplicate. *, p

Techniques Used: Inhibition, Activity Assay, Western Blot, Expressing, Quantitative RT-PCR, Staining, FACS

5) Product Images from "Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells"

Article Title: Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0598-5

Knockdown of HDAC 1 and HDAC 2 results in reduced cell proliferation. a HDAC 1 and HDAC 2 expression was estimated by qRT-PCR in U-87 MG and LN18 cells after gene silencing using specific siRNAs. b Western blot analysis shows efficacy of HDAC 1 and HDAC 2 knockdown at protein level. c Western blot for acetylated histones H3 and H4 (H3Ac, H4Ac) in HDAC 1 and HDAC 2 depleted U-87MG and LN18 cells 48 h after siRNA transfection. d MTT metabolism test for cell viability 24, 48, and 72 h after transfection with HDAC 1 or/and HDAC 2 siRNAs or a control siRNA. e BrdU incorporation test for cell proliferation 48 h after knockdown of HDAC 1 or/and HDAC 2 in U-87MG and LN18 cells. The respective p values were calculated using type 2 two-tailed t test, and p
Figure Legend Snippet: Knockdown of HDAC 1 and HDAC 2 results in reduced cell proliferation. a HDAC 1 and HDAC 2 expression was estimated by qRT-PCR in U-87 MG and LN18 cells after gene silencing using specific siRNAs. b Western blot analysis shows efficacy of HDAC 1 and HDAC 2 knockdown at protein level. c Western blot for acetylated histones H3 and H4 (H3Ac, H4Ac) in HDAC 1 and HDAC 2 depleted U-87MG and LN18 cells 48 h after siRNA transfection. d MTT metabolism test for cell viability 24, 48, and 72 h after transfection with HDAC 1 or/and HDAC 2 siRNAs or a control siRNA. e BrdU incorporation test for cell proliferation 48 h after knockdown of HDAC 1 or/and HDAC 2 in U-87MG and LN18 cells. The respective p values were calculated using type 2 two-tailed t test, and p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, MTT Assay, BrdU Incorporation Assay, Two Tailed Test

6) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

7) Product Images from "SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis"

Article Title: SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

Journal: Leukemia

doi: 10.1038/leu.2017.183

SETD2 protein and H3K36Me3 deficiency in SM. ( a ) representative western blot results for SETD2 protein and H3K36Me3 levels in SM patients as compared to a pool of healthy donors (HDs). One of three independent experiments is shown. ( b and c ) Box and whiskers plots of SETD2 and H3K36Me3 level estimates obtained by densitometric analysis of western blots. Median, interquartile range, minimum, maximum and outliers are indicated. SETD2 and H3K36Me3 signal intensities in single blots obtained from three individual experiments were normalized to those of beta-actin and H3 histone, respectively, and averaged. Normalized SETD2 and H3K36Me3 levels calculated in SM patients were then expressed in comparison to normalized SETD2 and H3K36Me3 levels detected in a pool of HDs, conventionally set to 1. The asterisks indicate that MCL and ASM had significantly lower levels of SETD2 protein ( P
Figure Legend Snippet: SETD2 protein and H3K36Me3 deficiency in SM. ( a ) representative western blot results for SETD2 protein and H3K36Me3 levels in SM patients as compared to a pool of healthy donors (HDs). One of three independent experiments is shown. ( b and c ) Box and whiskers plots of SETD2 and H3K36Me3 level estimates obtained by densitometric analysis of western blots. Median, interquartile range, minimum, maximum and outliers are indicated. SETD2 and H3K36Me3 signal intensities in single blots obtained from three individual experiments were normalized to those of beta-actin and H3 histone, respectively, and averaged. Normalized SETD2 and H3K36Me3 levels calculated in SM patients were then expressed in comparison to normalized SETD2 and H3K36Me3 levels detected in a pool of HDs, conventionally set to 1. The asterisks indicate that MCL and ASM had significantly lower levels of SETD2 protein ( P

Techniques Used: Western Blot

SETD2 loss of function mutations in the index MCL case (MCL1). ( a ) Sanger sequencing chromatograms with the frameshift (top) and nonsense (bottom) mutations identified by whole-exome sequencing. ( b ) Localization of the mutations with respect to the key functional domains of the SETD2 protein. The SRI domain is necessary for histone H3 lysine 36 trimethylation (H3K36Me3) and mediates SETD2 interaction with the phosphorylated C-terminal domain of the RNA polymerase II large subunit (RNA pol II) and with the heterogeneous nuclear ribonucleoprotein-L (HnRNP), thus coupling H3K36Me3 with transcription elongation and splicing. ( c ) from top to bottom: western blotting (WB) showing the truncated SETD2 (tSETD2) protein as compared to full-length SETD2 detectable in a pool of proteins from mononuclear cells of healthy donors; co-immunoprecipitation experiments performed by using: an anti-RNA pol II, an anti-hnRNP and an anti-histone H3, respectively, to isolate the proteins of interest and then an anti-SETD2 as primary antibody to label the PVDF membrane on which the immunoprecipitates were transferred; WB for H3K36Me3. Histone H3 and actin were used as loading controls.
Figure Legend Snippet: SETD2 loss of function mutations in the index MCL case (MCL1). ( a ) Sanger sequencing chromatograms with the frameshift (top) and nonsense (bottom) mutations identified by whole-exome sequencing. ( b ) Localization of the mutations with respect to the key functional domains of the SETD2 protein. The SRI domain is necessary for histone H3 lysine 36 trimethylation (H3K36Me3) and mediates SETD2 interaction with the phosphorylated C-terminal domain of the RNA polymerase II large subunit (RNA pol II) and with the heterogeneous nuclear ribonucleoprotein-L (HnRNP), thus coupling H3K36Me3 with transcription elongation and splicing. ( c ) from top to bottom: western blotting (WB) showing the truncated SETD2 (tSETD2) protein as compared to full-length SETD2 detectable in a pool of proteins from mononuclear cells of healthy donors; co-immunoprecipitation experiments performed by using: an anti-RNA pol II, an anti-hnRNP and an anti-histone H3, respectively, to isolate the proteins of interest and then an anti-SETD2 as primary antibody to label the PVDF membrane on which the immunoprecipitates were transferred; WB for H3K36Me3. Histone H3 and actin were used as loading controls.

Techniques Used: Sequencing, Functional Assay, Western Blot, Immunoprecipitation

8) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

9) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

10) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

11) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

12) Product Images from "Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase"

Article Title: Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02858

An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of histone H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p
Figure Legend Snippet: An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of histone H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p

Techniques Used: Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Activity Assay

ACLY inhibitors suppress IL-4-induced gene expression in ACLY knockout THP-1 cells. (A,B) Western blot analysis of ACLY protein expression (A) and growth curves (B) of control and ACLY knockout (ACLY KO) THP-1 cells. (C) Western blot analysis of histone H3 acetylation at K9, K23, K27, and K14 in control or ACLY KO THP-1 macrophages. (D,E) Q-PCR analysis of IL-4—induced mRNA expression of CCL13 and F13A1 in control or ACLY KO THP-1 macrophages. (F) Western blot analysis of STAT6 phosphorylation in control or ACLY KO THP-1 macrophages. (G,H) Q-PCR analysis of mRNA expression of CCL13 and F13A1 in ACLY KO THP-1 macrophages stimulated with IL-4 in the presence of BMS 303141, SB 204990, MEDICA16 or HC. * p
Figure Legend Snippet: ACLY inhibitors suppress IL-4-induced gene expression in ACLY knockout THP-1 cells. (A,B) Western blot analysis of ACLY protein expression (A) and growth curves (B) of control and ACLY knockout (ACLY KO) THP-1 cells. (C) Western blot analysis of histone H3 acetylation at K9, K23, K27, and K14 in control or ACLY KO THP-1 macrophages. (D,E) Q-PCR analysis of IL-4—induced mRNA expression of CCL13 and F13A1 in control or ACLY KO THP-1 macrophages. (F) Western blot analysis of STAT6 phosphorylation in control or ACLY KO THP-1 macrophages. (G,H) Q-PCR analysis of mRNA expression of CCL13 and F13A1 in ACLY KO THP-1 macrophages stimulated with IL-4 in the presence of BMS 303141, SB 204990, MEDICA16 or HC. * p

Techniques Used: Expressing, Knock-Out, Western Blot, Polymerase Chain Reaction

Effects of ACLY inhibitors on acetyl-CoA levels, histone acetylation, and IL-4-induced STAT6 and STAT3 activation in human MDMs. (A) LC-MS analysis of acetyl-CoA levels in MDMs treated with 5 μM BMS 303141 and 10 μM SB 204990 for 24 h. (B) Western blot analysis of histone H3 acetylation at K14 and K27 in MDMs treated with indicated concentrations of BMS 303141 and SB 204990 for 24 h. (C–F) Q-PCR analysis of mRNA expression of ALOX15 and CCL17 in MDMs pre-treated with acetate, octanoate and TOFA for 1 h prior to treatments with SB 204490, hydroxycitrate, and IL-4 for 24 h. (G,H) Q-PCR analysis of mRNA expression of ALOX15 in MDMs pre-treated with indicated concentrations of BTA (G) or CTPi (H) for 1 h prior to treatment with IL-4 for 24 h. (I) Western blot analysis of STAT3 and STAT6 tyrosine phosphorylation in MDMs pre-treated with ACLY inhibitors 1 h prior to 0.5 h-treatment with IL-4. * p
Figure Legend Snippet: Effects of ACLY inhibitors on acetyl-CoA levels, histone acetylation, and IL-4-induced STAT6 and STAT3 activation in human MDMs. (A) LC-MS analysis of acetyl-CoA levels in MDMs treated with 5 μM BMS 303141 and 10 μM SB 204990 for 24 h. (B) Western blot analysis of histone H3 acetylation at K14 and K27 in MDMs treated with indicated concentrations of BMS 303141 and SB 204990 for 24 h. (C–F) Q-PCR analysis of mRNA expression of ALOX15 and CCL17 in MDMs pre-treated with acetate, octanoate and TOFA for 1 h prior to treatments with SB 204490, hydroxycitrate, and IL-4 for 24 h. (G,H) Q-PCR analysis of mRNA expression of ALOX15 in MDMs pre-treated with indicated concentrations of BTA (G) or CTPi (H) for 1 h prior to treatment with IL-4 for 24 h. (I) Western blot analysis of STAT3 and STAT6 tyrosine phosphorylation in MDMs pre-treated with ACLY inhibitors 1 h prior to 0.5 h-treatment with IL-4. * p

Techniques Used: Activation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Polymerase Chain Reaction, Expressing

IL-4 does not induce ACLY phosphorylation in human MDMs. (A–C) Western blot analysis of ACLY and Akt phosphorylation (A) , nuclear ACLY levels (B) , and histone H3 acetylation at K27 and K14 (C) in MDMs treated with 20 ng/mL IL-4 for indicated times. Data represent mean values ± SE of 3–4 independent experiments. * p
Figure Legend Snippet: IL-4 does not induce ACLY phosphorylation in human MDMs. (A–C) Western blot analysis of ACLY and Akt phosphorylation (A) , nuclear ACLY levels (B) , and histone H3 acetylation at K27 and K14 (C) in MDMs treated with 20 ng/mL IL-4 for indicated times. Data represent mean values ± SE of 3–4 independent experiments. * p

Techniques Used: Western Blot

13) Product Images from "RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure"

Article Title: RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117845

Depletion of RAD18 suppressed entry into the M phase from the G2 phase after IR exposure. (A) HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 1 or 2 Gy IR, and then lysed at the indicated time points, after irradiation. Samples were analyzed by western blotting with the indicated antibodies. (B) Cells were exposed to 1 Gy of IR, fixed with ethanol at the indicated time points after irradiation, and then immunostained with phospho-histone H3 and propidium iodide (PI). The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments. (C) Cells were exposed to various doses of IR and then fixed with ethanol 60 min after irradiation. The fixed cells were immunostained with phosphor-histone H3 and PI. The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments.
Figure Legend Snippet: Depletion of RAD18 suppressed entry into the M phase from the G2 phase after IR exposure. (A) HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 1 or 2 Gy IR, and then lysed at the indicated time points, after irradiation. Samples were analyzed by western blotting with the indicated antibodies. (B) Cells were exposed to 1 Gy of IR, fixed with ethanol at the indicated time points after irradiation, and then immunostained with phospho-histone H3 and propidium iodide (PI). The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments. (C) Cells were exposed to various doses of IR and then fixed with ethanol 60 min after irradiation. The fixed cells were immunostained with phosphor-histone H3 and PI. The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments.

Techniques Used: Transfection, Irradiation, Western Blot, Flow Cytometry, Cytometry, Standard Deviation

14) Product Images from "The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells"

Article Title: The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-015-1967-5

Panobinostat treatment leads to the deposition of activating histone marks which can be enhanced by erlotinib. Non-cytotoxic concentrations of PS induced acetylation of histone H3, which is further increased by combination with erlotinib in both adenocarcinoma cell lines HCC827 and A549. Also, the activating mono-, di- and tri-methylated H3K4 marks were at least additively induced after combination treatment in HCC827 and A549 compared to single agent treatment with PS or erlotinib. In NCI-H460, erlotinib had no additional effect on PS-induced histone acetylation and methylation
Figure Legend Snippet: Panobinostat treatment leads to the deposition of activating histone marks which can be enhanced by erlotinib. Non-cytotoxic concentrations of PS induced acetylation of histone H3, which is further increased by combination with erlotinib in both adenocarcinoma cell lines HCC827 and A549. Also, the activating mono-, di- and tri-methylated H3K4 marks were at least additively induced after combination treatment in HCC827 and A549 compared to single agent treatment with PS or erlotinib. In NCI-H460, erlotinib had no additional effect on PS-induced histone acetylation and methylation

Techniques Used: Methylation

15) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

16) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

17) Product Images from "The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells"

Article Title: The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-015-1967-5

Panobinostat treatment leads to the deposition of activating histone marks which can be enhanced by erlotinib. Non-cytotoxic concentrations of PS induced acetylation of histone H3, which is further increased by combination with erlotinib in both adenocarcinoma cell lines HCC827 and A549. Also, the activating mono-, di- and tri-methylated H3K4 marks were at least additively induced after combination treatment in HCC827 and A549 compared to single agent treatment with PS or erlotinib. In NCI-H460, erlotinib had no additional effect on PS-induced histone acetylation and methylation
Figure Legend Snippet: Panobinostat treatment leads to the deposition of activating histone marks which can be enhanced by erlotinib. Non-cytotoxic concentrations of PS induced acetylation of histone H3, which is further increased by combination with erlotinib in both adenocarcinoma cell lines HCC827 and A549. Also, the activating mono-, di- and tri-methylated H3K4 marks were at least additively induced after combination treatment in HCC827 and A549 compared to single agent treatment with PS or erlotinib. In NCI-H460, erlotinib had no additional effect on PS-induced histone acetylation and methylation

Techniques Used: Methylation

18) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

19) Product Images from "Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells"

Article Title: Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0598-5

Knockdown of HDAC 1 and HDAC 2 results in reduced cell proliferation. a HDAC 1 and HDAC 2 expression was estimated by qRT-PCR in U-87 MG and LN18 cells after gene silencing using specific siRNAs. b Western blot analysis shows efficacy of HDAC 1 and HDAC 2 knockdown at protein level. c Western blot for acetylated histones H3 and H4 (H3Ac, H4Ac) in HDAC 1 and HDAC 2 depleted U-87MG and LN18 cells 48 h after siRNA transfection. d MTT metabolism test for cell viability 24, 48, and 72 h after transfection with HDAC 1 or/and HDAC 2 siRNAs or a control siRNA. e BrdU incorporation test for cell proliferation 48 h after knockdown of HDAC 1 or/and HDAC 2 in U-87MG and LN18 cells. The respective p values were calculated using type 2 two-tailed t test, and p
Figure Legend Snippet: Knockdown of HDAC 1 and HDAC 2 results in reduced cell proliferation. a HDAC 1 and HDAC 2 expression was estimated by qRT-PCR in U-87 MG and LN18 cells after gene silencing using specific siRNAs. b Western blot analysis shows efficacy of HDAC 1 and HDAC 2 knockdown at protein level. c Western blot for acetylated histones H3 and H4 (H3Ac, H4Ac) in HDAC 1 and HDAC 2 depleted U-87MG and LN18 cells 48 h after siRNA transfection. d MTT metabolism test for cell viability 24, 48, and 72 h after transfection with HDAC 1 or/and HDAC 2 siRNAs or a control siRNA. e BrdU incorporation test for cell proliferation 48 h after knockdown of HDAC 1 or/and HDAC 2 in U-87MG and LN18 cells. The respective p values were calculated using type 2 two-tailed t test, and p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, MTT Assay, BrdU Incorporation Assay, Two Tailed Test

20) Product Images from "Deguelin, an Aurora B Kinase Inhibitor, Exhibits Potent Anti-Tumor Effect in Human Esophageal Squamous Cell Carcinoma"

Article Title: Deguelin, an Aurora B Kinase Inhibitor, Exhibits Potent Anti-Tumor Effect in Human Esophageal Squamous Cell Carcinoma

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2017.10.030

The effect of deguelin on Aurora A in vitro kinase activity. An inactive histone 3.3 protein was used as the substrate for an in vitro kinase assay with active Aurora A and 100 μM ATP as indicated. Proteins were resolved by SDS-PAGE and detected by Western blotting.
Figure Legend Snippet: The effect of deguelin on Aurora A in vitro kinase activity. An inactive histone 3.3 protein was used as the substrate for an in vitro kinase assay with active Aurora A and 100 μM ATP as indicated. Proteins were resolved by SDS-PAGE and detected by Western blotting.

Techniques Used: In Vitro, Activity Assay, Kinase Assay, SDS Page, Western Blot

Deguelin inhibits Aurora B kinase activity both in vitro and ex vivo . (a) Computational predicted binding mode of deguelin with Aurora B. Left, The ligand is shown in sticks with red oxygen atoms and cyan carbon atoms. Left, binding pocket. The kinase domain of Aurora B is shown as a rainbow colored cartoon. Right, binding mode. The pocket is colored in gray, of which the key residues are labeled and shown as sticks. Yellow dashed lines represent hydrogen bonds. The figure is generated by PyMOL. (b) Deguelin inhibits Aurora B in vitro kinase activity in a dose-dependent manner. An inactive histone 3.3 protein was used as the substrate for an in vitro kinase assay with active Aurora B and 100 μM ATP as indicated. Proteins were resolved by SDS-PAGE and detected by Western blotting. (c) KYSE150 (left) and Eca109 (right) cells were treated with deguelin (0, 1, 2 and 5 μM) for 24 h, whole cell extract and histone proteins were subjected to Western blot analysis. (d) KYSE150 (left) and Eca109 (right) cells were treated with 5 μM deguelin for various time points as indicated, and histone proteins were subjected to Western blot analysis.
Figure Legend Snippet: Deguelin inhibits Aurora B kinase activity both in vitro and ex vivo . (a) Computational predicted binding mode of deguelin with Aurora B. Left, The ligand is shown in sticks with red oxygen atoms and cyan carbon atoms. Left, binding pocket. The kinase domain of Aurora B is shown as a rainbow colored cartoon. Right, binding mode. The pocket is colored in gray, of which the key residues are labeled and shown as sticks. Yellow dashed lines represent hydrogen bonds. The figure is generated by PyMOL. (b) Deguelin inhibits Aurora B in vitro kinase activity in a dose-dependent manner. An inactive histone 3.3 protein was used as the substrate for an in vitro kinase assay with active Aurora B and 100 μM ATP as indicated. Proteins were resolved by SDS-PAGE and detected by Western blotting. (c) KYSE150 (left) and Eca109 (right) cells were treated with deguelin (0, 1, 2 and 5 μM) for 24 h, whole cell extract and histone proteins were subjected to Western blot analysis. (d) KYSE150 (left) and Eca109 (right) cells were treated with 5 μM deguelin for various time points as indicated, and histone proteins were subjected to Western blot analysis.

Techniques Used: Activity Assay, In Vitro, Ex Vivo, Binding Assay, Labeling, Generated, Kinase Assay, SDS Page, Western Blot

21) Product Images from "Induction of apoptosis by HBI‐8000 in adult T‐cell leukemia/lymphoma is associated with activation of Bim and NLRP3"

Article Title: Induction of apoptosis by HBI‐8000 in adult T‐cell leukemia/lymphoma is associated with activation of Bim and NLRP3

Journal: Cancer Science

doi: 10.1111/cas.12971

Analysis of acetylated histones, tumor suppressor genes, and antibody array in adult T‐cell leukemia/lymphoma ( ATL ) cell lines and primary ATL cells. (a–d) ATL cell lines (2–5 × 10 5 /mL), primary ATL cells, and normal CD 4 lymphocytes (1 × 10 6 /mL) were treated with either the vehicle or the indicated concentrations of HBI ‐8000 for 48 h, then collected. Whole‐cell lysates were prepared and Western blotting was carried out. In the membrane for CD 4 lymphocytes, KOB cells treated with HBI ‐8000 were used as positive control.
Figure Legend Snippet: Analysis of acetylated histones, tumor suppressor genes, and antibody array in adult T‐cell leukemia/lymphoma ( ATL ) cell lines and primary ATL cells. (a–d) ATL cell lines (2–5 × 10 5 /mL), primary ATL cells, and normal CD 4 lymphocytes (1 × 10 6 /mL) were treated with either the vehicle or the indicated concentrations of HBI ‐8000 for 48 h, then collected. Whole‐cell lysates were prepared and Western blotting was carried out. In the membrane for CD 4 lymphocytes, KOB cells treated with HBI ‐8000 were used as positive control.

Techniques Used: Ab Array, Western Blot, Positive Control

22) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

23) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

24) Product Images from "The Effect of Hydroxamic Siderophores Structure on Acetylation of Histone H3 and Alpha Tubulin in Pinus sylvestris Root Cells"

Article Title: The Effect of Hydroxamic Siderophores Structure on Acetylation of Histone H3 and Alpha Tubulin in Pinus sylvestris Root Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20236099

The effect of metabolites of pathogenic ( Fusarium oxysporum ) vs. mycorrhizal ( Hebeloma crustulinofrome ) fungi on histone acetylation in P. sylvestris root cells. Control roots were only treated with filter paper. ( a ) The proportion of acetylated histone H3 to non-acetylated histone H3. Bars and whiskers represent the mean ± SE, respectively. ( b ) Western-blot analysis of the effect of different siderophores on the proportion of acetylated histone H3 vs. non-acetylated histone H3 (the uncut gels can be found in the Supplementary ).
Figure Legend Snippet: The effect of metabolites of pathogenic ( Fusarium oxysporum ) vs. mycorrhizal ( Hebeloma crustulinofrome ) fungi on histone acetylation in P. sylvestris root cells. Control roots were only treated with filter paper. ( a ) The proportion of acetylated histone H3 to non-acetylated histone H3. Bars and whiskers represent the mean ± SE, respectively. ( b ) Western-blot analysis of the effect of different siderophores on the proportion of acetylated histone H3 vs. non-acetylated histone H3 (the uncut gels can be found in the Supplementary ).

Techniques Used: Western Blot

The effect of structurally different siderophores (ferrioxamine—FO, ferricrocin—FCR and triacetylfusarinine C—TAFC) chelated with iron and their iron free forms (desferrioxamine—DFO, desferricrocin—DES-FCR and destriacetylfusarinine C—DES-TAFC) on histone acetylation in P. sylvestris root cells. Control roots were treated with distilled water. ( a ) The proportion of acetylated histone H3 to non-acetylated histone H3. Bars and whiskers represent the mean ± SE, respectively. ( b ) Western-blot analysis of the effect of different siderophores on acetylated histone H3 and non-acetylated histone H3 (the uncut gels can be found in the Supplementary ).
Figure Legend Snippet: The effect of structurally different siderophores (ferrioxamine—FO, ferricrocin—FCR and triacetylfusarinine C—TAFC) chelated with iron and their iron free forms (desferrioxamine—DFO, desferricrocin—DES-FCR and destriacetylfusarinine C—DES-TAFC) on histone acetylation in P. sylvestris root cells. Control roots were treated with distilled water. ( a ) The proportion of acetylated histone H3 to non-acetylated histone H3. Bars and whiskers represent the mean ± SE, respectively. ( b ) Western-blot analysis of the effect of different siderophores on acetylated histone H3 and non-acetylated histone H3 (the uncut gels can be found in the Supplementary ).

Techniques Used: Western Blot

25) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbagrm.2018.03.007

Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

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

26) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

27) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

28) Product Images from "Enhanced Solubility, Permeability and Anticancer Activity of Vorinostat Using Tailored Mesoporous Silica Nanoparticles"

Article Title: Enhanced Solubility, Permeability and Anticancer Activity of Vorinostat Using Tailored Mesoporous Silica Nanoparticles

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics10040283

HDAC inhibition and anti-tumour activity of vorinostat dissolved in media, DMSO or encapsulated within nanoparticles. ( a ) Western blot of acetylated histone H3 and total histone H3 following treatment of HCT116 CRC cells with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) for 1 h. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. ( b ) mRNA expression of ATF3 and JUN determined by qRT-PCR following 24 h treatment with escalating doses of vorinostat dissolved in DMSO or encapsulated within nanoparticles (MCM-41-NH 2 -VOR). ( c ) Apoptosis induction of HCT116 cells following 24 h treatment with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) was determined by propidium-iodide staining and FACS analysis. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. Values shown are mean ± SEM of a representative experiment performed in triplicate. *, p
Figure Legend Snippet: HDAC inhibition and anti-tumour activity of vorinostat dissolved in media, DMSO or encapsulated within nanoparticles. ( a ) Western blot of acetylated histone H3 and total histone H3 following treatment of HCT116 CRC cells with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) for 1 h. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. ( b ) mRNA expression of ATF3 and JUN determined by qRT-PCR following 24 h treatment with escalating doses of vorinostat dissolved in DMSO or encapsulated within nanoparticles (MCM-41-NH 2 -VOR). ( c ) Apoptosis induction of HCT116 cells following 24 h treatment with escalating doses of vorinostat dissolved in media, DMSO, or encapsulated within nanoparticles (MCM-41-NH 2 -VOR) was determined by propidium-iodide staining and FACS analysis. Cells treated with corresponding amounts of empty nanoparticles (MCM-41-NH 2 ) served as controls. Values shown are mean ± SEM of a representative experiment performed in triplicate. *, p

Techniques Used: Inhibition, Activity Assay, Western Blot, Expressing, Quantitative RT-PCR, Staining, FACS

29) Product Images from "The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells"

Article Title: The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-015-1967-5

Panobinostat treatment leads to the deposition of activating histone marks which can be enhanced by erlotinib. Non-cytotoxic concentrations of PS induced acetylation of histone H3, which is further increased by combination with erlotinib in both adenocarcinoma cell lines HCC827 and A549. Also, the activating mono-, di- and tri-methylated H3K4 marks were at least additively induced after combination treatment in HCC827 and A549 compared to single agent treatment with PS or erlotinib. In NCI-H460, erlotinib had no additional effect on PS-induced histone acetylation and methylation
Figure Legend Snippet: Panobinostat treatment leads to the deposition of activating histone marks which can be enhanced by erlotinib. Non-cytotoxic concentrations of PS induced acetylation of histone H3, which is further increased by combination with erlotinib in both adenocarcinoma cell lines HCC827 and A549. Also, the activating mono-, di- and tri-methylated H3K4 marks were at least additively induced after combination treatment in HCC827 and A549 compared to single agent treatment with PS or erlotinib. In NCI-H460, erlotinib had no additional effect on PS-induced histone acetylation and methylation

Techniques Used: Methylation

30) Product Images from "Knockdown of lncRNA MIR31HG inhibits adipocyte differentiation of human adipose-derived stem cells via histone modification of FABP4"

Article Title: Knockdown of lncRNA MIR31HG inhibits adipocyte differentiation of human adipose-derived stem cells via histone modification of FABP4

Journal: Scientific Reports

doi: 10.1038/s41598-017-08131-6

Schematics showing the regulation of adipogenesis by MIR31HG . MIR31HG promoted adipogenic differentiation of stem cells. In mechanism, MIR31HG contributed to histone H3 lysine 4 trimethylation and H3 acetylation at the FABP4 gene locus and promoted its transcription, leading to the promotion of adipogenesis.
Figure Legend Snippet: Schematics showing the regulation of adipogenesis by MIR31HG . MIR31HG promoted adipogenic differentiation of stem cells. In mechanism, MIR31HG contributed to histone H3 lysine 4 trimethylation and H3 acetylation at the FABP4 gene locus and promoted its transcription, leading to the promotion of adipogenesis.

Techniques Used:

31) Product Images from "SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis"

Article Title: SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

Journal: Leukemia

doi: 10.1038/leu.2017.183

SETD2 protein and H3K36Me3 deficiency in SM. ( a ) representative western blot results for SETD2 protein and H3K36Me3 levels in SM patients as compared to a pool of healthy donors (HDs). One of three independent experiments is shown. ( b and c ) Box and whiskers plots of SETD2 and H3K36Me3 level estimates obtained by densitometric analysis of western blots. Median, interquartile range, minimum, maximum and outliers are indicated. SETD2 and H3K36Me3 signal intensities in single blots obtained from three individual experiments were normalized to those of beta-actin and H3 histone, respectively, and averaged. Normalized SETD2 and H3K36Me3 levels calculated in SM patients were then expressed in comparison to normalized SETD2 and H3K36Me3 levels detected in a pool of HDs, conventionally set to 1. The asterisks indicate that MCL and ASM had significantly lower levels of SETD2 protein ( P
Figure Legend Snippet: SETD2 protein and H3K36Me3 deficiency in SM. ( a ) representative western blot results for SETD2 protein and H3K36Me3 levels in SM patients as compared to a pool of healthy donors (HDs). One of three independent experiments is shown. ( b and c ) Box and whiskers plots of SETD2 and H3K36Me3 level estimates obtained by densitometric analysis of western blots. Median, interquartile range, minimum, maximum and outliers are indicated. SETD2 and H3K36Me3 signal intensities in single blots obtained from three individual experiments were normalized to those of beta-actin and H3 histone, respectively, and averaged. Normalized SETD2 and H3K36Me3 levels calculated in SM patients were then expressed in comparison to normalized SETD2 and H3K36Me3 levels detected in a pool of HDs, conventionally set to 1. The asterisks indicate that MCL and ASM had significantly lower levels of SETD2 protein ( P

Techniques Used: Western Blot

SETD2 loss of function mutations in the index MCL case (MCL1). ( a ) Sanger sequencing chromatograms with the frameshift (top) and nonsense (bottom) mutations identified by whole-exome sequencing. ( b ) Localization of the mutations with respect to the key functional domains of the SETD2 protein. The SRI domain is necessary for histone H3 lysine 36 trimethylation (H3K36Me3) and mediates SETD2 interaction with the phosphorylated C-terminal domain of the RNA polymerase II large subunit (RNA pol II) and with the heterogeneous nuclear ribonucleoprotein-L (HnRNP), thus coupling H3K36Me3 with transcription elongation and splicing. ( c ) from top to bottom: western blotting (WB) showing the truncated SETD2 (tSETD2) protein as compared to full-length SETD2 detectable in a pool of proteins from mononuclear cells of healthy donors; co-immunoprecipitation experiments performed by using: an anti-RNA pol II, an anti-hnRNP and an anti-histone H3, respectively, to isolate the proteins of interest and then an anti-SETD2 as primary antibody to label the PVDF membrane on which the immunoprecipitates were transferred; WB for H3K36Me3. Histone H3 and actin were used as loading controls.
Figure Legend Snippet: SETD2 loss of function mutations in the index MCL case (MCL1). ( a ) Sanger sequencing chromatograms with the frameshift (top) and nonsense (bottom) mutations identified by whole-exome sequencing. ( b ) Localization of the mutations with respect to the key functional domains of the SETD2 protein. The SRI domain is necessary for histone H3 lysine 36 trimethylation (H3K36Me3) and mediates SETD2 interaction with the phosphorylated C-terminal domain of the RNA polymerase II large subunit (RNA pol II) and with the heterogeneous nuclear ribonucleoprotein-L (HnRNP), thus coupling H3K36Me3 with transcription elongation and splicing. ( c ) from top to bottom: western blotting (WB) showing the truncated SETD2 (tSETD2) protein as compared to full-length SETD2 detectable in a pool of proteins from mononuclear cells of healthy donors; co-immunoprecipitation experiments performed by using: an anti-RNA pol II, an anti-hnRNP and an anti-histone H3, respectively, to isolate the proteins of interest and then an anti-SETD2 as primary antibody to label the PVDF membrane on which the immunoprecipitates were transferred; WB for H3K36Me3. Histone H3 and actin were used as loading controls.

Techniques Used: Sequencing, Functional Assay, Western Blot, Immunoprecipitation

32) Product Images from "Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex"

Article Title: Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157305

Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.
Figure Legend Snippet: Direct binding and phosphorylation dependent Aurora-C activation through conserved IN box domain of INCENP. (A) Schematic represents INCENP full-length and carboxyl terminal deletion mutants (C3-C8) [ 5 ]. The deduced regions of INCENP involved in Aurora-B and Aurora-C binding (aa 822–897) and activation (aa 822–892), as well as the domain serving as a substrate for phosphorylation (aa 893–919) are indicated. 3A indicates three alanine mutants of TSS motif (T893, S894, S895) for Aurora-B and Aurora-C phosphorylation sites. (B) 35 S-labeled in vitro translated Aurora-C was incubated with beads bound to either GST or GST-ΔINCENP deletions. Bound Aurora-C and GST-ΔINCENP deletions were visualized by autoradiography and CBB staining, respectively. Relative Aurora-C binding to GST-INCENP deletions was measured and quantified by ImageJ software. Arrowheads show GST fusion proteins. (C) GST-Aurora-C was incubated with GST-ΔINCENP deletions and histone H3 in vitro . Protein amount was determined by CBB stain and kinase activity was measured using histone H3 as substrate. [γ 32 P]-ATP incorporation in proteins was visualized by autoradiography. (D) GST-Aurora-C WT or KD mutants were incubated with histone H3, either WT or 3A mutant of GST-ΔINCENP C3 (left panel) or full length His-INCENP (right panel) in vitro , and analyzed as in C. (E) GST-Aurora-C or GST-Aurora-B was co-infected with His-INCENP and GST-Survivin in Sf-9 cells and proteins were isolated on glutathione sepharose. Proteins were detected by immunoblot with anti-INCENP antibody or CBB stain, and kinase activity was measured using histone H3 as substrate as in C. Absence or presence of each protein are indicated with (-) and (+) signs, respectively. (F) GST-Aurora-C WT, T198A or T202A mutants were incubated with full-length His-INCENP and histone H3 in vitro , and analyzed as in C. Experiments were repeated twice and representative images are shown in each figure.

Techniques Used: Binding Assay, Activation Assay, Labeling, In Vitro, Incubation, Autoradiography, Staining, Software, Activity Assay, Mutagenesis, Infection, Isolation

Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.
Figure Legend Snippet: Aurora-C phosphorylates Survivin on Serine 20. (A) Fragmentation of DHRIpSTFK on the TofTof. Inset summarizes ions found. A key ion is the b5, mainly observed as the -98 neutral-loss ion (loss of phosphoric acid), indicating it as the primary site of phosphorylation. Essentially complete sequence ion coverage was observed considering both “b” and “y” type ions. Detection of the Ile-specific d4 fragment ions provides confirmation that the overall sequence assignment is correct. (B) Sf-9 cells were co-infected with GST-Survivin WT or S20A mutant and GST-Aurora-C WT or KD (left panel) or GST-Aurora-B WT or KD (right panel). 48 h after infection, proteins were isolated on glutathione sepharose, co-precipitated GST-Aurora-C, GST-Aurora-B, and GST-Survivin were incubated with [γ 32 P]-ATP and histone H3 as substrate. The reactions were resolved by SDS-PAGE and visualized by autoradiography (top) or CBB stained (bottom). Relative [γ 32 P]-ATP incorporation to histone H3 shown was normalized by total histone H3 and quantified by ImageJ software. Experiments were repeated three times and showed reproducible results. (C) 293T cells were co-transfected with HA-INCENP, Flag-Aurora-C, and His-Survivin plasmids respectively. 24 h after transfection, cells were lysed and immunoprecipitated by anti-Flag antibody. Immunoprecipitates were immunoblotted with indicated antibodies. Arrow indicates slower mobility sift band of Survivin. (D) HeLa cells stably expressing GFP-Survivin WT, S20A, or S20E mutant were treated with 40 ng/ml nocodazole for 16 h. Cells were lysed and immunoblotted with indicated antibodies. (E) Analysis of cellular and mitotic defects in HeLa cell clones stably expressing GFP-Survivin WT, S20A, or S20E mutant. Representative images of GFP-Survivin S20A expressing cells with abnormal chromosome alignment, abnormal chromosome separation, and multinucleation are shown on the left. Green fluorescence shows distribution of Survivin and DNA is stained with DAPI (blue). The percentages of chromosome misalignment, chromosome missegregation, and multinucleated giant abnormal cells in the indicated transfected clones are shown in the bar graph on the right. At least 200 cells in each of the clones were analyzed for this assay. Data represent mean values ± SD from two clones (n = 2). Scale bar indicates 10 μm. Experiments were repeated twice with two biological replicates in each experiment. Figures show the representative images.

Techniques Used: Sequencing, Infection, Mutagenesis, Isolation, Incubation, SDS Page, Autoradiography, Staining, Software, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Clone Assay, Fluorescence

33) Product Images from "Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells"

Article Title: Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152591

The effect of nicotine on cell cycle arrest in human tubular epithelial cells. The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. * P
Figure Legend Snippet: The effect of nicotine on cell cycle arrest in human tubular epithelial cells. The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. * P

Techniques Used: Expressing

Related Articles

Immunoprecipitation:

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation
Article Snippet: .. Antibodies against acetylated histone H3 (06-599, Merck Millipore, Billerica, MA, USA), H3K27me3 (07-499, Merck Millipore), or normal rabbit IgG (12-370, Merck Millipore) were used for immunoprecipitation. .. 5 μl of purified immunoprecipitated-DNA was amplified using real-time PCR amplification with primers (0.5 μM) designed specifically for the COX -2 promoter region (set A and B, A) and the KAPA SYBR® FAST qPCR Kit (KK4602, Roche Diagnostics).

Incubation:

Article Title: Addition of a histone deacetylase inhibitor increases recombinant protein expression in Medicago truncatula cell cultures
Article Snippet: .. If no signal was detected, the membrane was washed three times for 5 minutes each with PBS-T and incubated with 1:25000 anti-histone H3 (07–690, Merck Millipore, USA) in PBS-T, at 4 °C, overnight, with gentle shaking. .. Following incubation for two hours in agitation at room temperature with 1:8000 anti-rabbit HRP conjugated, histone H3 was detected as already described for acetyl-histone H3.

Inhibition:

Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation
Article Snippet: .. The inhibition of histone acetylation at the COX - 2 promoter by SAHA is consistent with a previous study showing inhibition of histone H3 and H4 acetylation at the osteopontin gene promoter by another HDAC inhibitor trichostatin A (TSA) [ ]. ..

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    Merck KGaA monoclonal antibodies against γ h2ax
    The yield of <t>γ-H2AX</t> foci in Chinese hamster V79 cells exposed to γ-rays at high (400 mGy/min), medium (10 mGy/min) and low (1 mGy/min) dose rates.
    Monoclonal Antibodies Against γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA rabbit anti acetyl histone h3 antibodies
    The Nrf2 promoter is epigenetically repressed in cortical neurons. ( a ) ChIP analysis of acetylated <t>histone</t> H3 (Ac-H3) occupancy at the Nrf2 (left) and β-actin (right) promoters in neurons and astrocytes, normalized to input and expressed relative to each other. * P
    Rabbit Anti Acetyl Histone H3 Antibodies, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA trophozoite stage p falciparum 3d7 parasites
    Inhibition of deacetylase activity in P. falciparum nuclear extracts. The HDAC inhibitors vorinostat, belinostat, panobinostat, romidepsin and TSA were assessed for the inhibition of deacetylase activity in nuclear extracts prepared from trophozoite-stage P. falciparum <t>3D7</t> parasites using the Ac-RGK(Ac)-AMC fluorogenic peptide substrate. The mean percent inhibition (±standard error of the mean [SEM]) for three independent assays is shown.
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    Merck KGaA phosphorylated histone h3
    MEC proliferation and survival rates are not altered in Cdc42-overexpressing mammary glands. (A) Representative images of pHH3 immunostaining on tissue sections from line 4 and control mammary glands treated for 1 week with dox. Scale bar = 50 μm. Graph shows average percentage of pHH3-positive cells ± SEM in TEBs after 1 week and 3 weeks of dox treatment (n = 3,3;3,4, P = 0.43;0.77). (B) Representative images of CC3 immunostaining on tissue sections from line 4 and control mammary glands treated for 1 week with dox. Scale bar = 50 μm. Graph depicts average percentage of CC3-positive cells ± SEM in TEBs after 1 week and 3 weeks of dox treatment in vivo (n = 5,5;3,4 P = 0.53;0.59). (C) Histograms represent flow cytometry analysis of isolated primary control and Cdc42-OE MECs stained with propidium iodide (PI) after 1 week of dox treatment in vivo. Table shows the percentage of cells in each phase of the cell cycle out of total live cells (≥67,000 events) demonstrating that control and Cdc42 OE MECs have similar cell cycle profiles. Data are representative of two independent experiments (n = 5 mice per genotype). CC3, cleaved caspase-3; Cdc42, cell division cycle 42; MEC, mammary epithelial cell; OE, overexpressing; pHH3, phosphorylated <t>histone-H3;</t> TEB, terminal end bud.
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    Image Search Results


    The yield of γ-H2AX foci in Chinese hamster V79 cells exposed to γ-rays at high (400 mGy/min), medium (10 mGy/min) and low (1 mGy/min) dose rates.

    Journal: International Journal of Molecular Sciences

    Article Title: Changes in the Number of Double-Strand DNA Breaks in Chinese Hamster V79 Cells Exposed to ?-Radiation with Different Dose Rates

    doi: 10.3390/ijms140713719

    Figure Lengend Snippet: The yield of γ-H2AX foci in Chinese hamster V79 cells exposed to γ-rays at high (400 mGy/min), medium (10 mGy/min) and low (1 mGy/min) dose rates.

    Article Snippet: Slides were incubated with monoclonal antibodies against γ-H2AX (Anti-phospho-Histone H2AX Rabbit Monoclonal, Merck-Millipore, Darmstadt, Germany) at 4 °C over night.

    Techniques:

    The Nrf2 promoter is epigenetically repressed in cortical neurons. ( a ) ChIP analysis of acetylated histone H3 (Ac-H3) occupancy at the Nrf2 (left) and β-actin (right) promoters in neurons and astrocytes, normalized to input and expressed relative to each other. * P

    Journal: Nature Communications

    Article Title: Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2

    doi: 10.1038/ncomms8066

    Figure Lengend Snippet: The Nrf2 promoter is epigenetically repressed in cortical neurons. ( a ) ChIP analysis of acetylated histone H3 (Ac-H3) occupancy at the Nrf2 (left) and β-actin (right) promoters in neurons and astrocytes, normalized to input and expressed relative to each other. * P

    Article Snippet: For immunoprecipitation, a portion of the sheared chromatin sample was diluted tenfold with dilution buffer and then immunoprecipitated via 2 h incubation with Dynabeads Protein A/G pre-coupled to either rabbit anti-acetyl-Histone H3 antibodies (AcH3, 2.5 μg per sample, Merck Millipore) or normal IgG.

    Techniques: Chromatin Immunoprecipitation

    Inhibition of deacetylase activity in P. falciparum nuclear extracts. The HDAC inhibitors vorinostat, belinostat, panobinostat, romidepsin and TSA were assessed for the inhibition of deacetylase activity in nuclear extracts prepared from trophozoite-stage P. falciparum 3D7 parasites using the Ac-RGK(Ac)-AMC fluorogenic peptide substrate. The mean percent inhibition (±standard error of the mean [SEM]) for three independent assays is shown.

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    Article Title: Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites

    doi: 10.1016/j.ijpddr.2015.05.004

    Figure Lengend Snippet: Inhibition of deacetylase activity in P. falciparum nuclear extracts. The HDAC inhibitors vorinostat, belinostat, panobinostat, romidepsin and TSA were assessed for the inhibition of deacetylase activity in nuclear extracts prepared from trophozoite-stage P. falciparum 3D7 parasites using the Ac-RGK(Ac)-AMC fluorogenic peptide substrate. The mean percent inhibition (±standard error of the mean [SEM]) for three independent assays is shown.

    Article Snippet: 2.7 Deacetylase activity assays Nuclear extracts were prepared from trophozoite stage P. falciparum 3D7 parasites using a nuclear protein extraction kit (Merck Millipore), according to the manufacturer's instructions.

    Techniques: Inhibition, Histone Deacetylase Assay, Activity Assay

    Hyperacetylation of P. falciparum proteins by HDAC inhibitors. Trophozoite-stage P. falciparum 3D7 parasites were treated with 3× IC 50 of different HDAC inhibitors (vorinostat, panobinostat, belinostat or romidepsin) or controls (DMSO vehicle or chloroquine) for 3 h followed by preparation of protein lysates and Western blot analysis using rabbit antibodies specific to tetra-acetylated H4 (A) , histone H3 lysine 9 (B) , histone H3 lysine acetylated N-terminus (C) , or (pan) acetyl lysine (D) . Each membrane was stripped and re-probed with anti-RAP2 monoclonal antibody to allow loading on the same gel to be ascertained. Membranes were imaged using Odyssey Classic (Li-Cor Biosciences). Densitometry analysis was performed and each sample was normalised to the RAP2 loading control. Results are expressed as the fold change in relative density compared to the control, set to one.

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    Article Title: Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites

    doi: 10.1016/j.ijpddr.2015.05.004

    Figure Lengend Snippet: Hyperacetylation of P. falciparum proteins by HDAC inhibitors. Trophozoite-stage P. falciparum 3D7 parasites were treated with 3× IC 50 of different HDAC inhibitors (vorinostat, panobinostat, belinostat or romidepsin) or controls (DMSO vehicle or chloroquine) for 3 h followed by preparation of protein lysates and Western blot analysis using rabbit antibodies specific to tetra-acetylated H4 (A) , histone H3 lysine 9 (B) , histone H3 lysine acetylated N-terminus (C) , or (pan) acetyl lysine (D) . Each membrane was stripped and re-probed with anti-RAP2 monoclonal antibody to allow loading on the same gel to be ascertained. Membranes were imaged using Odyssey Classic (Li-Cor Biosciences). Densitometry analysis was performed and each sample was normalised to the RAP2 loading control. Results are expressed as the fold change in relative density compared to the control, set to one.

    Article Snippet: 2.7 Deacetylase activity assays Nuclear extracts were prepared from trophozoite stage P. falciparum 3D7 parasites using a nuclear protein extraction kit (Merck Millipore), according to the manufacturer's instructions.

    Techniques: Western Blot

    MEC proliferation and survival rates are not altered in Cdc42-overexpressing mammary glands. (A) Representative images of pHH3 immunostaining on tissue sections from line 4 and control mammary glands treated for 1 week with dox. Scale bar = 50 μm. Graph shows average percentage of pHH3-positive cells ± SEM in TEBs after 1 week and 3 weeks of dox treatment (n = 3,3;3,4, P = 0.43;0.77). (B) Representative images of CC3 immunostaining on tissue sections from line 4 and control mammary glands treated for 1 week with dox. Scale bar = 50 μm. Graph depicts average percentage of CC3-positive cells ± SEM in TEBs after 1 week and 3 weeks of dox treatment in vivo (n = 5,5;3,4 P = 0.53;0.59). (C) Histograms represent flow cytometry analysis of isolated primary control and Cdc42-OE MECs stained with propidium iodide (PI) after 1 week of dox treatment in vivo. Table shows the percentage of cells in each phase of the cell cycle out of total live cells (≥67,000 events) demonstrating that control and Cdc42 OE MECs have similar cell cycle profiles. Data are representative of two independent experiments (n = 5 mice per genotype). CC3, cleaved caspase-3; Cdc42, cell division cycle 42; MEC, mammary epithelial cell; OE, overexpressing; pHH3, phosphorylated histone-H3; TEB, terminal end bud.

    Journal: Breast Cancer Research : BCR

    Article Title: Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration

    doi: 10.1186/bcr3487

    Figure Lengend Snippet: MEC proliferation and survival rates are not altered in Cdc42-overexpressing mammary glands. (A) Representative images of pHH3 immunostaining on tissue sections from line 4 and control mammary glands treated for 1 week with dox. Scale bar = 50 μm. Graph shows average percentage of pHH3-positive cells ± SEM in TEBs after 1 week and 3 weeks of dox treatment (n = 3,3;3,4, P = 0.43;0.77). (B) Representative images of CC3 immunostaining on tissue sections from line 4 and control mammary glands treated for 1 week with dox. Scale bar = 50 μm. Graph depicts average percentage of CC3-positive cells ± SEM in TEBs after 1 week and 3 weeks of dox treatment in vivo (n = 5,5;3,4 P = 0.53;0.59). (C) Histograms represent flow cytometry analysis of isolated primary control and Cdc42-OE MECs stained with propidium iodide (PI) after 1 week of dox treatment in vivo. Table shows the percentage of cells in each phase of the cell cycle out of total live cells (≥67,000 events) demonstrating that control and Cdc42 OE MECs have similar cell cycle profiles. Data are representative of two independent experiments (n = 5 mice per genotype). CC3, cleaved caspase-3; Cdc42, cell division cycle 42; MEC, mammary epithelial cell; OE, overexpressing; pHH3, phosphorylated histone-H3; TEB, terminal end bud.

    Article Snippet: IHC/IF: Ki67 1:5000 (Abcam, Cambridge, UK, ab15580); BrdU 1:1000 (Thermo Fisher Scientific, MA3-071); CC3 1:1000 (Cell Signaling, 9661); phosphorylated histone-H3 1:5000 (Merck Millipore, Darmstadt, Germany, 06-570); F4/80 1:50, no antigen retrieval (Invitrogen, MF48000); phosphorylated ERM 1:1000 (Cell Signaling, 3141); E-cadherin 1:250 (BD Transduction, 610181); K14 1:400 (Covance, Leeds, UK, PRB-155P); K8 1:250 (Developmental Studies Hybridoma Bank, TROMA-I).

    Techniques: Immunostaining, In Vivo, Flow Cytometry, Cytometry, Isolation, Staining, Mouse Assay