p21 Cell Signaling Technology Inc Search Results


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  • 94
    Cell Signaling Technology Inc cdkn1a p21
    Negative effects of CXCR2 on romidepsin-induced <t>p21</t> protein expression via Akt-Mdm2 axis in a p53-independent manner ( A ) Dose-dependent effects of romidepsin on Akt, Mdm2 and p21 protein expression in p53 null SKOV-3 cells. Cells was treated with 0, 4, 8, 16, 32 and 64 nM romidepsin for 24 h. ( B ) Effects of silencing Akt1 and MDM2 on romidepsin-induced p21 protein expression in SKCXCR2 cells. ( C ) Effects of overexpressed Akt1 on romidepsin-induced p21 protein expression in SKOV-3 cells. β-actin was detected as an internal loading control of cell lysates. Cells was treated with 64 nM romidepsin for 24 h. ( D ) Schematic representation of molecular mechanism of CXCR2-mediated Akt-Mdm2 axis on cell cycle inhibitor p21 regulation in p53-dependent and independent manner in ovarian cancer cells. A representative result is shown from duplicated experiments.
    Cdkn1a P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21 waf1 cip1
    p-BQ-induced activation of MAPK, c-Myc and cell cycle deregulation . ( A ) Immunoblots showing expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc. ( B ) Quantitation of expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc after 8 and 24 weeks of p-BQ treatment. ( C ) Immunoblots showing protein expression of Cyclin D1, <t>p21</t> <t>waf1/Cip1</t> and hyperphosphorylated Retinoblastoma (pRb) at Ser 807/811. ( D ) Quantitation of protein expression of cell cycle regulatory proteins after 8 and 24 weeks of treatment. C, sham control; −vit C, vitamin C-restricted guinea pigs; + vit C, vitamin C-supplemented guinea pigs. Data represented as mean ± SD (n = 4). Significance is shown as *P ≤ 0.05, **P ≤ 0.005.
    P21 Waf1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti p21waf1 cip1 cdkn1a
    p-BQ-induced activation of MAPK, c-Myc and cell cycle deregulation . ( A ) Immunoblots showing expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc. ( B ) Quantitation of expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc after 8 and 24 weeks of p-BQ treatment. ( C ) Immunoblots showing protein expression of Cyclin D1, <t>p21</t> <t>waf1/Cip1</t> and hyperphosphorylated Retinoblastoma (pRb) at Ser 807/811. ( D ) Quantitation of protein expression of cell cycle regulatory proteins after 8 and 24 weeks of treatment. C, sham control; −vit C, vitamin C-restricted guinea pigs; + vit C, vitamin C-supplemented guinea pigs. Data represented as mean ± SD (n = 4). Significance is shown as *P ≤ 0.05, **P ≤ 0.005.
    Anti P21waf1 Cip1 Cdkn1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21 cdkn1a antibody
    CITED1 silencing restrains cell cycle progression and reduces cell viability. (A) A bar chart showing the % change in cell cycle distribution in #1 siCITED1 treated HT144 cells relative to siNEG treated HT144 cells. The reduction in the total S-phase is shown at 33 h, 48 h and 72 h post-transfection in addition to the corresponding increase in the diploid G1 fraction. (B) Western blots showing upregulation of <t>CDKN1A/P21</t> following CITED1 silencing in HT144 cells and suppression of CDKN1C/P57 following CITED1 overexpression in A2058 cells. (C) An Alamar Blue based metabolic assay shows a reduction in cell viability over 5 days in HT144 cells treated with siCITED1 relative to those treated with siNEG. Stars indicate significance for siNEG vs. #1 siCITED1 where ∗∗∗ p
    P21 Cdkn1a Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc signalsilence p21 waf1 cip1 sirna
    <t>P21</t> deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control <t>siRNA</t> or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi)
    Signalsilence P21 Waf1 Cip1 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21 waf1 cip1 12d1 1 1000
    <t>P21</t> deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control <t>siRNA</t> or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi)
    P21 Waf1 Cip1 12d1 1 1000, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc monoclonal p21 waf1 cip1
    Altered MITF, <t>p21</t> and E2F1 expression precedes G1 cell cycle arrest
    Monoclonal P21 Waf1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 76/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc human p21 waf1 cip1
    Altered MITF, <t>p21</t> and E2F1 expression precedes G1 cell cycle arrest
    Human P21 Waf1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit monoclonal anti p21
    Idasanutlin activates p53 and inhibits the growth of p53 wt cells. ( a – c ) Three p53 wt cell lines, SJSA-1, U-2 OS, and MCF-7, and one p53 del cell line, SAOS-2, were treated for 8 or 24 h with 0.05, 0.5, or 5 μM idasanutlin, or DMSO as a control. Western blot detection of p53, <t>p21,</t> and GAPDH was performed for the cells treated for 8 and 24 h. Cell cycle analysis was performed following 24 h of treatment with BrdU pulse-labeling for the last hour. The cells were fixed and stained with propidium iodide (PI) and FITC-anti-BrdU antibody (BrdU-FITC). The results presented on panels ( a – c ) are representative of at least three independent experiments. ( d ) SJSA-1, U-2 OS, MCF-7, and SAOS-2 cells were treated for five days with increasing concentrations of idasanutlin or an equivalent volume of DMSO, before assessment of cell viability. The data were analyzed with Dr-Fit software [ 26 ] and the best fitting model (red line) was chosen based on the Bayesian Information Criterion (BIC) ( Supplementary Table S1 ). EC 50 values of the growth inhibitory/stimulatory effects determined by the Dr-Fit software are indicated (see also Supplementary Table S1 ). Data points show means ± SD from three independent experiments.
    Rabbit Monoclonal Anti P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cyclin dependent kinase inhibitor cdkn1a p21
    Idasanutlin activates p53 and inhibits the growth of p53 wt cells. ( a – c ) Three p53 wt cell lines, SJSA-1, U-2 OS, and MCF-7, and one p53 del cell line, SAOS-2, were treated for 8 or 24 h with 0.05, 0.5, or 5 μM idasanutlin, or DMSO as a control. Western blot detection of p53, <t>p21,</t> and GAPDH was performed for the cells treated for 8 and 24 h. Cell cycle analysis was performed following 24 h of treatment with BrdU pulse-labeling for the last hour. The cells were fixed and stained with propidium iodide (PI) and FITC-anti-BrdU antibody (BrdU-FITC). The results presented on panels ( a – c ) are representative of at least three independent experiments. ( d ) SJSA-1, U-2 OS, MCF-7, and SAOS-2 cells were treated for five days with increasing concentrations of idasanutlin or an equivalent volume of DMSO, before assessment of cell viability. The data were analyzed with Dr-Fit software [ 26 ] and the best fitting model (red line) was chosen based on the Bayesian Information Criterion (BIC) ( Supplementary Table S1 ). EC 50 values of the growth inhibitory/stimulatory effects determined by the Dr-Fit software are indicated (see also Supplementary Table S1 ). Data points show means ± SD from three independent experiments.
    Cyclin Dependent Kinase Inhibitor Cdkn1a P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit monoclonal anti p21 waf1 cip1
    In lung tumor, miR-106b-25 cluster and MCM7 host gene are overexpressed while <t>p21</t> is downregulated compared to normal tissue. ( A ) Boxplots representing the relative abundance of mi-R25, 93, 106b and MCM7 transcript in tumoral compared to normal tissue in lung TCGA casuistry. N patients= 470 T, 56 N. (B) Pathways predicted to be affected by miR-106b-25 cluster. −10*log 10 (FDR) was used as a relevance score (see Supplementary Table 3, available at Carcinogenesis Online). ( C ) Boxplot comparing p21 transcript abundance in tumoral compared to normal tissue in lung TCGA casuistry.
    Rabbit Monoclonal Anti P21 Waf1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse monoclonal anti p21 cip1 waf1
    Synergistic effect of 5-fluorouracil (5-FU) and human interferon-beta (IFN-β) for treating MDA-MB-231 cells MDA-MB-231 cells were co-treated with 5-FU 1.0 μg/ml and IFN-β 500 Unit/ml for different lengths of time. Cell lysates were used for immunoblotting analysis and incubated with primary antibodies, BAX, <t>p21</t> <t>(Cip1/Waf1),</t> p53, and phospho-p38. ( A ) Expression of BAX protein. ( B ) The relative value of BAX protein. ( C ) Expression of p21 <t>(Cip1/Waf1)</t> protein. ( D ) The relative value of p21 (Cip1/Waf1) protein. ( E ) Expression of p53 and pp38 protein. ( F ) The relative value of p53 and pp38 protein. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (1.0 μg/ml), U: 5-FU treatment (1.0 μg/ml), B: IFN-β treatment (500 Unit/ml), U + B (5-FU 1.0 μg/ml and IFN-β 500 Unit/ml co-treatment). Data are presented as the mean ± SD of three different experiments each performed in triplicate. * p
    Mouse Monoclonal Anti P21 Cip1 Waf1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cdkn1a promoter
    Synergistic effect of 5-fluorouracil (5-FU) and human interferon-beta (IFN-β) for treating MDA-MB-231 cells MDA-MB-231 cells were co-treated with 5-FU 1.0 μg/ml and IFN-β 500 Unit/ml for different lengths of time. Cell lysates were used for immunoblotting analysis and incubated with primary antibodies, BAX, <t>p21</t> <t>(Cip1/Waf1),</t> p53, and phospho-p38. ( A ) Expression of BAX protein. ( B ) The relative value of BAX protein. ( C ) Expression of p21 <t>(Cip1/Waf1)</t> protein. ( D ) The relative value of p21 (Cip1/Waf1) protein. ( E ) Expression of p53 and pp38 protein. ( F ) The relative value of p53 and pp38 protein. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (1.0 μg/ml), U: 5-FU treatment (1.0 μg/ml), B: IFN-β treatment (500 Unit/ml), U + B (5-FU 1.0 μg/ml and IFN-β 500 Unit/ml co-treatment). Data are presented as the mean ± SD of three different experiments each performed in triplicate. * p
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    Cell Signaling Technology Inc cdkn1a
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
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    Cell Signaling Technology Inc rabbit anti p21 waf1 cip1
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
    Rabbit Anti P21 Waf1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc р21waf1 cip1 protein
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
    р21waf1 Cip1 Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc human senescent proteins p21 waf1 cip1
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
    Human Senescent Proteins P21 Waf1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21waf cip1
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
    P21waf Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti p21wif1 cip1
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
    Anti P21wif1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti human p21 waf cip1 12d1
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
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    Cell Signaling Technology Inc anti p21wap1 cip1
    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing <t>CDKN1A</t> expression.
    Anti P21wap1 Cip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21 sirna cells
    A-B. <t>p21</t> but not p27 is involved in TNF-α induced inhibition of proliferation . (A) LN-18 cells were transfected with p21 <t>siRNA</t> oligonucleotides (100 nM) or (B) p27 antisense (As) oligonucleotides (200 nM) for 6 hr and cultured as monolayers or spheroids for 18 hr. Cells were treated with TNF-α (10 ng/ml) for 24 hr, and tritiated thymidine incorporation assay was done as described in Fig. 1. * p
    P21 Sirna Cells, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21 2946
    A-B. <t>p21</t> but not p27 is involved in TNF-α induced inhibition of proliferation . (A) LN-18 cells were transfected with p21 <t>siRNA</t> oligonucleotides (100 nM) or (B) p27 antisense (As) oligonucleotides (200 nM) for 6 hr and cultured as monolayers or spheroids for 18 hr. Cells were treated with TNF-α (10 ng/ml) for 24 hr, and tritiated thymidine incorporation assay was done as described in Fig. 1. * p
    P21 2946, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti cdkn1a
    Validation of top gene hits in ESCC cell lines. A. Cell proliferation assay of ESCC cells overexpressed <t>CDKN1A,</t> ELAVL2 or TSPAN4 under the treatment of PTX and DDP. B. Annexin V staining of ESCC cells overexpressed CDKN1A, ELAVL2 or TSPAN4 under the treatment of PTX and DDP. C. Western blot analysis of CDKN1A, ELAVL2, TSPAN4, Bax, P53 and GAPDH in ESCC cells overexpressed CDKN1A or ELAVL2. * P
    Rabbit Anti Cdkn1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cdkn1a expression
    CDK inhibitors are upregulated in breast cancer cells following treatment with HDAC inhibitors (A) CDK inhibitor genes CDKN1C, <t>CDKN1A,</t> CDKN2B, and CDKN2D were significantly (p value
    Cdkn1a Expression, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p21 waf1 cell signal
    CDK inhibitors are upregulated in breast cancer cells following treatment with HDAC inhibitors (A) CDK inhibitor genes CDKN1C, <t>CDKN1A,</t> CDKN2B, and CDKN2D were significantly (p value
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    Image Search Results


    Negative effects of CXCR2 on romidepsin-induced p21 protein expression via Akt-Mdm2 axis in a p53-independent manner ( A ) Dose-dependent effects of romidepsin on Akt, Mdm2 and p21 protein expression in p53 null SKOV-3 cells. Cells was treated with 0, 4, 8, 16, 32 and 64 nM romidepsin for 24 h. ( B ) Effects of silencing Akt1 and MDM2 on romidepsin-induced p21 protein expression in SKCXCR2 cells. ( C ) Effects of overexpressed Akt1 on romidepsin-induced p21 protein expression in SKOV-3 cells. β-actin was detected as an internal loading control of cell lysates. Cells was treated with 64 nM romidepsin for 24 h. ( D ) Schematic representation of molecular mechanism of CXCR2-mediated Akt-Mdm2 axis on cell cycle inhibitor p21 regulation in p53-dependent and independent manner in ovarian cancer cells. A representative result is shown from duplicated experiments.

    Journal: Oncotarget

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    doi: 10.18632/oncotarget.24231

    Figure Lengend Snippet: Negative effects of CXCR2 on romidepsin-induced p21 protein expression via Akt-Mdm2 axis in a p53-independent manner ( A ) Dose-dependent effects of romidepsin on Akt, Mdm2 and p21 protein expression in p53 null SKOV-3 cells. Cells was treated with 0, 4, 8, 16, 32 and 64 nM romidepsin for 24 h. ( B ) Effects of silencing Akt1 and MDM2 on romidepsin-induced p21 protein expression in SKCXCR2 cells. ( C ) Effects of overexpressed Akt1 on romidepsin-induced p21 protein expression in SKOV-3 cells. β-actin was detected as an internal loading control of cell lysates. Cells was treated with 64 nM romidepsin for 24 h. ( D ) Schematic representation of molecular mechanism of CXCR2-mediated Akt-Mdm2 axis on cell cycle inhibitor p21 regulation in p53-dependent and independent manner in ovarian cancer cells. A representative result is shown from duplicated experiments.

    Article Snippet: Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Resistant effects of CXCR2 on the anti-tumor potential of romidepsin in p53-null cells ( A ) Inhibitory effects of CXCR2 on romidepsin-induced p21 protein in SKA and SKCXCR2cells. Cells were treated with romidepsin (0, 32 and 64 nM) for 24 h. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. ( B ) IC50 of romidepsin in SKA and SKCXCR2 cells. Cells were treated with 10–100 nM romidepsin for 48 h and then cell proliferation assay was performed. All data are shown as mean ± SE from triplicated experiments. * indicates a significant increase ( p ≤ 0.05) by Student’s t -test. ( C ) Clonogenic survival assay in SKA and SKCXCR2 after treatment of romidepsin (0, 32 and 64 nM) for 48 h. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. All data are shown as mean ± SE from triplicated experiments. Each SE is located within circles.

    Journal: Oncotarget

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    doi: 10.18632/oncotarget.24231

    Figure Lengend Snippet: Resistant effects of CXCR2 on the anti-tumor potential of romidepsin in p53-null cells ( A ) Inhibitory effects of CXCR2 on romidepsin-induced p21 protein in SKA and SKCXCR2cells. Cells were treated with romidepsin (0, 32 and 64 nM) for 24 h. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. ( B ) IC50 of romidepsin in SKA and SKCXCR2 cells. Cells were treated with 10–100 nM romidepsin for 48 h and then cell proliferation assay was performed. All data are shown as mean ± SE from triplicated experiments. * indicates a significant increase ( p ≤ 0.05) by Student’s t -test. ( C ) Clonogenic survival assay in SKA and SKCXCR2 after treatment of romidepsin (0, 32 and 64 nM) for 48 h. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. All data are shown as mean ± SE from triplicated experiments. Each SE is located within circles.

    Article Snippet: Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Proliferation Assay, Clonogenic Cell Survival Assay

    The p53-dependent effects of nutlin-3 on cell proliferation and p21 levels in p53WT, mutant and null cells ( A ) The p53-dependent effect of nutlin-3 on cell proliferation in CXCR2-negative vs. positive cells with different p53 status. All data are shown as mean ± SE from triplicated experiments. # and * indicate a significant decrease and increase ( p ≤ 0.05) by ANOVA and Student’s t -test, respectively. ( B ) Time-dependent effect of nutlin-3 on p53 and its downstream p21 expression in p53WT A2780 cells. ( C ) Comparative induction of nutlin-3 on p53 and p21 protein levels in p53WT cells (AA/ACXCR2), p53-mutant (OVA/OVCXCR2) and p53-null (SKA/SKCXCR2) cells. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments.

    Journal: Oncotarget

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    doi: 10.18632/oncotarget.24231

    Figure Lengend Snippet: The p53-dependent effects of nutlin-3 on cell proliferation and p21 levels in p53WT, mutant and null cells ( A ) The p53-dependent effect of nutlin-3 on cell proliferation in CXCR2-negative vs. positive cells with different p53 status. All data are shown as mean ± SE from triplicated experiments. # and * indicate a significant decrease and increase ( p ≤ 0.05) by ANOVA and Student’s t -test, respectively. ( B ) Time-dependent effect of nutlin-3 on p53 and its downstream p21 expression in p53WT A2780 cells. ( C ) Comparative induction of nutlin-3 on p53 and p21 protein levels in p53WT cells (AA/ACXCR2), p53-mutant (OVA/OVCXCR2) and p53-null (SKA/SKCXCR2) cells. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments.

    Article Snippet: Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Mutagenesis, Expressing

    Negative effects of CXCR2 on p21 promoter activity in a p53-dependent manner ( A ) Differential effects of CXCR2 on p21 promoter activity in p53WT A2780 cells, p53-mutant OVCAR-3 and p53-null SKOV-3 cells. ( B ) The p53-dependent inhibition of CXCR2 on p21 promoter activity in deleted constructs of p21 promoter in p53WT A2780 cells. All data are shown as mean ± SE from triplicated experiments. ( C ) Induced effects of CXCR2 on Mdm2 protein in p53-dependent p21 regulation. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. # indicates a significant increase ( p ≤ 0.05) in each pair by Student’s t -test.

    Journal: Oncotarget

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    doi: 10.18632/oncotarget.24231

    Figure Lengend Snippet: Negative effects of CXCR2 on p21 promoter activity in a p53-dependent manner ( A ) Differential effects of CXCR2 on p21 promoter activity in p53WT A2780 cells, p53-mutant OVCAR-3 and p53-null SKOV-3 cells. ( B ) The p53-dependent inhibition of CXCR2 on p21 promoter activity in deleted constructs of p21 promoter in p53WT A2780 cells. All data are shown as mean ± SE from triplicated experiments. ( C ) Induced effects of CXCR2 on Mdm2 protein in p53-dependent p21 regulation. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. # indicates a significant increase ( p ≤ 0.05) in each pair by Student’s t -test.

    Article Snippet: Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Activity Assay, Mutagenesis, Inhibition, Construct

    Inhibitory effects of CXCR2-derived Akt-Mdm2 axis on p53-dependent p21 protein expression in p53WT cells ( A ) Knockdown of Mdm2 increased p21 promoter activity and recovered the CXCR2-mediated downregulation of p21. ( B ) Confirmation of the knockdown of Mdm2 protein through western blot and the effect of knockdown of Mdm2 protein on p53-dependent p21 protein expression. ( C ) Comparison of Akt activation in AA and ACXCR2 cells by confocal imaging analysis. ( D ) Knockdown of Akt1 increased p21 luciferase activity and recovered the CXCR2-mediated downregulation of p21. ( E ) Confirmation of the knockdown of Akt1 through western blot and the effect of knockdown of Akt1 on Mdm2, p53 and p21 protein expression levels. ( F ) Overexpression of Akt1 decreased the p21 luciferase activity and further abated the CXCR2-mediated downregulation of p21. ( G ) Confirmation of the overexpression of Akt1 through western blotting and the effect of silencing Akt1 on Mdm2, p53 and p21 protein expression. β-actin was detected as an internal loading control of cell lysates. All data are shown as mean ± SE from triplicated experiments (A, D, F) and a representative result is shown from duplicated experiments (B, C, E, G). * and # indicate a significant increase and decrease ( p ≤ 0.05), respectively, by Student’s t -test.

    Journal: Oncotarget

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    doi: 10.18632/oncotarget.24231

    Figure Lengend Snippet: Inhibitory effects of CXCR2-derived Akt-Mdm2 axis on p53-dependent p21 protein expression in p53WT cells ( A ) Knockdown of Mdm2 increased p21 promoter activity and recovered the CXCR2-mediated downregulation of p21. ( B ) Confirmation of the knockdown of Mdm2 protein through western blot and the effect of knockdown of Mdm2 protein on p53-dependent p21 protein expression. ( C ) Comparison of Akt activation in AA and ACXCR2 cells by confocal imaging analysis. ( D ) Knockdown of Akt1 increased p21 luciferase activity and recovered the CXCR2-mediated downregulation of p21. ( E ) Confirmation of the knockdown of Akt1 through western blot and the effect of knockdown of Akt1 on Mdm2, p53 and p21 protein expression levels. ( F ) Overexpression of Akt1 decreased the p21 luciferase activity and further abated the CXCR2-mediated downregulation of p21. ( G ) Confirmation of the overexpression of Akt1 through western blotting and the effect of silencing Akt1 on Mdm2, p53 and p21 protein expression. β-actin was detected as an internal loading control of cell lysates. All data are shown as mean ± SE from triplicated experiments (A, D, F) and a representative result is shown from duplicated experiments (B, C, E, G). * and # indicate a significant increase and decrease ( p ≤ 0.05), respectively, by Student’s t -test.

    Article Snippet: Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Derivative Assay, Expressing, Activity Assay, Western Blot, Activation Assay, Imaging, Luciferase, Over Expression

    Effects of romidepsin on p21 promoter activity in p53-null SKOV-3 cells ( A ) Effects of CXCR2 on romidepsin-induced p21 promoter activity in SKOV-3 cells. ( B ) Dose-dependent effects of romidepsin on p21 luciferase activity in SKOV-3 cells. Experiments were performed in triplicate and all data are shown as mean ± S.E. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. ( C ) Effects of romidepsin on p21 promoter activity in deleted constructs of p21 promoter p53 response element in p53-null SKOV-3 cells. All data are shown as mean ± SE from triplicated experiments. * indicates a statistical significance ( p ≤ 0.05) by Student’s t -test.

    Journal: Oncotarget

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    doi: 10.18632/oncotarget.24231

    Figure Lengend Snippet: Effects of romidepsin on p21 promoter activity in p53-null SKOV-3 cells ( A ) Effects of CXCR2 on romidepsin-induced p21 promoter activity in SKOV-3 cells. ( B ) Dose-dependent effects of romidepsin on p21 luciferase activity in SKOV-3 cells. Experiments were performed in triplicate and all data are shown as mean ± S.E. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. ( C ) Effects of romidepsin on p21 promoter activity in deleted constructs of p21 promoter p53 response element in p53-null SKOV-3 cells. All data are shown as mean ± SE from triplicated experiments. * indicates a statistical significance ( p ≤ 0.05) by Student’s t -test.

    Article Snippet: Antibodies were purchased as follows: Akt, p21 (waf1/cip1), Akt isoform and Akt phosphorylated form (pAkt, Ser473) from Cell Signaling Technology (Beverly, MA, USA) and p53, Mdm2, CXCR2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Activity Assay, Luciferase, Construct

    Regulation of the CDKN1A/p21/WAF mRNA and protein by EVI1 , etoposide, and daunorubicin. A, B) qRT-PCR on RxNA from U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide (A) or 30 nM daunorubicin (B) for 48 h. CDKN1A levels were normalized to those of the housekeeping gene B2M using the ΔΔct method [48] , with untreated U937_vec cells as a calibrator. Shown are means+SEMs from 3 independent experiments. *, p

    Journal: PLoS ONE

    Article Title: EVI1 Inhibits Apoptosis Induced by Antileukemic Drugs via Upregulation of CDKN1A/p21/WAF in Human Myeloid Cells

    doi: 10.1371/journal.pone.0056308

    Figure Lengend Snippet: Regulation of the CDKN1A/p21/WAF mRNA and protein by EVI1 , etoposide, and daunorubicin. A, B) qRT-PCR on RxNA from U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide (A) or 30 nM daunorubicin (B) for 48 h. CDKN1A levels were normalized to those of the housekeeping gene B2M using the ΔΔct method [48] , with untreated U937_vec cells as a calibrator. Shown are means+SEMs from 3 independent experiments. *, p

    Article Snippet: Primary antibodies directed against EVI1 (rabbit mAb anti-EVI1 C50E12, Cell Signaling Technology, Danvers, MA, USA) and p21 (rabbit mAb anti-p21Waf1/Cip1 12D1, Cell Signaling Technology) were used at a 1∶1000 dilution; the antibody against the nuclear marker protein RCC1 (mouse mAb anti-RCC1 E-6, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a dilution of 1∶500; and the antibody against the housekeeping gene β-tubulin (mouse mAb anti-β-tubulin clone TUB 2.1, Sigma-Aldrich, Seelze, Germany) was used at a dilution of 1∶2.500.

    Techniques: Quantitative RT-PCR

    CDKN1A/p21/WAF overexpression partially mimics the chemotherapy resistance phenotype of EVI1 ove rexpression. A) Immunoblot analysis confirming overexpressi on of p21 in U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. β-Tubulin was used as loading control. Detection of endogenous p21 would require longer exposure times. B) Immunoblot analysis of cytoplasmatic (cyto) and nuclear (nu) extracts from U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. The same amount of protein was loaded in each lane (corresponding to up to twice as many cell equivalents for nuclear versus cytoplasmatic extracts). The cytoplasmatic protein β-tubulin and the nuclear protein RCC1 were used as loading controls. C) Cell cycle distribution of CDKN1A overexpressing and control cells. Shown are means+SEMs from 3 independent experiments. *, p

    Journal: PLoS ONE

    Article Title: EVI1 Inhibits Apoptosis Induced by Antileukemic Drugs via Upregulation of CDKN1A/p21/WAF in Human Myeloid Cells

    doi: 10.1371/journal.pone.0056308

    Figure Lengend Snippet: CDKN1A/p21/WAF overexpression partially mimics the chemotherapy resistance phenotype of EVI1 ove rexpression. A) Immunoblot analysis confirming overexpressi on of p21 in U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. β-Tubulin was used as loading control. Detection of endogenous p21 would require longer exposure times. B) Immunoblot analysis of cytoplasmatic (cyto) and nuclear (nu) extracts from U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. The same amount of protein was loaded in each lane (corresponding to up to twice as many cell equivalents for nuclear versus cytoplasmatic extracts). The cytoplasmatic protein β-tubulin and the nuclear protein RCC1 were used as loading controls. C) Cell cycle distribution of CDKN1A overexpressing and control cells. Shown are means+SEMs from 3 independent experiments. *, p

    Article Snippet: Primary antibodies directed against EVI1 (rabbit mAb anti-EVI1 C50E12, Cell Signaling Technology, Danvers, MA, USA) and p21 (rabbit mAb anti-p21Waf1/Cip1 12D1, Cell Signaling Technology) were used at a 1∶1000 dilution; the antibody against the nuclear marker protein RCC1 (mouse mAb anti-RCC1 E-6, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a dilution of 1∶500; and the antibody against the housekeeping gene β-tubulin (mouse mAb anti-β-tubulin clone TUB 2.1, Sigma-Aldrich, Seelze, Germany) was used at a dilution of 1∶2.500.

    Techniques: Over Expression, Infection

    CMTM5 inhibits tumour growth and metastasis through regulating PI3K-AKT signalling. a The related proteins of PI3K/Akt pathway, including AKT, pAKT, PI3K p85 subunit, p21, cyclinD1, cyclinE, Bcl2, Bax, Bad, cleaved caspase 3, MMP2 and MMP9 in CMTM5 overexpressed Huh7 cells were examined by western blotting. b The relative kinase activity of PI3K in CMTM5 overexpressed Huh7 cells were examined by PI3K kinase ELISA assay. c The proteins levels of AKT, pAKT and P85 in Huh7-CMTM5 cells after treatment of PI3K inhibitor LY294002 (10 μM). d – g Huh7-CMTM5 cells were treated with or without LY294002, Cell growth ( d ), cell apoptosis ( e ) and cell metastatic and invasion ( f , g ) were examined. h – j The expression of PI3K p85 and pAKT (Ser473) in 76 paired tumour and adjacent normal liver tissues were examined by IHC staining and scored from 0 to 9 as described in the method. The left plots ( h ) were representative results of IHC staining against indicated proteins. The right plots were the statistical analysis of the correlation of CMTM5 expression with p85 ( i ) and pAKT ( j ) in HCC tissues (Spearman correlation test). Data are shown as mean ± SD from three independent experiments. * P

    Journal: Cancer Cell International

    Article Title: CMTM5 is downregulated and suppresses tumour growth in hepatocellular carcinoma through regulating PI3K-AKT signalling

    doi: 10.1186/s12935-017-0485-8

    Figure Lengend Snippet: CMTM5 inhibits tumour growth and metastasis through regulating PI3K-AKT signalling. a The related proteins of PI3K/Akt pathway, including AKT, pAKT, PI3K p85 subunit, p21, cyclinD1, cyclinE, Bcl2, Bax, Bad, cleaved caspase 3, MMP2 and MMP9 in CMTM5 overexpressed Huh7 cells were examined by western blotting. b The relative kinase activity of PI3K in CMTM5 overexpressed Huh7 cells were examined by PI3K kinase ELISA assay. c The proteins levels of AKT, pAKT and P85 in Huh7-CMTM5 cells after treatment of PI3K inhibitor LY294002 (10 μM). d – g Huh7-CMTM5 cells were treated with or without LY294002, Cell growth ( d ), cell apoptosis ( e ) and cell metastatic and invasion ( f , g ) were examined. h – j The expression of PI3K p85 and pAKT (Ser473) in 76 paired tumour and adjacent normal liver tissues were examined by IHC staining and scored from 0 to 9 as described in the method. The left plots ( h ) were representative results of IHC staining against indicated proteins. The right plots were the statistical analysis of the correlation of CMTM5 expression with p85 ( i ) and pAKT ( j ) in HCC tissues (Spearman correlation test). Data are shown as mean ± SD from three independent experiments. * P

    Article Snippet: Membrane was blocked with 5% non-fat milk in TBS and then incubated with antibodies against CMTM5 (ab96077, Abcam), AKT (4691, CST), pAKT (4051, CST), PI3K (ab86714, Abcam), p21 (2946, CST), CyclinD1 (2978, CST), CyclinE (4129, CST), Bcl2 (ab117115, Abcam), Bax (AB32503, Abcam), Bad (ab32445, Abcam), cleaved caspase 3 (9661, CST), MMP2 (13132, CST), MMP9 (13667, CST) overnight at 4 °C.

    Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Staining

    miR-1260b regulated cell proliferation, cell cycle, apoptosis, and senescence. a Representative profile of EdU cell growth in SPCA1 cells and H1299 cells after transfection with Lv-miR-1260b and Sh-miR-1260b respectively compared with the control. b Effects of miR-1260b alteration on cell cycle distribution of cells. c FACS analysis of the effect of miR-1260b expression alteration on cell apoptosis. d SA-β-gal staining analysis of the effect of miR-1260b expression alteration on cell senescence. e Expression levels of Cyclin D1, Ki67, Bcl-2, Caspase-3, and p21 were detected by western blot. GAPDH was used as an internal control. All experiments are performed three times independently. The data expressed as the mean ± SD (* P

    Journal: Cell Death & Disease

    Article Title: miR-1260b, mediated by YY1, activates KIT signaling by targeting SOCS6 to regulate cell proliferation and apoptosis in NSCLC

    doi: 10.1038/s41419-019-1390-y

    Figure Lengend Snippet: miR-1260b regulated cell proliferation, cell cycle, apoptosis, and senescence. a Representative profile of EdU cell growth in SPCA1 cells and H1299 cells after transfection with Lv-miR-1260b and Sh-miR-1260b respectively compared with the control. b Effects of miR-1260b alteration on cell cycle distribution of cells. c FACS analysis of the effect of miR-1260b expression alteration on cell apoptosis. d SA-β-gal staining analysis of the effect of miR-1260b expression alteration on cell senescence. e Expression levels of Cyclin D1, Ki67, Bcl-2, Caspase-3, and p21 were detected by western blot. GAPDH was used as an internal control. All experiments are performed three times independently. The data expressed as the mean ± SD (* P

    Article Snippet: Primary antibodies used in Western blotting were Cyclin D1 (Cell Signaling Technology, 2978), Bcl-2 (Cell Signaling Technology, 4223), Ki67 (Abcam, ab92742), Caspase-3 (Cell Signaling Technology, 9665), p21 (Cell Signaling Technology, 8831), SOCS6 (Abcam, ab197335), KIT (Cell Signaling Technology, 3392), p38 (Cell Signaling Technology, 14451), phospho-p38 (Cell Signaling Technology, 4511), ERK (Cell Signaling Technology, 9102), phospho-ERK (Cell Signaling Technology, 8544), GAPDH (Cell Signaling Technology, 5174).

    Techniques: Transfection, FACS, Expressing, Staining, Western Blot

    p21 was specifically up-regulated in IDH1 R132H -expressing U87 cells. A: Quantitative reverse transcription-PCR analysis of p21, p53, and MDM2 mRNA levels in IDH1 wt - and IDH1 R132H -transfectedU87 cells. p21 mRNA was significantly increased in IDH1 R132H

    Journal: Neurologia Medico-Chirurgica

    Article Title: An R132H Mutation in Isocitrate Dehydrogenase 1 Enhances p21 Expression and Inhibits Phosphorylation of Retinoblastoma Protein in Glioma Cells

    doi: 10.2176/nmc.oa2012-0409

    Figure Lengend Snippet: p21 was specifically up-regulated in IDH1 R132H -expressing U87 cells. A: Quantitative reverse transcription-PCR analysis of p21, p53, and MDM2 mRNA levels in IDH1 wt - and IDH1 R132H -transfectedU87 cells. p21 mRNA was significantly increased in IDH1 R132H

    Article Snippet: ELISA kits were used for the measurement of p21 (#7167; Cell Signaling Technologies) and p53 protein (CY-7049; CycLex, Ina, Nagano).

    Techniques: Expressing, Polymerase Chain Reaction

    III. IDHR132H up-regulates p21 independent of p53

    Journal: Neurologia Medico-Chirurgica

    Article Title: An R132H Mutation in Isocitrate Dehydrogenase 1 Enhances p21 Expression and Inhibits Phosphorylation of Retinoblastoma Protein in Glioma Cells

    doi: 10.2176/nmc.oa2012-0409

    Figure Lengend Snippet: III. IDHR132H up-regulates p21 independent of p53

    Article Snippet: ELISA kits were used for the measurement of p21 (#7167; Cell Signaling Technologies) and p53 protein (CY-7049; CycLex, Ina, Nagano).

    Techniques:

    MP induces apoptosis and G2/M phase cell cycle arrest in SAS and SCC9 cells. Cells were treated with different concentration of MP (0, 2, 4 and 8 μM) for 24 h. ( A ) Morphological changes in cells including nuclei condensation and fragmentation were observed by DAPI staining under a fluorescence microscope. ( B ) Cell cycle was analysed by PI staining and flow cytometry. Sub-G1, G0/G1, S and G2/M indicate different cell cycle phases. ( C ) Apoptotic cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. ( D ) The left panel shows representative Western blots for the effect of MP on expression of cell cycle regulatory proteins (Cdc2, Cyclin A, Cyclin B1, p21 Cip1, and p27 Kip1). Bar graphs represent the relative density of each band normalized to GAPDH (right panel). ( E ) The left panel shows representative Western blots for the effect of MP on expression of apoptosis-related proteins (cleaved caspase-3, -8, -9 and cleaved PARP). Bar graphs represent the relative density of each band normalized to GAPDH (right panel). Values represent the mean ± SD of 3 independent experiments. * P

    Journal: Scientific Reports

    Article Title: Inhibition of cathepsin S confers sensitivity to methyl protodioscin in oral cancer cells via activation of p38 MAPK/JNK signaling pathways

    doi: 10.1038/srep45039

    Figure Lengend Snippet: MP induces apoptosis and G2/M phase cell cycle arrest in SAS and SCC9 cells. Cells were treated with different concentration of MP (0, 2, 4 and 8 μM) for 24 h. ( A ) Morphological changes in cells including nuclei condensation and fragmentation were observed by DAPI staining under a fluorescence microscope. ( B ) Cell cycle was analysed by PI staining and flow cytometry. Sub-G1, G0/G1, S and G2/M indicate different cell cycle phases. ( C ) Apoptotic cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. ( D ) The left panel shows representative Western blots for the effect of MP on expression of cell cycle regulatory proteins (Cdc2, Cyclin A, Cyclin B1, p21 Cip1, and p27 Kip1). Bar graphs represent the relative density of each band normalized to GAPDH (right panel). ( E ) The left panel shows representative Western blots for the effect of MP on expression of apoptosis-related proteins (cleaved caspase-3, -8, -9 and cleaved PARP). Bar graphs represent the relative density of each band normalized to GAPDH (right panel). Values represent the mean ± SD of 3 independent experiments. * P

    Article Snippet: Antibody against Cdc2, Cyclin A, Cyclin B1, p21 Cip1, p27 Kip1, cleaved caspase-3, -8, and -9, cleaved poly (ADP-ribose) polymerase (PARP), LC3B, p62, Beclin1, Cathepsin S, p-AKT, AKT, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Concentration Assay, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Western Blot, Expressing

    Palmitic acid induces cellular senescence and accumulation of lipid in hepatocytes. ( a ) HepG2 cells were cultured in the absence or presence of 0.1 mM palmitic acid, and the number of lipid droplets and the lipid area were quantified. Average lipid area was shown as a bar chart. ( b ) Relative expression of ACAA1, ACOX1, ACAA2, and HADHA in HepG2 cells cultured in the absence or presence of 0.1 mM palmitic acid. PPAR-dependent transcription ( c ) and transcriptional activation ability of PGC-1α ( d ) were assessed. ( e ) SMARCD1 relative gene expression in HepG2 cells in the absence or presence of 0.1 mM palmitic acid (PA). Activity and expression of senescence markers (F, SA-β-Gal; G, p16; H, p21; I, phosphor p38; J, γH2AX) were measured in HepG2 cells cultured in the absence or presence of 0.1 mM PA. Data were analyzed by two-sided Student’s t -test and are expressed as mean ± SD. P

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: SMARCD1 regulates senescence-associated lipid accumulation in hepatocytes

    doi: 10.1038/s41514-017-0011-1

    Figure Lengend Snippet: Palmitic acid induces cellular senescence and accumulation of lipid in hepatocytes. ( a ) HepG2 cells were cultured in the absence or presence of 0.1 mM palmitic acid, and the number of lipid droplets and the lipid area were quantified. Average lipid area was shown as a bar chart. ( b ) Relative expression of ACAA1, ACOX1, ACAA2, and HADHA in HepG2 cells cultured in the absence or presence of 0.1 mM palmitic acid. PPAR-dependent transcription ( c ) and transcriptional activation ability of PGC-1α ( d ) were assessed. ( e ) SMARCD1 relative gene expression in HepG2 cells in the absence or presence of 0.1 mM palmitic acid (PA). Activity and expression of senescence markers (F, SA-β-Gal; G, p16; H, p21; I, phosphor p38; J, γH2AX) were measured in HepG2 cells cultured in the absence or presence of 0.1 mM PA. Data were analyzed by two-sided Student’s t -test and are expressed as mean ± SD. P

    Article Snippet: Immunofluorescence Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with ice-cold methanol at −20 °C for 10 min. After washing the cells, cells were blocked with blocking solution (5% goat serum, 0.3% Triton-X 100) for 1 h. After removing the solution, cells were incubated with primary antibodies (anti-p16INK4a (ab81278; Abcam, Cambridge, MA, USA); ant-p21 (#2947; Cell Signaling, Beverly, MA, USA); anti-phospho-p38 MAPK (#4511; Cell Signaling); anti-phospho-histone H2A.X (#9718; Cell Signaling)) at 4 °C overnight.

    Techniques: Cell Culture, Expressing, Activation Assay, Pyrolysis Gas Chromatography, Activity Assay

    Lipid accumulation and senescence induction in the SMARCD1 -overexpressing HepG2 cells cultured in the absence or presence of 0.1 mM PA. ( a ) Relative SMARCD1 expression in SMARCD1-overexpressing HepG2 cells. PPAR-dependent transcription ( b ) and transcriptional activation ability of PGC-1α ( c ) were determined in SMARCD1-overexpressing HepG2 cells. ( d ) Relative expression of FAO genes in mock-transduced and SMARCD1 -overexpressing HepG2 cells in the absence or presence of PA. ( e ) The number of lipid droplets and lipid area in mock-transduced and SMARCD1 -overexpressing HepG2 cells in the absence or presence of PA. Activity and expression of senescence markers (F, SA-β-Gal; G, p16; H, p21; I, phosphorylated p38; J, γH2AX) were measured in mock-transduced and SMARCD1 -overexpressing HepG2 cells. Data are expressed as mean ± SD ( * P

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: SMARCD1 regulates senescence-associated lipid accumulation in hepatocytes

    doi: 10.1038/s41514-017-0011-1

    Figure Lengend Snippet: Lipid accumulation and senescence induction in the SMARCD1 -overexpressing HepG2 cells cultured in the absence or presence of 0.1 mM PA. ( a ) Relative SMARCD1 expression in SMARCD1-overexpressing HepG2 cells. PPAR-dependent transcription ( b ) and transcriptional activation ability of PGC-1α ( c ) were determined in SMARCD1-overexpressing HepG2 cells. ( d ) Relative expression of FAO genes in mock-transduced and SMARCD1 -overexpressing HepG2 cells in the absence or presence of PA. ( e ) The number of lipid droplets and lipid area in mock-transduced and SMARCD1 -overexpressing HepG2 cells in the absence or presence of PA. Activity and expression of senescence markers (F, SA-β-Gal; G, p16; H, p21; I, phosphorylated p38; J, γH2AX) were measured in mock-transduced and SMARCD1 -overexpressing HepG2 cells. Data are expressed as mean ± SD ( * P

    Article Snippet: Immunofluorescence Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with ice-cold methanol at −20 °C for 10 min. After washing the cells, cells were blocked with blocking solution (5% goat serum, 0.3% Triton-X 100) for 1 h. After removing the solution, cells were incubated with primary antibodies (anti-p16INK4a (ab81278; Abcam, Cambridge, MA, USA); ant-p21 (#2947; Cell Signaling, Beverly, MA, USA); anti-phospho-p38 MAPK (#4511; Cell Signaling); anti-phospho-histone H2A.X (#9718; Cell Signaling)) at 4 °C overnight.

    Techniques: Cell Culture, Expressing, Activation Assay, Pyrolysis Gas Chromatography, Activity Assay

    Smarcd1 roles in high-fat diet-induced hepatic lipid accumulation. ( a ) Senescence induction in scr-shRNA-transduced and Smarcd1-silenced normal mouse primary hepatocytes. Hematoxylin-eosin staining of liver sections obtained from 20-week-old mice fed with normal chow diet (NCD) ( b ) or high-fat diet (HFD) ( c ). Scale bar, 50 μm. Relative gene expression of Smarcd1 ( d ), p16 ( e ), p21 ( f ), and FAO genes ( g – j ) in livers obtained from NCD or HFD mice. Number indicates the individual mice. ( k ) Relative expression of SMARCD1 gene in peripheral blood mononuclear cells derived from young and old volunteers. Data are expressed as mean ± SD. P

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: SMARCD1 regulates senescence-associated lipid accumulation in hepatocytes

    doi: 10.1038/s41514-017-0011-1

    Figure Lengend Snippet: Smarcd1 roles in high-fat diet-induced hepatic lipid accumulation. ( a ) Senescence induction in scr-shRNA-transduced and Smarcd1-silenced normal mouse primary hepatocytes. Hematoxylin-eosin staining of liver sections obtained from 20-week-old mice fed with normal chow diet (NCD) ( b ) or high-fat diet (HFD) ( c ). Scale bar, 50 μm. Relative gene expression of Smarcd1 ( d ), p16 ( e ), p21 ( f ), and FAO genes ( g – j ) in livers obtained from NCD or HFD mice. Number indicates the individual mice. ( k ) Relative expression of SMARCD1 gene in peripheral blood mononuclear cells derived from young and old volunteers. Data are expressed as mean ± SD. P

    Article Snippet: Immunofluorescence Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with ice-cold methanol at −20 °C for 10 min. After washing the cells, cells were blocked with blocking solution (5% goat serum, 0.3% Triton-X 100) for 1 h. After removing the solution, cells were incubated with primary antibodies (anti-p16INK4a (ab81278; Abcam, Cambridge, MA, USA); ant-p21 (#2947; Cell Signaling, Beverly, MA, USA); anti-phospho-p38 MAPK (#4511; Cell Signaling); anti-phospho-histone H2A.X (#9718; Cell Signaling)) at 4 °C overnight.

    Techniques: shRNA, Staining, Mouse Assay, Expressing, Derivative Assay

    Combination of ceritinib with CGM097 leads to increased antitumor activity in TP53 wild-type neuroblastoma xenograft tumors harboring ALK aberrations. ( A ) The improved in vivo efficacy in the NB-1, SH-SY5Y and NB-1643 xenograft mouse models that harbor wild-type TP53 and ALK aberrations when ceritinib was combined with CGM097 and lack of antitumor activity of this combination in the IMR-32 xenograft tumors that harbor wild-type TP53 and ALK . The neuroblastoma cell lines NB-1, SH-SY5Y and IMR-32 were implanted into the flanks of nude mice and NB-1643 in SCID mice. Animals were randomized into four groups when the average tumor volume was 200–300 mm 3 and received vehicle, ceritinib (50 mg/kg), CGM097 (50 mg/kg) or both inhibitors in combination. Combination of ceritinib with CGM097 was withdrawn on day 60 and day 45 for NB-1 and SH-SY5Y, respectively, to allow tumor regrowth. Tumor dimensions and body weights were measured at the time of randomization and twice weekly thereafter for the study duration. Average tumor volume and SEM are shown as a function of time. ( B ) Inhibition of phospho-ALK, induction of p21 and increased levels of cleaved PARP in NB-1, SH-SY5Y and NB-1643 xenograft tissues treated with ceritinib in combination with CGM097. Animals were treated with vehicle, ceritinib (50 mg/kg), CGM097 (50 mg/kg) or both inhibitors in combination for 3 days. Tumor tissues were recovered 4 hr after the last dose treatment and analyzed by Western blotting. DOI: http://dx.doi.org/10.7554/eLife.17137.005 10.7554/eLife.17137.006 Details of human neuroblastoma cell line xenograft studies. DOI: http://dx.doi.org/10.7554/eLife.17137.006

    Journal: eLife

    Article Title: Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models

    doi: 10.7554/eLife.17137

    Figure Lengend Snippet: Combination of ceritinib with CGM097 leads to increased antitumor activity in TP53 wild-type neuroblastoma xenograft tumors harboring ALK aberrations. ( A ) The improved in vivo efficacy in the NB-1, SH-SY5Y and NB-1643 xenograft mouse models that harbor wild-type TP53 and ALK aberrations when ceritinib was combined with CGM097 and lack of antitumor activity of this combination in the IMR-32 xenograft tumors that harbor wild-type TP53 and ALK . The neuroblastoma cell lines NB-1, SH-SY5Y and IMR-32 were implanted into the flanks of nude mice and NB-1643 in SCID mice. Animals were randomized into four groups when the average tumor volume was 200–300 mm 3 and received vehicle, ceritinib (50 mg/kg), CGM097 (50 mg/kg) or both inhibitors in combination. Combination of ceritinib with CGM097 was withdrawn on day 60 and day 45 for NB-1 and SH-SY5Y, respectively, to allow tumor regrowth. Tumor dimensions and body weights were measured at the time of randomization and twice weekly thereafter for the study duration. Average tumor volume and SEM are shown as a function of time. ( B ) Inhibition of phospho-ALK, induction of p21 and increased levels of cleaved PARP in NB-1, SH-SY5Y and NB-1643 xenograft tissues treated with ceritinib in combination with CGM097. Animals were treated with vehicle, ceritinib (50 mg/kg), CGM097 (50 mg/kg) or both inhibitors in combination for 3 days. Tumor tissues were recovered 4 hr after the last dose treatment and analyzed by Western blotting. DOI: http://dx.doi.org/10.7554/eLife.17137.005 10.7554/eLife.17137.006 Details of human neuroblastoma cell line xenograft studies. DOI: http://dx.doi.org/10.7554/eLife.17137.006

    Article Snippet: Antibodies against phospho and total ALK, ERK, AKT, total p21, p27, PUMA, BIM and PARP were obtained from Cell Signaling, p53 from Santa Cruz Biotechnology, MYCN from Proteintech Group and MDM2 and GAPDH from EMD Millipore.

    Techniques: Activity Assay, In Vivo, Mouse Assay, Inhibition, Western Blot

    p-BQ-induced activation of MAPK, c-Myc and cell cycle deregulation . ( A ) Immunoblots showing expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc. ( B ) Quantitation of expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc after 8 and 24 weeks of p-BQ treatment. ( C ) Immunoblots showing protein expression of Cyclin D1, p21 waf1/Cip1 and hyperphosphorylated Retinoblastoma (pRb) at Ser 807/811. ( D ) Quantitation of protein expression of cell cycle regulatory proteins after 8 and 24 weeks of treatment. C, sham control; −vit C, vitamin C-restricted guinea pigs; + vit C, vitamin C-supplemented guinea pigs. Data represented as mean ± SD (n = 4). Significance is shown as *P ≤ 0.05, **P ≤ 0.005.

    Journal: Toxicology Reports

    Article Title: p-Benzoquinone initiates non-invasive urothelial cancer through aberrant tyrosine phosphorylation of EGFR, MAP kinase activation and cell cycle deregulation: Prevention by vitamin C

    doi: 10.1016/j.toxrep.2017.06.005

    Figure Lengend Snippet: p-BQ-induced activation of MAPK, c-Myc and cell cycle deregulation . ( A ) Immunoblots showing expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc. ( B ) Quantitation of expression of GTPase Hras, p44/p42 MAPK, phospho-p44/p42 MAPK, c-Myc and phospho(ser62)c-Myc after 8 and 24 weeks of p-BQ treatment. ( C ) Immunoblots showing protein expression of Cyclin D1, p21 waf1/Cip1 and hyperphosphorylated Retinoblastoma (pRb) at Ser 807/811. ( D ) Quantitation of protein expression of cell cycle regulatory proteins after 8 and 24 weeks of treatment. C, sham control; −vit C, vitamin C-restricted guinea pigs; + vit C, vitamin C-supplemented guinea pigs. Data represented as mean ± SD (n = 4). Significance is shown as *P ≤ 0.05, **P ≤ 0.005.

    Article Snippet: The membrane was blocked in 5% milk for 1 h and then incubated with primary antibody (1:1000 dilution) of p53, phospho p53, Bax, Bcl-2, caspase 3, cleaved caspase 3, PARP, p44/p42 MAPK, phospho p44/p42 MAPK, c-Myc, phospho (serine 62) c-Myc, p21 waf1/cip1 , cyclin D1, phospho Rb (807/811) (Cell Signalling Technologies), anti-GTPase Hras (Abcam), respectively, as needed.

    Techniques: Activation Assay, Western Blot, Expressing, Quantitation Assay

    Effect of Bcl-xL and Bcl-xL phosphorylation mutant expression on senescence-associated biomarkers in BJ cells. IF-revealed expression level of (A) p21Waf1/Cip1, (B) γH2A.X, and (C) Ki-67 in early versus late population doubling of control BJ cells and BJ cells expressing empty lentivirus vector or lentivirus vectors encoding Bcl-xL(wt) and various Bcl-xL phosphorylation mutants. Left panels, X axis: The BJ cell population is indicated with population doubling range (DP [range]) and the numbers of individual cells analysed and represented in the histographs ( n ). Data were collected from a multitude of independent micrographs. Right panels: Typical micrographs showing all cell populations at early (29 to 32) and late (50 to 55) population doubling. ( D ) Expression kinetics of Bcl-xL, HA-Bcl-xL (and phosphorylation mutants), p53 and p21Waf1/Cip1 in the control BJ cell population and BJ cells expressing empty lentivirus vector or lentivirus vectors encoding Bcl-xL(wt) and the various Bcl-xL phosphorylation mutants at early (29 to 32), middle (40 to 45) and late (50 to 55) population doublings, revealed by Western blotting; β-actin expression is shown as control. SDS-PAGE was run on 9–18% gradient gels.

    Journal: PLoS ONE

    Article Title: Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts

    doi: 10.1371/journal.pone.0159091

    Figure Lengend Snippet: Effect of Bcl-xL and Bcl-xL phosphorylation mutant expression on senescence-associated biomarkers in BJ cells. IF-revealed expression level of (A) p21Waf1/Cip1, (B) γH2A.X, and (C) Ki-67 in early versus late population doubling of control BJ cells and BJ cells expressing empty lentivirus vector or lentivirus vectors encoding Bcl-xL(wt) and various Bcl-xL phosphorylation mutants. Left panels, X axis: The BJ cell population is indicated with population doubling range (DP [range]) and the numbers of individual cells analysed and represented in the histographs ( n ). Data were collected from a multitude of independent micrographs. Right panels: Typical micrographs showing all cell populations at early (29 to 32) and late (50 to 55) population doubling. ( D ) Expression kinetics of Bcl-xL, HA-Bcl-xL (and phosphorylation mutants), p53 and p21Waf1/Cip1 in the control BJ cell population and BJ cells expressing empty lentivirus vector or lentivirus vectors encoding Bcl-xL(wt) and the various Bcl-xL phosphorylation mutants at early (29 to 32), middle (40 to 45) and late (50 to 55) population doublings, revealed by Western blotting; β-actin expression is shown as control. SDS-PAGE was run on 9–18% gradient gels.

    Article Snippet: The antibodies (Abs) in this study were Bcl-xL (54H6) rabbit monoclonal Ab (mAb), Ki-67(8D5) mouse mAb, p21Waf1/Cip1(12D1) rabbit mAb, p16/INK4A rabbit polyclonal Ab (pAb) and p53(1C12) mouse mAb obtained from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques: Mutagenesis, Expressing, Plasmid Preparation, Western Blot, SDS Page

    CITED1 silencing restrains cell cycle progression and reduces cell viability. (A) A bar chart showing the % change in cell cycle distribution in #1 siCITED1 treated HT144 cells relative to siNEG treated HT144 cells. The reduction in the total S-phase is shown at 33 h, 48 h and 72 h post-transfection in addition to the corresponding increase in the diploid G1 fraction. (B) Western blots showing upregulation of CDKN1A/P21 following CITED1 silencing in HT144 cells and suppression of CDKN1C/P57 following CITED1 overexpression in A2058 cells. (C) An Alamar Blue based metabolic assay shows a reduction in cell viability over 5 days in HT144 cells treated with siCITED1 relative to those treated with siNEG. Stars indicate significance for siNEG vs. #1 siCITED1 where ∗∗∗ p

    Journal: PeerJ

    Article Title: Loss of CITED1, an MITF regulator, drives a phenotype switch in vitro and can predict clinical outcome in primary melanoma tumours

    doi: 10.7717/peerj.788

    Figure Lengend Snippet: CITED1 silencing restrains cell cycle progression and reduces cell viability. (A) A bar chart showing the % change in cell cycle distribution in #1 siCITED1 treated HT144 cells relative to siNEG treated HT144 cells. The reduction in the total S-phase is shown at 33 h, 48 h and 72 h post-transfection in addition to the corresponding increase in the diploid G1 fraction. (B) Western blots showing upregulation of CDKN1A/P21 following CITED1 silencing in HT144 cells and suppression of CDKN1C/P57 following CITED1 overexpression in A2058 cells. (C) An Alamar Blue based metabolic assay shows a reduction in cell viability over 5 days in HT144 cells treated with siCITED1 relative to those treated with siNEG. Stars indicate significance for siNEG vs. #1 siCITED1 where ∗∗∗ p

    Article Snippet: Antibodies and immunoblotting The following antibodies were used: anti-CITED1, #AB15096 from Abcam; anti-MITF (C5 clone), # MA5-14146 from ThermoScientific; anti-MITF (D5 clone) from Dako, #M3621, (used in ); anti-CDKN1A/P21, #2947 and anti-CDKN1C/P57, #2557 were purchased from CellSignaling Technology and anti- β -Actin (AC-15), #A5441 from Sigma-Aldrich.

    Techniques: Transfection, Western Blot, Over Expression, Metabolic Assay

    Effect of β-sitosterol (ST) on G0/G1 phase cell cycle regulators and mitogenic and survival signaling in breast cancer cells. MDA-MB-231 cells were treated with either DMSO control or various doses of β-Sitosterol (60 and 90 μM) for 48 h. At the end of these treatments, cell lysate was prepared and western blot analysis was performed. Membranes were probed with (A) anti-cyclin D1, CDK-4, p21/Cip1, p27/Kip1, and (B) anti-p-Erk1/2, Erk1/2, p-Akt and Akt antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system. Membranes were striped and re-probed with anti-β actin for loading control.

    Journal: BMC Complementary and Alternative Medicine

    Article Title: ?-sitosterol induces G1 arrest and causes depolarization of mitochondrial membrane potential in breast carcinoma MDA-MB-231 cells

    doi: 10.1186/1472-6882-13-280

    Figure Lengend Snippet: Effect of β-sitosterol (ST) on G0/G1 phase cell cycle regulators and mitogenic and survival signaling in breast cancer cells. MDA-MB-231 cells were treated with either DMSO control or various doses of β-Sitosterol (60 and 90 μM) for 48 h. At the end of these treatments, cell lysate was prepared and western blot analysis was performed. Membranes were probed with (A) anti-cyclin D1, CDK-4, p21/Cip1, p27/Kip1, and (B) anti-p-Erk1/2, Erk1/2, p-Akt and Akt antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system. Membranes were striped and re-probed with anti-β actin for loading control.

    Article Snippet: Anti-CDK4, cyclin D1 antibodies were from Santa Cruz Biotechnology (CA, USA); anti-beta-actin antibody was from Sigma; and anti-p21/Cip1, p27/Kip1, phospho and total Erk1/2 and Akt, and HRP-conjugated secondary antibodies were from Cell Signaling Technology (MA, USA).

    Techniques: Multiple Displacement Amplification, Western Blot

    Binding of hPer2 to hp53 prevents hp21 from being expressed. H1299 cells were transfected with pCS2+FLAG-hp53, -hp53(ch)GST, -NLS-hp53(ch)GST, -hp53(ch)hPer2(356-574/683-872) (labeled #), or -NLS-hp53(ch)hPer2(356-574/683-872) and harvested 24 h later. Cell lysates (∼40 μg) were resolved by SDS–PAGE and recombinant (top left and right) and endogenous proteins (middle and bottom left and right) detected by immunoblotting using α-p21, -FLAG, and -tubulin antibodies.

    Journal: Molecular Biology of the Cell

    Article Title: Association of the circadian factor Period 2 to p53 influences p53's function in DNA-damage signaling

    doi: 10.1091/mbc.E14-05-0994

    Figure Lengend Snippet: Binding of hPer2 to hp53 prevents hp21 from being expressed. H1299 cells were transfected with pCS2+FLAG-hp53, -hp53(ch)GST, -NLS-hp53(ch)GST, -hp53(ch)hPer2(356-574/683-872) (labeled #), or -NLS-hp53(ch)hPer2(356-574/683-872) and harvested 24 h later. Cell lysates (∼40 μg) were resolved by SDS–PAGE and recombinant (top left and right) and endogenous proteins (middle and bottom left and right) detected by immunoblotting using α-p21, -FLAG, and -tubulin antibodies.

    Article Snippet: Finally, endogenous hp21 expression was monitored in extracts (30–40 μg) from H1299 cells transfected with FLAG-hp53, FLAG-hp53(ch)GST, FLAG-NLS-hp53(ch)GST, FLAG-hp53(ch)hPer2(356-574/683-872), NLS-FLAG-hp53(ch)hPer2(356-574/683-872), FLAG-hp53(ch)hPer2, FLAG-hp53(ch)hPer2, or EV using an α-p21 antibody (Cell Signaling).

    Techniques: Binding Assay, Transfection, Labeling, SDS Page, Recombinant

    The hPer2 protein maintains hp53 transcriptionally inactive when complexed. (A) Schematic representation of the approach followed. In all cases, H1299 cells were harvested after transfection and before irradiation (γ-IR, 5 Gy; t = 0). (B, C) Cells harvested 2 h after irradiation. (D) Time points taken at 2-h intervals after irradiation. (B) H1299 cells were cotransfected with the reporter hp21 WAF1/CIP1 -luc construct, pCS2+FLAG-hp53, pCS2+FLAG-hp53(ch)GST, pCS2+FLAG-hp53(ch)hPer2, or EV, with pCMV-β-gal as internal control. Extracts from cells treated (+γ-IR) or not (–γ-IR) with radiation were assayed for luciferase and β-galactosidase activities. The experiment was replicated thrice; error bars indicate SEM, and data were evaluated by ANOVA using Bonferroni posthoc test (SPSS). ### p ≤ 0.001. (C) H1299 cells were transfected with either pCS2+FLAG-hp53 or pCS2+FLAG-hp53(ch)hPer2 and treated (+γ-IR) or not (–γ-IR) with radiation. The qRT-PCR data were normalized to the levels of expression in untreated FLAG-hp53–transfected cells (–γ-IR). Data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were evaluated by ANOVA using Bonferroni or Games-Howell posthoc analyses when needed (SPSS). NS, not significant; # p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001. (D) H1299 cells were transfected with pCS2+FLAG-hp53, pCS2+FLAG-hp53(ch)GST, or pCS2+FLAG-hp53(ch)hPer2 and treated (+γ-IR) or not (–γ-IR) with radiation as indicated in A and Materials and Methods . Aliquots of lysates taken at different times (20 μg) were blotted using specific antibodies (α-Chk1 and α-p21 for endogenous Chk1 kinase and hp21 WAF1/CIP1 , respectively; α-FLAG for FLAG-hp53, FLAG-hp53(ch)GST, and hp53(ch)hPer2; α-Chk1-Ser 345 for phosphorylation in Ser 345 of endogenous Chk1; and α-p53-Ser 15 for phosphorylation in Ser 15 in FLAG-hp53, FLAG-hp53(ch)GST, and hp53(ch)hPer2). Tubulin was used as a loading control (bottom). Asterisk indicates a nonspecific signal.

    Journal: Molecular Biology of the Cell

    Article Title: Association of the circadian factor Period 2 to p53 influences p53's function in DNA-damage signaling

    doi: 10.1091/mbc.E14-05-0994

    Figure Lengend Snippet: The hPer2 protein maintains hp53 transcriptionally inactive when complexed. (A) Schematic representation of the approach followed. In all cases, H1299 cells were harvested after transfection and before irradiation (γ-IR, 5 Gy; t = 0). (B, C) Cells harvested 2 h after irradiation. (D) Time points taken at 2-h intervals after irradiation. (B) H1299 cells were cotransfected with the reporter hp21 WAF1/CIP1 -luc construct, pCS2+FLAG-hp53, pCS2+FLAG-hp53(ch)GST, pCS2+FLAG-hp53(ch)hPer2, or EV, with pCMV-β-gal as internal control. Extracts from cells treated (+γ-IR) or not (–γ-IR) with radiation were assayed for luciferase and β-galactosidase activities. The experiment was replicated thrice; error bars indicate SEM, and data were evaluated by ANOVA using Bonferroni posthoc test (SPSS). ### p ≤ 0.001. (C) H1299 cells were transfected with either pCS2+FLAG-hp53 or pCS2+FLAG-hp53(ch)hPer2 and treated (+γ-IR) or not (–γ-IR) with radiation. The qRT-PCR data were normalized to the levels of expression in untreated FLAG-hp53–transfected cells (–γ-IR). Data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were evaluated by ANOVA using Bonferroni or Games-Howell posthoc analyses when needed (SPSS). NS, not significant; # p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001. (D) H1299 cells were transfected with pCS2+FLAG-hp53, pCS2+FLAG-hp53(ch)GST, or pCS2+FLAG-hp53(ch)hPer2 and treated (+γ-IR) or not (–γ-IR) with radiation as indicated in A and Materials and Methods . Aliquots of lysates taken at different times (20 μg) were blotted using specific antibodies (α-Chk1 and α-p21 for endogenous Chk1 kinase and hp21 WAF1/CIP1 , respectively; α-FLAG for FLAG-hp53, FLAG-hp53(ch)GST, and hp53(ch)hPer2; α-Chk1-Ser 345 for phosphorylation in Ser 345 of endogenous Chk1; and α-p53-Ser 15 for phosphorylation in Ser 15 in FLAG-hp53, FLAG-hp53(ch)GST, and hp53(ch)hPer2). Tubulin was used as a loading control (bottom). Asterisk indicates a nonspecific signal.

    Article Snippet: Finally, endogenous hp21 expression was monitored in extracts (30–40 μg) from H1299 cells transfected with FLAG-hp53, FLAG-hp53(ch)GST, FLAG-NLS-hp53(ch)GST, FLAG-hp53(ch)hPer2(356-574/683-872), NLS-FLAG-hp53(ch)hPer2(356-574/683-872), FLAG-hp53(ch)hPer2, FLAG-hp53(ch)hPer2, or EV using an α-p21 antibody (Cell Signaling).

    Techniques: Transfection, Irradiation, Construct, Luciferase, Quantitative RT-PCR, Expressing

    GPX7 regulates the protein levels of tumor suppressor genes A–B) Western blot analysis is shown for FLO-1 and OE33 OAC cells that are stably expressing GPX7 (5A) or following infection with GPX7-expressing adenoviral particles (5B). An increase in the levels of p73, p27, p21, and p16 and a decrease in phosphorylated RB (p-RB) were detected (FLO-1 cell line is silent for p16).

    Journal: Gut

    Article Title: Glutathione peroxidase 7 has potential tumor suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma

    doi: 10.1136/gutjnl-2013-304612

    Figure Lengend Snippet: GPX7 regulates the protein levels of tumor suppressor genes A–B) Western blot analysis is shown for FLO-1 and OE33 OAC cells that are stably expressing GPX7 (5A) or following infection with GPX7-expressing adenoviral particles (5B). An increase in the levels of p73, p27, p21, and p16 and a decrease in phosphorylated RB (p-RB) were detected (FLO-1 cell line is silent for p16).

    Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, ProteinTech Group, Chicago, Illinois, USA), anti-p73 antibody (rabbit, 1:500, Bethyl, Montgomery, Texas, USA), anti-p21 antibody (mouse, 1:1000, Cell Signaling, Danvers, Massachusetts, USA), anti-p27 antibody (rabbit, 1:1000, Cell Signaling), anti-p16 antibody (rabbit, 1:1000, Cell Signaling), anti-RB antibody (mouse, 1:1000, Cell Signaling), anti-phospho-RB, ser780 (rabbit, 1:1000, Cell Signaling), anti-phospho-RB, ser807 (rabbit, 1:1000, Cell Signaling), and anti-actin antibody (rabbit, 1:1000, Cell Signaling).

    Techniques: Western Blot, Stable Transfection, Expressing, Infection

    Knockdown of KIF4A restrains the tumor formation of CRC cells in vivo. a KIF4A knockdown in HCT116 stable cell line was confirmed at the protein level by western blotting. b General observation of the subcutaneous tumors in nude mice formed by HCT116 cells transfected with shKIF4A and shCtrl lentivirus ( n = 10 in each group). c Tumor volumes of xenografts in nude mice. Xenografts volumes were calculated with the following formula: V = ( L × W 2 )/2. d Twenty days after injection, the mice were killed and the xenograft tumors were collected. e The weight of the xenograft tumors was analyzed. f The tumor sections were performed immunochemistry staining by antibody against KIF4A, p21, and Ki67; representative images were shown (×400 magnification). *** P

    Journal: Cell Death & Disease

    Article Title: KIF4A facilitates cell proliferation via induction of p21-mediated cell cycle progression and promotes metastasis in colorectal cancer

    doi: 10.1038/s41419-018-0550-9

    Figure Lengend Snippet: Knockdown of KIF4A restrains the tumor formation of CRC cells in vivo. a KIF4A knockdown in HCT116 stable cell line was confirmed at the protein level by western blotting. b General observation of the subcutaneous tumors in nude mice formed by HCT116 cells transfected with shKIF4A and shCtrl lentivirus ( n = 10 in each group). c Tumor volumes of xenografts in nude mice. Xenografts volumes were calculated with the following formula: V = ( L × W 2 )/2. d Twenty days after injection, the mice were killed and the xenograft tumors were collected. e The weight of the xenograft tumors was analyzed. f The tumor sections were performed immunochemistry staining by antibody against KIF4A, p21, and Ki67; representative images were shown (×400 magnification). *** P

    Article Snippet: Antibodies against p21 (#2947), p27 (#3686), cyclin D1 (#2978), cyclin E2 (#4132), Cdk2 (#2546), cleaved caspase 3 (#9661P), cleaved caspase 7 (#9491P), and cleaved caspase 9 (#9501P) were obtained from Cell Signaling Technology.

    Techniques: In Vivo, Stable Transfection, Western Blot, Mouse Assay, Transfection, Injection, Staining

    KIF4A inhibits the transcription of p21. a Analysis of the relationship between KIF4A expression and p21 mRNA levels in CRC patient tissues from GSE41258. b The p21 promoter (−2400/+11) activity was increased by KIF4A knockdown in HCT116 cells. c Full sequence of the human p21 promoter. p1–6 shows the regions of p21 promoter detected by the paired primers. d ChIP-qPCR analysis of KIF4A binding at p1, p2, p3, p4, p5, and p6 loci. e Western blot detection of KIF4A in the ChIP assay. Mean ± standard deviation of triplicate experiments are shown. * P

    Journal: Cell Death & Disease

    Article Title: KIF4A facilitates cell proliferation via induction of p21-mediated cell cycle progression and promotes metastasis in colorectal cancer

    doi: 10.1038/s41419-018-0550-9

    Figure Lengend Snippet: KIF4A inhibits the transcription of p21. a Analysis of the relationship between KIF4A expression and p21 mRNA levels in CRC patient tissues from GSE41258. b The p21 promoter (−2400/+11) activity was increased by KIF4A knockdown in HCT116 cells. c Full sequence of the human p21 promoter. p1–6 shows the regions of p21 promoter detected by the paired primers. d ChIP-qPCR analysis of KIF4A binding at p1, p2, p3, p4, p5, and p6 loci. e Western blot detection of KIF4A in the ChIP assay. Mean ± standard deviation of triplicate experiments are shown. * P

    Article Snippet: Antibodies against p21 (#2947), p27 (#3686), cyclin D1 (#2978), cyclin E2 (#4132), Cdk2 (#2546), cleaved caspase 3 (#9661P), cleaved caspase 7 (#9491P), and cleaved caspase 9 (#9501P) were obtained from Cell Signaling Technology.

    Techniques: Expressing, Activity Assay, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot, Standard Deviation

    Downregulation of p21 reverses the inhibition of proliferation, migration, and invasion induced by KIF4A knockdown. a KIF4A and p21 knockdown in HCT116 and DLD1 stable cell line was confirmed at the protein level by western blotting. b – d The inhibition of proliferation, migration, and invasion caused by KIF4A knockdown was regressed by transient transfection of p21 siRNA in HCT116 and DLD1 stable cell lines. Data are shown as mean ± standard deviations from three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: KIF4A facilitates cell proliferation via induction of p21-mediated cell cycle progression and promotes metastasis in colorectal cancer

    doi: 10.1038/s41419-018-0550-9

    Figure Lengend Snippet: Downregulation of p21 reverses the inhibition of proliferation, migration, and invasion induced by KIF4A knockdown. a KIF4A and p21 knockdown in HCT116 and DLD1 stable cell line was confirmed at the protein level by western blotting. b – d The inhibition of proliferation, migration, and invasion caused by KIF4A knockdown was regressed by transient transfection of p21 siRNA in HCT116 and DLD1 stable cell lines. Data are shown as mean ± standard deviations from three independent experiments. * P

    Article Snippet: Antibodies against p21 (#2947), p27 (#3686), cyclin D1 (#2978), cyclin E2 (#4132), Cdk2 (#2546), cleaved caspase 3 (#9661P), cleaved caspase 7 (#9491P), and cleaved caspase 9 (#9501P) were obtained from Cell Signaling Technology.

    Techniques: Inhibition, Migration, Stable Transfection, Western Blot, Transfection

    Effects of hypoxia and Hif-1α stabilization on the growth of MSCs in mass population. ( a ) BrdU incorporation analysis of MSCs under normoxic or hypoxic conditions. Cells were added with 10 μ M of BrdU in the culture medium for 2 h. MSCs were then stained with antibody against BrdU and propidium iodide (PI) for flow cytometric analysis. Shown are the representative plots and percentage of cells in each cell cycle fraction. ( b ) Effects of hypoxia on cell cycle inhibitory proteins in MSCs. MSCs were cultured under 21 or 1% O 2 for 2 days and analyzed for protein levels of each indicated gene by using antibody specific to p21 or p27. ( c ) MSCs were maintained under hypoxic (1% O 2 ) or normoxic (21% O 2 ) for at least two passages and plated in the dish (5 × 10 4 /100 mm dish density) at equal density. Eight days after plating, the cell numbers were counted. Shown are the mean±s.e.m. values from three experiments. ( d ) Doubling times were calculated as t / n , where t is the duration of culture and n is the number of population doublings calculated by using the formula n =log(NH−NI)/log2 (where NI is the number of cells originally plated and NH the number of cells harvested at the time of counting). Shown are the mean±s.e.m. values from three experiments. APC, allophycocyanin.

    Journal: Experimental & Molecular Medicine

    Article Title: Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state

    doi: 10.1038/emm.2013.87

    Figure Lengend Snippet: Effects of hypoxia and Hif-1α stabilization on the growth of MSCs in mass population. ( a ) BrdU incorporation analysis of MSCs under normoxic or hypoxic conditions. Cells were added with 10 μ M of BrdU in the culture medium for 2 h. MSCs were then stained with antibody against BrdU and propidium iodide (PI) for flow cytometric analysis. Shown are the representative plots and percentage of cells in each cell cycle fraction. ( b ) Effects of hypoxia on cell cycle inhibitory proteins in MSCs. MSCs were cultured under 21 or 1% O 2 for 2 days and analyzed for protein levels of each indicated gene by using antibody specific to p21 or p27. ( c ) MSCs were maintained under hypoxic (1% O 2 ) or normoxic (21% O 2 ) for at least two passages and plated in the dish (5 × 10 4 /100 mm dish density) at equal density. Eight days after plating, the cell numbers were counted. Shown are the mean±s.e.m. values from three experiments. ( d ) Doubling times were calculated as t / n , where t is the duration of culture and n is the number of population doublings calculated by using the formula n =log(NH−NI)/log2 (where NI is the number of cells originally plated and NH the number of cells harvested at the time of counting). Shown are the mean±s.e.m. values from three experiments. APC, allophycocyanin.

    Article Snippet: Antibody against Hif-1α was purchased from Cayman Chemical (Ann Arbor, MI, USA), antibody against NSE from Abcam (Cambridge, UK), antibodies against TAZ from Santa Cruz Biotech (Santa Cruz, CA, USA), antibodies against ARNT from Upstate (Lake Placid, NY, USA), antibody against pan-neuronal neurofilament from Covance (Emeryville, CA, USA), antibody against tubulin b-III from Chemicon (Lake Placid), antibody against p21 (Cell Signaling, Danvers, MA, USA), antibody against p27 (Cell Signaling) and antibody against actin (Millipore).

    Techniques: BrdU Incorporation Assay, Staining, Flow Cytometry, Cell Culture

    P21 deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control siRNA or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi)

    Journal: EBioMedicine

    Article Title: MDM2 Antagonists Counteract Drug-Induced DNA Damage

    doi: 10.1016/j.ebiom.2017.09.016

    Figure Lengend Snippet: P21 deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control siRNA or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi)

    Article Snippet: The next day SignalSilence p21 Waf1/Cip1 siRNA (#6456) or SignalSilence control siRNA were added to culture media (100 nM final concentration, all from Cell Signaling, Danvers, MA) along with 6 μL of Lipofectamine RNAi MAX (Invitrogen, part of the Thermo Fisher Scientific, Waltham, MA). siRNA transfection was repeated on the following day to increase knockdown efficiency.

    Techniques: Transfection

    Altered MITF, p21 and E2F1 expression precedes G1 cell cycle arrest

    Journal: Cancer research

    Article Title: SOX10 ablation arrests the cell cycle, induces senescence and suppresses melanomagenesis

    doi: 10.1158/0008-5472.CAN-12-4620

    Figure Lengend Snippet: Altered MITF, p21 and E2F1 expression precedes G1 cell cycle arrest

    Article Snippet: Primary antibodies were: monoclonal SOX10 (R & D Systems #MAB2864, Minneapolis, MN), monoclonal alpha-Tubulin (Calbiochem #CP06, San Diego, CA), total RB and Phospho-RB Ser807/811 antibody kit (Cell Signaling #9969, Danvers, MA), polyclonal E2F1 (Cell Signaling #3742), monoclonal p16 (F-12 Santa Cruz Biotechnology #sc-1661), monoclonal p21 Waf1/Cip1 (Cell Signaling #2946), monoclonal cyclin D1 (BD Pharmingen #G124-326, Franklin Lakes, NJ), monoclonal p27 (Santa Cruz Biotechnology #sc-1641), polyclonal CDK2, CDK4 and CDK6 (Santa Cruz Biotechnology #sc-163, sc-601, and sc-177, respectively) and monoclonal MITF (generously provided by Heinz Arnheiter).

    Techniques: Expressing

    Susceptibility of HCT116 wild-type (HCT116) and HCT116 p21 knockout (p21−/−) colorectal cancer cell lines towards 5FU treatment. ( a ) Effects of 5FU (10 μM) on the induction of apoptosis and necrosis in HCT116 and p21−/− cells after 48 h of incubation as assessed by the Annexin-PI assay. The experiment was carried out in technical and biological duplicate. One representative experiment is shown. ( b ) Fractions of live, early-apoptotic, late-apoptotic and necrotic cells in 5FU-treated and control HCT116 and p21−/− cell populations as determined from the Annexin-PI assay after 48 h of incubation. Values of one representative experiment are shown. ( c ) Cell viability of HCT116 and p21−/− cells after treatment with 5FU (10 μM) for 24 h and 48 h with respect to DMSO controls. Values represent means ± SD of three independent experiments. ** p

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: Susceptibility of HCT116 wild-type (HCT116) and HCT116 p21 knockout (p21−/−) colorectal cancer cell lines towards 5FU treatment. ( a ) Effects of 5FU (10 μM) on the induction of apoptosis and necrosis in HCT116 and p21−/− cells after 48 h of incubation as assessed by the Annexin-PI assay. The experiment was carried out in technical and biological duplicate. One representative experiment is shown. ( b ) Fractions of live, early-apoptotic, late-apoptotic and necrotic cells in 5FU-treated and control HCT116 and p21−/− cell populations as determined from the Annexin-PI assay after 48 h of incubation. Values of one representative experiment are shown. ( c ) Cell viability of HCT116 and p21−/− cells after treatment with 5FU (10 μM) for 24 h and 48 h with respect to DMSO controls. Values represent means ± SD of three independent experiments. ** p

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: Knock-Out, Incubation

    Localization of p21 in 5FU-resistant HCT116 cells. ( a ) HCT116 cells were treated with various concentrations of 5FU for 48 h and the expression of p21 was determined by immunofluorescence staining using mouse anti-p21 monoclonal antibodies followed by an Alexa Fluor 555-labeled secondary antibody to visualize p21 expression (red) and the nuclei (Hoechst 33342, blue). Scale bar: 50 μm. ( b ) The expression level of phosphorylated-p21 (p-p21 T145 ) in 5FU-resistant HCT116 cells was determined by Western Blot analysis. Cells were treated with various concentrations of 5FU for 48 h. After incubation, dead cells were discarded by washing the plates 3 times with PBS and the remaining resistant cells were collected to prepare protein lysates as mentioned in the Material and Methods section. The blots were re-probed with GAPDH to confirm equal loading of the samples. ( c ) Ex ovo images and overviews of H E-stained sections of CAM micro-tumors. For this, HCT116 cells were treated with 15 μM of 5FU for 48 h. Then the 5FU-resistant HCT116 cells were subjected to the CAM. After 5 days, tumor tissues were collected, tumor size was measured, and xenografts were subjected to standard histological and immunohistochemical procedures. ( d ) H E staining and immunohistochemical staining of p21 protein in vital areas of tumor slices. ( e ) Immunoscore of p21 regarding its cytoplasmic and nuclear localization in xenografts of 5FU-treated HCT116 and control cells. *** p

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: Localization of p21 in 5FU-resistant HCT116 cells. ( a ) HCT116 cells were treated with various concentrations of 5FU for 48 h and the expression of p21 was determined by immunofluorescence staining using mouse anti-p21 monoclonal antibodies followed by an Alexa Fluor 555-labeled secondary antibody to visualize p21 expression (red) and the nuclei (Hoechst 33342, blue). Scale bar: 50 μm. ( b ) The expression level of phosphorylated-p21 (p-p21 T145 ) in 5FU-resistant HCT116 cells was determined by Western Blot analysis. Cells were treated with various concentrations of 5FU for 48 h. After incubation, dead cells were discarded by washing the plates 3 times with PBS and the remaining resistant cells were collected to prepare protein lysates as mentioned in the Material and Methods section. The blots were re-probed with GAPDH to confirm equal loading of the samples. ( c ) Ex ovo images and overviews of H E-stained sections of CAM micro-tumors. For this, HCT116 cells were treated with 15 μM of 5FU for 48 h. Then the 5FU-resistant HCT116 cells were subjected to the CAM. After 5 days, tumor tissues were collected, tumor size was measured, and xenografts were subjected to standard histological and immunohistochemical procedures. ( d ) H E staining and immunohistochemical staining of p21 protein in vital areas of tumor slices. ( e ) Immunoscore of p21 regarding its cytoplasmic and nuclear localization in xenografts of 5FU-treated HCT116 and control cells. *** p

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: Expressing, Immunofluorescence, Staining, Labeling, Western Blot, Incubation, Chick Chorioallantoic Membrane Assay, Immunohistochemistry

    Interaction between p21 and Chk2 proteins. Cells were treated with 100 μM 5FU for 48 h and the protein–protein interaction of p21-p-Chk2 T68 was analyzed by proximity ligation assay (HT29) (red signals indicate the protein–protein interaction between p21 and p-Chk2 T68 . ( a ) Fluorescence images of untreated control HT29 cells (40× magnification) and 5FU-treated HT29 cells (40× and 100× magnification) (scale bar: 50 μm); ( b ) confocal images. ( c ) 5FU-treated HCT116 cell lysates were prepared and immunoprecipitated with an anti-p-chk2 T68 antibody. The resulting immune complexes were then analyzed for p21 by Western Blot using an anti-p21 antibody. ( d ) Complex of p21 and Chk2 obtained by structural modelling; p21 (cyan) was modelled using two PDB structures 4I58 and 5EOU as templates using MODELLER [ 39 ]. Chk2 (magenta) was obtained from its crystal structure 2WTC after removing the coordinates of the inhibitor. The structures were energy-minimized and docked using ClusPro [ 40 ] and rendered using Chimera [ 40 ]. ( e ) Complex of phosphorylated p21 and Chek2: the energy-minimized stable structure of p21 (cyan) was phosphorylated at Threonine residue at position 145 using Chimera [ 41 ] and was docked with the structural model of Chek2 (magenta) using ClusPro [ 40 ]. The nuclear localization signal (NLS) region of Chk2 is shown in red. The insert shows the phosphorylation of T145 of p21 in the model. The interactions between the proteins were identified using the Protein Interaction Calculator (PIC) [ 42 ]. ( f ) Complex of p21 and phosphorylated Chk2: the energy-minimized stable structure of Chk2 (magenta) was phosphorylated at Threonine residue at position 68 using Chimera [ 41 ] and was docked with the structural model of p21 (cyan) using ClusPro [ 40 ]. The NLS region of Chk2 is shown in red and the residues around and in the NLS are labelled. The insert shows the phosphorylation of T68 of Chk2 in the model. The interactions between the proteins were identified using the PIC [ 42 ]. ( g ) Complex of phosphorylated p21 (T145) and phosphorylated Chek2 (T68): individual structural models of p21 phosphorylated at T145 and Chek2 phosphorylated at T68 were docked using ClusPro [ 40 ] and rendered using Chimera [ 41 ]. The NLS of Chek2 is shown in red and the nuclear export signal (NES) is shown in green. The interaction profile of the p-p21/p-Chk2 complex identified by the PIC [ 42 ] shows that no amino acid in the NES region of p21 interacts with p-Chk2, hence showing that the NES is free from any interactions facilitating export of the complex to the cytoplasm.

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: Interaction between p21 and Chk2 proteins. Cells were treated with 100 μM 5FU for 48 h and the protein–protein interaction of p21-p-Chk2 T68 was analyzed by proximity ligation assay (HT29) (red signals indicate the protein–protein interaction between p21 and p-Chk2 T68 . ( a ) Fluorescence images of untreated control HT29 cells (40× magnification) and 5FU-treated HT29 cells (40× and 100× magnification) (scale bar: 50 μm); ( b ) confocal images. ( c ) 5FU-treated HCT116 cell lysates were prepared and immunoprecipitated with an anti-p-chk2 T68 antibody. The resulting immune complexes were then analyzed for p21 by Western Blot using an anti-p21 antibody. ( d ) Complex of p21 and Chk2 obtained by structural modelling; p21 (cyan) was modelled using two PDB structures 4I58 and 5EOU as templates using MODELLER [ 39 ]. Chk2 (magenta) was obtained from its crystal structure 2WTC after removing the coordinates of the inhibitor. The structures were energy-minimized and docked using ClusPro [ 40 ] and rendered using Chimera [ 40 ]. ( e ) Complex of phosphorylated p21 and Chek2: the energy-minimized stable structure of p21 (cyan) was phosphorylated at Threonine residue at position 145 using Chimera [ 41 ] and was docked with the structural model of Chek2 (magenta) using ClusPro [ 40 ]. The nuclear localization signal (NLS) region of Chk2 is shown in red. The insert shows the phosphorylation of T145 of p21 in the model. The interactions between the proteins were identified using the Protein Interaction Calculator (PIC) [ 42 ]. ( f ) Complex of p21 and phosphorylated Chk2: the energy-minimized stable structure of Chk2 (magenta) was phosphorylated at Threonine residue at position 68 using Chimera [ 41 ] and was docked with the structural model of p21 (cyan) using ClusPro [ 40 ]. The NLS region of Chk2 is shown in red and the residues around and in the NLS are labelled. The insert shows the phosphorylation of T68 of Chk2 in the model. The interactions between the proteins were identified using the PIC [ 42 ]. ( g ) Complex of phosphorylated p21 (T145) and phosphorylated Chek2 (T68): individual structural models of p21 phosphorylated at T145 and Chek2 phosphorylated at T68 were docked using ClusPro [ 40 ] and rendered using Chimera [ 41 ]. The NLS of Chek2 is shown in red and the nuclear export signal (NES) is shown in green. The interaction profile of the p-p21/p-Chk2 complex identified by the PIC [ 42 ] shows that no amino acid in the NES region of p21 interacts with p-Chk2, hence showing that the NES is free from any interactions facilitating export of the complex to the cytoplasm.

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: Proximity Ligation Assay, Fluorescence, Immunoprecipitation, Western Blot

    Schematic model of p21/Chk2-mediated 5FU resistance. Black stars mark where experimental evidence is given, and orange star marks in silico analysis. The fate of p-Chk2 in the cytoplasm after detachment of the complex is unclear.

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: Schematic model of p21/Chk2-mediated 5FU resistance. Black stars mark where experimental evidence is given, and orange star marks in silico analysis. The fate of p-Chk2 in the cytoplasm after detachment of the complex is unclear.

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: In Silico

    p21 inhibits pro-apoptotic effect of Chk2. ( a , b ) Expression levels of phospho-proteins in transfected cells. HCT116 cells were transfected with hyperphosphorylated p21 T145D . After 48 h of transfection, cells lysates were prepared and subjected to the Human Phospho-Kinase Array Kit (R D systems). The expression levels of phosphorylated Chk2 (p-Chk2 T68 ) and Chk2 in 5FU-resistant HCT116 ( c ), HT29 ( d ), and SW837 ( e ) cells were determined by Western Blot. (The same lysates were loaded on two different membranes for Chk2 and pChk2 detection). For this, cells were treated with various concentrations of 5FU for 48 h. Dead cells were removed by washing with PBS and the remaining viable cells were collected to prepare protein lysates as mentioned in the Material and Methods sections. The blots were re-probed with GAPDH to confirm equal loading of the samples. ( f ) The expression levels of phosphorylated Chk2 (p-Chk2 T68 ), Chk2, and p-E2F1 in HCT116 cells transfected with hyperphosphorylated p21 T145D and unphosphorylatable p21 T145A in response to 5FU treatment were determined by Western Blot analysis. After 24 h transfection, cells were left untreated or were treated with 5FU (25 μM) for further 48 h. Dead cells were removed by washing with PBS and the remaining viable cells were collected to prepare protein lysates. The expression of p-E2F1, p-Chk2 T68 and Chk2 were determined and the blots were re-probed with GAPDH to confirm equal loading of the samples. Immunohistochemical staining of p-chk2 T68 in CAM xenografts formed by HCT116 control ( g ) and HCT116 5FU-treated ( h ) cells. ( i ) p-chk2 T68 immunoscore in cytoplasmic and nuclear localizations in tumors of the HCT116 5FU-treated and untreated groups. * p

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: p21 inhibits pro-apoptotic effect of Chk2. ( a , b ) Expression levels of phospho-proteins in transfected cells. HCT116 cells were transfected with hyperphosphorylated p21 T145D . After 48 h of transfection, cells lysates were prepared and subjected to the Human Phospho-Kinase Array Kit (R D systems). The expression levels of phosphorylated Chk2 (p-Chk2 T68 ) and Chk2 in 5FU-resistant HCT116 ( c ), HT29 ( d ), and SW837 ( e ) cells were determined by Western Blot. (The same lysates were loaded on two different membranes for Chk2 and pChk2 detection). For this, cells were treated with various concentrations of 5FU for 48 h. Dead cells were removed by washing with PBS and the remaining viable cells were collected to prepare protein lysates as mentioned in the Material and Methods sections. The blots were re-probed with GAPDH to confirm equal loading of the samples. ( f ) The expression levels of phosphorylated Chk2 (p-Chk2 T68 ), Chk2, and p-E2F1 in HCT116 cells transfected with hyperphosphorylated p21 T145D and unphosphorylatable p21 T145A in response to 5FU treatment were determined by Western Blot analysis. After 24 h transfection, cells were left untreated or were treated with 5FU (25 μM) for further 48 h. Dead cells were removed by washing with PBS and the remaining viable cells were collected to prepare protein lysates. The expression of p-E2F1, p-Chk2 T68 and Chk2 were determined and the blots were re-probed with GAPDH to confirm equal loading of the samples. Immunohistochemical staining of p-chk2 T68 in CAM xenografts formed by HCT116 control ( g ) and HCT116 5FU-treated ( h ) cells. ( i ) p-chk2 T68 immunoscore in cytoplasmic and nuclear localizations in tumors of the HCT116 5FU-treated and untreated groups. * p

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: Expressing, Transfection, Western Blot, Immunohistochemistry, Staining, Chick Chorioallantoic Membrane Assay

    Co-localization of p21 and p-Chk2 T68 /Chk2 in 5FU-resistant cells. ( a , b ) HT29, ( c ) HCT116 and ( d ) SW837 cells were treated with various concentrations of 5FU for 48 h and the expression of p21 and p-Chk2 T68 was examined by immunofluorescence staining using mouse anti-p21 monoclonal antibodies followed by an Alexa Fluor 555-labeled secondary antibody to visualize p21 expression (red), rabbit anti-p-Chk2 T68 monoclonal antibodies followed by an Alexa Fluor 488-labeled secondary antibody to visualize p-Chk2 T68 expression (green) and cell nuclei (Hoechst 33342, blue). ( a ) Enlarged images of p21 and p-Chk2T68 co-localization in 5FU-resistant HT29 cells. Scale bar: 50 μm; ( e ) HCT116 cells were treated with 100 μM 5FU for 48 h and the expression of p21 and Chk2 was examined by immunofluorescence staining using mouse anti-p21 monoclonal antibodies followed by an Alexa Fluor 555-labeled secondary antibody to visualize p21 expression (red), rabbit anti-Chk2 monoclonal antibodies followed by an Alexa Fluor 488-labeled secondary antibody to visualize Chk2 expression (green) and cell nuclei (Hoechst 33342, blue). Scale bar: 50 μm; and ( f ) 2.5-fold computer-enlarged images from merge images in ( e ).

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: Co-localization of p21 and p-Chk2 T68 /Chk2 in 5FU-resistant cells. ( a , b ) HT29, ( c ) HCT116 and ( d ) SW837 cells were treated with various concentrations of 5FU for 48 h and the expression of p21 and p-Chk2 T68 was examined by immunofluorescence staining using mouse anti-p21 monoclonal antibodies followed by an Alexa Fluor 555-labeled secondary antibody to visualize p21 expression (red), rabbit anti-p-Chk2 T68 monoclonal antibodies followed by an Alexa Fluor 488-labeled secondary antibody to visualize p-Chk2 T68 expression (green) and cell nuclei (Hoechst 33342, blue). ( a ) Enlarged images of p21 and p-Chk2T68 co-localization in 5FU-resistant HT29 cells. Scale bar: 50 μm; ( e ) HCT116 cells were treated with 100 μM 5FU for 48 h and the expression of p21 and Chk2 was examined by immunofluorescence staining using mouse anti-p21 monoclonal antibodies followed by an Alexa Fluor 555-labeled secondary antibody to visualize p21 expression (red), rabbit anti-Chk2 monoclonal antibodies followed by an Alexa Fluor 488-labeled secondary antibody to visualize Chk2 expression (green) and cell nuclei (Hoechst 33342, blue). Scale bar: 50 μm; and ( f ) 2.5-fold computer-enlarged images from merge images in ( e ).

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: Expressing, Immunofluorescence, Staining, Labeling

    Cytoplasmic p21 mediates 5FU resistance in colorectal cancer cells. ( a – f ) The expression levels of PARP, cleaved PARP (cl. PARP), phosphorylated-p21 (p-p21 T145 ) and p21 in resistant cells (R) and dead cells (D) were determined by Western Blot analysis after 48 h of treatment with 5FU. For this, HCT116, HT29, and SW837 cells were treated with various concentrations of 5FU. After 48 h, resistant cells (R) and dead cells (D) were collected separately and protein lysates were prepared. The blots were re-probed with GAPDH to confirm equal loading of the samples. ( g ) Expression of p21 in viable and dead cells after 48 h of 5FU treatment. HCT116, HT29, and SW837 cells were treated with various concentrations of 5FU for 48 h and the expression levels of p21 were determined by immunofluorescence staining for p21 (red) and the cell nuclei (Hoechst 33342, blue). White arrow indicates apoptotic cells having condensed chromatin and/or fragmented nuclei. Scale bar: 50 μm. ( h ) 5FU susceptibility of HCT116 cells transfected with hyperphosphorylated p21 T145D and unphosphorylatable p21 T145A . After 24 h of transfection, cells were treated with 5FU (25 μM) for further 48 h. Untreated cells were used as controls. The expression levels of PARP, cleaved PARP (cl. PARP), and p21 were determined by Western Blot analysis. The blots were re-probed with GAPDH to confirm equal loading of the samples.

    Journal: Cancers

    Article Title: Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

    doi: 10.3390/cancers10100373

    Figure Lengend Snippet: Cytoplasmic p21 mediates 5FU resistance in colorectal cancer cells. ( a – f ) The expression levels of PARP, cleaved PARP (cl. PARP), phosphorylated-p21 (p-p21 T145 ) and p21 in resistant cells (R) and dead cells (D) were determined by Western Blot analysis after 48 h of treatment with 5FU. For this, HCT116, HT29, and SW837 cells were treated with various concentrations of 5FU. After 48 h, resistant cells (R) and dead cells (D) were collected separately and protein lysates were prepared. The blots were re-probed with GAPDH to confirm equal loading of the samples. ( g ) Expression of p21 in viable and dead cells after 48 h of 5FU treatment. HCT116, HT29, and SW837 cells were treated with various concentrations of 5FU for 48 h and the expression levels of p21 were determined by immunofluorescence staining for p21 (red) and the cell nuclei (Hoechst 33342, blue). White arrow indicates apoptotic cells having condensed chromatin and/or fragmented nuclei. Scale bar: 50 μm. ( h ) 5FU susceptibility of HCT116 cells transfected with hyperphosphorylated p21 T145D and unphosphorylatable p21 T145A . After 24 h of transfection, cells were treated with 5FU (25 μM) for further 48 h. Untreated cells were used as controls. The expression levels of PARP, cleaved PARP (cl. PARP), and p21 were determined by Western Blot analysis. The blots were re-probed with GAPDH to confirm equal loading of the samples.

    Article Snippet: Slides were incubated at 4 °C overnight with a primary monoclonal antibody p21 Waf1/Cip1 (12D1, mouse), 1:1.000 dilution (Cell Signaling), or pChk2T68 (2197, rabbit), 1:50 dilution (Cell Signaling).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Transfection

    Idasanutlin activates p53 and inhibits the growth of p53 wt cells. ( a – c ) Three p53 wt cell lines, SJSA-1, U-2 OS, and MCF-7, and one p53 del cell line, SAOS-2, were treated for 8 or 24 h with 0.05, 0.5, or 5 μM idasanutlin, or DMSO as a control. Western blot detection of p53, p21, and GAPDH was performed for the cells treated for 8 and 24 h. Cell cycle analysis was performed following 24 h of treatment with BrdU pulse-labeling for the last hour. The cells were fixed and stained with propidium iodide (PI) and FITC-anti-BrdU antibody (BrdU-FITC). The results presented on panels ( a – c ) are representative of at least three independent experiments. ( d ) SJSA-1, U-2 OS, MCF-7, and SAOS-2 cells were treated for five days with increasing concentrations of idasanutlin or an equivalent volume of DMSO, before assessment of cell viability. The data were analyzed with Dr-Fit software [ 26 ] and the best fitting model (red line) was chosen based on the Bayesian Information Criterion (BIC) ( Supplementary Table S1 ). EC 50 values of the growth inhibitory/stimulatory effects determined by the Dr-Fit software are indicated (see also Supplementary Table S1 ). Data points show means ± SD from three independent experiments.

    Journal: Cancers

    Article Title: Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells

    doi: 10.3390/cancers10110396

    Figure Lengend Snippet: Idasanutlin activates p53 and inhibits the growth of p53 wt cells. ( a – c ) Three p53 wt cell lines, SJSA-1, U-2 OS, and MCF-7, and one p53 del cell line, SAOS-2, were treated for 8 or 24 h with 0.05, 0.5, or 5 μM idasanutlin, or DMSO as a control. Western blot detection of p53, p21, and GAPDH was performed for the cells treated for 8 and 24 h. Cell cycle analysis was performed following 24 h of treatment with BrdU pulse-labeling for the last hour. The cells were fixed and stained with propidium iodide (PI) and FITC-anti-BrdU antibody (BrdU-FITC). The results presented on panels ( a – c ) are representative of at least three independent experiments. ( d ) SJSA-1, U-2 OS, MCF-7, and SAOS-2 cells were treated for five days with increasing concentrations of idasanutlin or an equivalent volume of DMSO, before assessment of cell viability. The data were analyzed with Dr-Fit software [ 26 ] and the best fitting model (red line) was chosen based on the Bayesian Information Criterion (BIC) ( Supplementary Table S1 ). EC 50 values of the growth inhibitory/stimulatory effects determined by the Dr-Fit software are indicated (see also Supplementary Table S1 ). Data points show means ± SD from three independent experiments.

    Article Snippet: The following antibodies and dilutions were used: rabbit polyclonal anti-p53 (1:200, Santa Cruz Biotechnology, cat. sc-6243, Dallas, TX, USA), rabbit monoclonal anti-p21 (1:1000, Cell Signaling Technology, CST, cat. 2947, Danvers, MA, USA), rabbit monoclonal anti-MDM2 (1:1000, Thermo Fisher Scientific, cat. 700555, Waltham, MA, USA), rabbit monoclonal anti-GAPDH (1:4000, CST, cat. 2118, Danvers, MA, USA) and goat peroxidase-conjugated anti-rabbit (1:2000, CST, cat. 7074, Danvers, MA, USA).

    Techniques: Western Blot, Cell Cycle Assay, Labeling, Staining, Software

    The selection of idasanutlin-resistant subpopulations of U-2 OS cells. ( a ) Selection strategy scheme. U-2 OS cells were seeded on 75 cm 2 cell culture flasks at sub-confluency. The next day, treatment with 5 µM idasanutlin was performed and repeated every 2–3 days, as indicated. After 21 days of the treatment, idasanutlin-sensitive cells (not-proliferating) were removed by mild trypsinization, and clones were picked and cultured individually. ( b ) Induction of p53 and p21 expression in idasanutlin-resistant clones. U-2 OS and SAOS-2 cells, as well as four idasanutlin-resistant clones (c.1.1, c.1.2, c.2.1, and c.2.2) were treated for 24 h with 0.5 or 5 µM idasanutlin, or DMSO as a control, followed by Western blot detection of p53, p21, and GAPDH expression. The presented results are representative of 3 independent experiments. ( c ) MTT cell survival test was performed on U-2 OS and SAOS-2 cells, and idasanutlin-resistant clones. The cells were treated for five days with increasing concentrations of idasanutlin or an equivalent volume of DMSO. The data represent mean ± SD values from three independent experiments, each performed in duplicates, and are presented as % of DMSO-treated control. ( d ) Cell cycle analysis of DMSO- or idasanutlin-treated U-2 OS cells, SAOS-2 cells and idasanutlin-resistant clones (c.2.1 and c.2.2) was performed after 24-h treatment with 5 µM idasanutlin or DMSO by propidium iodide (PI) staining. Graphs present cell cycle distribution as mean ± SD values from three independent experiments.

    Journal: Cancers

    Article Title: Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells

    doi: 10.3390/cancers10110396

    Figure Lengend Snippet: The selection of idasanutlin-resistant subpopulations of U-2 OS cells. ( a ) Selection strategy scheme. U-2 OS cells were seeded on 75 cm 2 cell culture flasks at sub-confluency. The next day, treatment with 5 µM idasanutlin was performed and repeated every 2–3 days, as indicated. After 21 days of the treatment, idasanutlin-sensitive cells (not-proliferating) were removed by mild trypsinization, and clones were picked and cultured individually. ( b ) Induction of p53 and p21 expression in idasanutlin-resistant clones. U-2 OS and SAOS-2 cells, as well as four idasanutlin-resistant clones (c.1.1, c.1.2, c.2.1, and c.2.2) were treated for 24 h with 0.5 or 5 µM idasanutlin, or DMSO as a control, followed by Western blot detection of p53, p21, and GAPDH expression. The presented results are representative of 3 independent experiments. ( c ) MTT cell survival test was performed on U-2 OS and SAOS-2 cells, and idasanutlin-resistant clones. The cells were treated for five days with increasing concentrations of idasanutlin or an equivalent volume of DMSO. The data represent mean ± SD values from three independent experiments, each performed in duplicates, and are presented as % of DMSO-treated control. ( d ) Cell cycle analysis of DMSO- or idasanutlin-treated U-2 OS cells, SAOS-2 cells and idasanutlin-resistant clones (c.2.1 and c.2.2) was performed after 24-h treatment with 5 µM idasanutlin or DMSO by propidium iodide (PI) staining. Graphs present cell cycle distribution as mean ± SD values from three independent experiments.

    Article Snippet: The following antibodies and dilutions were used: rabbit polyclonal anti-p53 (1:200, Santa Cruz Biotechnology, cat. sc-6243, Dallas, TX, USA), rabbit monoclonal anti-p21 (1:1000, Cell Signaling Technology, CST, cat. 2947, Danvers, MA, USA), rabbit monoclonal anti-MDM2 (1:1000, Thermo Fisher Scientific, cat. 700555, Waltham, MA, USA), rabbit monoclonal anti-GAPDH (1:4000, CST, cat. 2118, Danvers, MA, USA) and goat peroxidase-conjugated anti-rabbit (1:2000, CST, cat. 7074, Danvers, MA, USA).

    Techniques: Selection, Cell Culture, Clone Assay, Expressing, Western Blot, MTT Assay, Cell Cycle Assay, Staining

    In lung tumor, miR-106b-25 cluster and MCM7 host gene are overexpressed while p21 is downregulated compared to normal tissue. ( A ) Boxplots representing the relative abundance of mi-R25, 93, 106b and MCM7 transcript in tumoral compared to normal tissue in lung TCGA casuistry. N patients= 470 T, 56 N. (B) Pathways predicted to be affected by miR-106b-25 cluster. −10*log 10 (FDR) was used as a relevance score (see Supplementary Table 3, available at Carcinogenesis Online). ( C ) Boxplot comparing p21 transcript abundance in tumoral compared to normal tissue in lung TCGA casuistry.

    Journal: Carcinogenesis

    Article Title: MCM7 and its hosted miR-25, 93 and 106b cluster elicit YAP/TAZ oncogenic activity in lung cancer

    doi: 10.1093/carcin/bgw110

    Figure Lengend Snippet: In lung tumor, miR-106b-25 cluster and MCM7 host gene are overexpressed while p21 is downregulated compared to normal tissue. ( A ) Boxplots representing the relative abundance of mi-R25, 93, 106b and MCM7 transcript in tumoral compared to normal tissue in lung TCGA casuistry. N patients= 470 T, 56 N. (B) Pathways predicted to be affected by miR-106b-25 cluster. −10*log 10 (FDR) was used as a relevance score (see Supplementary Table 3, available at Carcinogenesis Online). ( C ) Boxplot comparing p21 transcript abundance in tumoral compared to normal tissue in lung TCGA casuistry.

    Article Snippet: Western blotting was performed using the following primary antibodies: rabbit polyclonal anti YAP (Santa Cruz, sc-15407), rabbit polyclonal anti TAZ (Sigma anti-WWTR1, HPA007415), mouse monoclonal anti B-actin (ACTBD11B7, Santa Cruz, sc-81178), rabbit monoclonal anti MCM7 (D10A11, Cell Signaling, 3735S), mouse monoclonal anti-TEF-1 (BD-Transduction Laboratories, 610923), rabbit monoclonal anti-p21 Waf1/Cip1 (Cell Signaling, 2947S), rabbit polyclonal anti Phospho-YAP Ser127 (Cell Signaling, 4911), mouse monoclonal anti-HSP90 (Santa Cruz, sc-13119), and rabbit polyclonal anti-p21 (Santa Cruz, sc-397).

    Techniques:

    In NSCLC cells, p21 is derepressed upon YAP/TAZ and TEAD1 knock-down. ( A ) Western blot representing YAP, TAZ and p21 protein abundance in H1299 cells, as measured by densitometry (Uvitec) and normalized to B-actin signal after YAP, TAZ, YAP/TAZ interference (left panel) and YAP, TEAD1, YAP/TEAD1 interference (right panel) compared to control counterparts. ( B ) Quantification by real time-PCR of p21 transcript levels, normalized to GAPDH, in siYAP, siTAZ, si YAP/TAZ, siTEAD1 and siYAP/TEAD1 H1299 cells respect to control counterparts. SEM is indicated. Asterisks represent statistically relevant results. P and n values: p21 siYAP#1/siGFP P = 0.005 n = 9; siTAZ#1/siGFP P = 0.03 n =3; si YAP#1TAZ#1/siGFP P =0.004 n = 6; siTEAD1#1/siGFP P = 0.03 n = 6; siYAP#1TEAD1#1/siGFP P = 0.01 n = 5. ( C , D ) As described in Figures (A, B) but for the H1975 cell line. SEM is indicated. P and n values: p21 siYAP#1/siGFP P = 0.01 n = 7; siTAZ#1/siGFP P = 0.002 n = 6; siYAP#1TAZ#1/siGFP P = 0.006 n = 5; siTEAD1#1/siGFP P = 0.02 n = 4; siYAP#1TEAD1#1/siGFP P = 0.01 n = 4. ( E )) and as shown in our study (mid panel).

    Journal: Carcinogenesis

    Article Title: MCM7 and its hosted miR-25, 93 and 106b cluster elicit YAP/TAZ oncogenic activity in lung cancer

    doi: 10.1093/carcin/bgw110

    Figure Lengend Snippet: In NSCLC cells, p21 is derepressed upon YAP/TAZ and TEAD1 knock-down. ( A ) Western blot representing YAP, TAZ and p21 protein abundance in H1299 cells, as measured by densitometry (Uvitec) and normalized to B-actin signal after YAP, TAZ, YAP/TAZ interference (left panel) and YAP, TEAD1, YAP/TEAD1 interference (right panel) compared to control counterparts. ( B ) Quantification by real time-PCR of p21 transcript levels, normalized to GAPDH, in siYAP, siTAZ, si YAP/TAZ, siTEAD1 and siYAP/TEAD1 H1299 cells respect to control counterparts. SEM is indicated. Asterisks represent statistically relevant results. P and n values: p21 siYAP#1/siGFP P = 0.005 n = 9; siTAZ#1/siGFP P = 0.03 n =3; si YAP#1TAZ#1/siGFP P =0.004 n = 6; siTEAD1#1/siGFP P = 0.03 n = 6; siYAP#1TEAD1#1/siGFP P = 0.01 n = 5. ( C , D ) As described in Figures (A, B) but for the H1975 cell line. SEM is indicated. P and n values: p21 siYAP#1/siGFP P = 0.01 n = 7; siTAZ#1/siGFP P = 0.002 n = 6; siYAP#1TAZ#1/siGFP P = 0.006 n = 5; siTEAD1#1/siGFP P = 0.02 n = 4; siYAP#1TEAD1#1/siGFP P = 0.01 n = 4. ( E )) and as shown in our study (mid panel).

    Article Snippet: Western blotting was performed using the following primary antibodies: rabbit polyclonal anti YAP (Santa Cruz, sc-15407), rabbit polyclonal anti TAZ (Sigma anti-WWTR1, HPA007415), mouse monoclonal anti B-actin (ACTBD11B7, Santa Cruz, sc-81178), rabbit monoclonal anti MCM7 (D10A11, Cell Signaling, 3735S), mouse monoclonal anti-TEF-1 (BD-Transduction Laboratories, 610923), rabbit monoclonal anti-p21 Waf1/Cip1 (Cell Signaling, 2947S), rabbit polyclonal anti Phospho-YAP Ser127 (Cell Signaling, 4911), mouse monoclonal anti-HSP90 (Santa Cruz, sc-13119), and rabbit polyclonal anti-p21 (Santa Cruz, sc-397).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction

    YAP/TAZ elicit their oncogenic function by simultaneously regulating MCM7 gene and hosted miR-25, 93, 106b cluster that in turn inhibit p21 and affect cell proliferation. ( A ) Western blot showing MCM7 and p21 protein abundance, normalized to B-actin, in cells knocked down for MCM7 with two alternative siRNAs (siMCM7#1 and siMCM7#2) compared to control siGFP H1299 cells. ( B ) Quantification by real time-PCR of p21 transcript levels, normalized to GAPDH, in cells knocked down for MCM7 compared to siGFP control cells. P and n values: H1299 p21 siMCM7#2 P = 0.04 n = 4. ( C , D ) Same as (A, B) but for H1975 cell line. P and n values: H1975 p21 siMCM7#1 P = 0.006 n = 3; siMCM7#2 P = 0.03 n = 3. ( E , F ) Western blot showing MCM7 and p21 protein abundance as measured by densitometry and normalized to B-actin (left panel), and real-time quantification of their transcripts normalized to GAPDH (right panel) in H1299 cells treated with LNA inhibitors for miR-25, 93 and 106b (E) and H1975 cell line (F). All the experiments were performed in triplicate. ( G ) Cartoon representing the newly characterized mechanism through which YAP/TAZ and TEAD by binding to MCM7 enhancer transcriptionally regulate both MCM7 gene and the hosted miR cluster miR-25, 93 and 106b, that in turn post-transcriptionally regulate p21 abundance eventually affecting cell proliferation (left panel). Treatment of cells with statin determines YAP/TAZ exclusion from nuclei, impairs transcription of MCM7 gene and hosted miRs cluster and derepresses p21 thus inhibiting cell proliferation (right panel).

    Journal: Carcinogenesis

    Article Title: MCM7 and its hosted miR-25, 93 and 106b cluster elicit YAP/TAZ oncogenic activity in lung cancer

    doi: 10.1093/carcin/bgw110

    Figure Lengend Snippet: YAP/TAZ elicit their oncogenic function by simultaneously regulating MCM7 gene and hosted miR-25, 93, 106b cluster that in turn inhibit p21 and affect cell proliferation. ( A ) Western blot showing MCM7 and p21 protein abundance, normalized to B-actin, in cells knocked down for MCM7 with two alternative siRNAs (siMCM7#1 and siMCM7#2) compared to control siGFP H1299 cells. ( B ) Quantification by real time-PCR of p21 transcript levels, normalized to GAPDH, in cells knocked down for MCM7 compared to siGFP control cells. P and n values: H1299 p21 siMCM7#2 P = 0.04 n = 4. ( C , D ) Same as (A, B) but for H1975 cell line. P and n values: H1975 p21 siMCM7#1 P = 0.006 n = 3; siMCM7#2 P = 0.03 n = 3. ( E , F ) Western blot showing MCM7 and p21 protein abundance as measured by densitometry and normalized to B-actin (left panel), and real-time quantification of their transcripts normalized to GAPDH (right panel) in H1299 cells treated with LNA inhibitors for miR-25, 93 and 106b (E) and H1975 cell line (F). All the experiments were performed in triplicate. ( G ) Cartoon representing the newly characterized mechanism through which YAP/TAZ and TEAD by binding to MCM7 enhancer transcriptionally regulate both MCM7 gene and the hosted miR cluster miR-25, 93 and 106b, that in turn post-transcriptionally regulate p21 abundance eventually affecting cell proliferation (left panel). Treatment of cells with statin determines YAP/TAZ exclusion from nuclei, impairs transcription of MCM7 gene and hosted miRs cluster and derepresses p21 thus inhibiting cell proliferation (right panel).

    Article Snippet: Western blotting was performed using the following primary antibodies: rabbit polyclonal anti YAP (Santa Cruz, sc-15407), rabbit polyclonal anti TAZ (Sigma anti-WWTR1, HPA007415), mouse monoclonal anti B-actin (ACTBD11B7, Santa Cruz, sc-81178), rabbit monoclonal anti MCM7 (D10A11, Cell Signaling, 3735S), mouse monoclonal anti-TEF-1 (BD-Transduction Laboratories, 610923), rabbit monoclonal anti-p21 Waf1/Cip1 (Cell Signaling, 2947S), rabbit polyclonal anti Phospho-YAP Ser127 (Cell Signaling, 4911), mouse monoclonal anti-HSP90 (Santa Cruz, sc-13119), and rabbit polyclonal anti-p21 (Santa Cruz, sc-397).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Binding Assay

    Synergistic effect of 5-fluorouracil (5-FU) and human interferon-beta (IFN-β) for treating MDA-MB-231 cells MDA-MB-231 cells were co-treated with 5-FU 1.0 μg/ml and IFN-β 500 Unit/ml for different lengths of time. Cell lysates were used for immunoblotting analysis and incubated with primary antibodies, BAX, p21 (Cip1/Waf1), p53, and phospho-p38. ( A ) Expression of BAX protein. ( B ) The relative value of BAX protein. ( C ) Expression of p21 (Cip1/Waf1) protein. ( D ) The relative value of p21 (Cip1/Waf1) protein. ( E ) Expression of p53 and pp38 protein. ( F ) The relative value of p53 and pp38 protein. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (1.0 μg/ml), U: 5-FU treatment (1.0 μg/ml), B: IFN-β treatment (500 Unit/ml), U + B (5-FU 1.0 μg/ml and IFN-β 500 Unit/ml co-treatment). Data are presented as the mean ± SD of three different experiments each performed in triplicate. * p

    Journal: Oncotarget

    Article Title: Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer

    doi: 10.18632/oncotarget.6719

    Figure Lengend Snippet: Synergistic effect of 5-fluorouracil (5-FU) and human interferon-beta (IFN-β) for treating MDA-MB-231 cells MDA-MB-231 cells were co-treated with 5-FU 1.0 μg/ml and IFN-β 500 Unit/ml for different lengths of time. Cell lysates were used for immunoblotting analysis and incubated with primary antibodies, BAX, p21 (Cip1/Waf1), p53, and phospho-p38. ( A ) Expression of BAX protein. ( B ) The relative value of BAX protein. ( C ) Expression of p21 (Cip1/Waf1) protein. ( D ) The relative value of p21 (Cip1/Waf1) protein. ( E ) Expression of p53 and pp38 protein. ( F ) The relative value of p53 and pp38 protein. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (1.0 μg/ml), U: 5-FU treatment (1.0 μg/ml), B: IFN-β treatment (500 Unit/ml), U + B (5-FU 1.0 μg/ml and IFN-β 500 Unit/ml co-treatment). Data are presented as the mean ± SD of three different experiments each performed in triplicate. * p

    Article Snippet: Next, the membrane was incubated with primary antibody, mouse monoclonal anti-p21 (Cip1/Waf1) (1:1,000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p53 (1:1,000 dilution, Santa Cruz Biotechnology, Inc. Dallas, TX, USA), anti-BAX (1:1,000 dilution, Cell Signaling Technology, Inc.), anti-GAPDH (1:1,000 dilution, Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-phospho-Erk1/2 (1:1,000 dilution, Cell Signaling Technology, Inc.), anti-phospho-Akt1/2/3 (1:1,000 dilution, Cell Signaling Technology, Inc.), anti-c-fos (1:2,000 dilution, Abcam plc., Cambridge, UK), or anti-phospho-p38 (1:1,000 dilution, Cell Signaling Technology, Inc.) in 1 × TBS with 3% (w/v) BSA (Sigma-Aldrich Co.) and 0.1% (v/v) Tween 20 overnight at 4°C.

    Techniques: Multiple Displacement Amplification, Incubation, Expressing, Negative Control

    Alteration of apoptosis and proliferation related protein expression in MDA-MB-231 cells after 5-fluorouracil (5-FU) treatment To confirm the effects of 5-FU, serially diluted 5-FU (0.5, 1.0, and 5.0 μg/ml) and 5-fluorocytosine (5-FC) at 5.0 μg/ml were applied to MDA-MB-231 cells, which were then cultured in 6-well plates. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with specific antibodies to BAX, c-fos, and p21 (Cip1/Waf1). ( A ) Expression of BAX and c-fos protein. ( B ) The relative value of BAX protein levels. (C) The relative value of c-fos protein levels. ( D ) The expression of p21 (Cip1/Waf1) protein. ( E ) Graph of p21 (Cip1/Waf1) protein levels. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (5.0 μg/ml), U: 5-FU treatment (0.5, 1.0, and 5.0 μg/ml, from the left). Data are presented as the mean ± SD of three different experiments each performed in triplicate. * p

    Journal: Oncotarget

    Article Title: Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer

    doi: 10.18632/oncotarget.6719

    Figure Lengend Snippet: Alteration of apoptosis and proliferation related protein expression in MDA-MB-231 cells after 5-fluorouracil (5-FU) treatment To confirm the effects of 5-FU, serially diluted 5-FU (0.5, 1.0, and 5.0 μg/ml) and 5-fluorocytosine (5-FC) at 5.0 μg/ml were applied to MDA-MB-231 cells, which were then cultured in 6-well plates. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with specific antibodies to BAX, c-fos, and p21 (Cip1/Waf1). ( A ) Expression of BAX and c-fos protein. ( B ) The relative value of BAX protein levels. (C) The relative value of c-fos protein levels. ( D ) The expression of p21 (Cip1/Waf1) protein. ( E ) Graph of p21 (Cip1/Waf1) protein levels. N: negative control (no treatment with 5-FU or 5-FC), C: 5-FC treatment (5.0 μg/ml), U: 5-FU treatment (0.5, 1.0, and 5.0 μg/ml, from the left). Data are presented as the mean ± SD of three different experiments each performed in triplicate. * p

    Article Snippet: Next, the membrane was incubated with primary antibody, mouse monoclonal anti-p21 (Cip1/Waf1) (1:1,000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p53 (1:1,000 dilution, Santa Cruz Biotechnology, Inc. Dallas, TX, USA), anti-BAX (1:1,000 dilution, Cell Signaling Technology, Inc.), anti-GAPDH (1:1,000 dilution, Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-phospho-Erk1/2 (1:1,000 dilution, Cell Signaling Technology, Inc.), anti-phospho-Akt1/2/3 (1:1,000 dilution, Cell Signaling Technology, Inc.), anti-c-fos (1:2,000 dilution, Abcam plc., Cambridge, UK), or anti-phospho-p38 (1:1,000 dilution, Cell Signaling Technology, Inc.) in 1 × TBS with 3% (w/v) BSA (Sigma-Aldrich Co.) and 0.1% (v/v) Tween 20 overnight at 4°C.

    Techniques: Expressing, Multiple Displacement Amplification, Cell Culture, SDS Page, Negative Control

    SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing CDKN1A expression.

    Journal: PLoS ONE

    Article Title: An Axis Involving SNAI1, microRNA-128 and SP1 Modulates Glioma Progression

    doi: 10.1371/journal.pone.0098651

    Figure Lengend Snippet: SNAI1/miR-128/SP1 axis regulates glioma progression. SNAI1 suppresses miR-128 expression and miR-128 restrains SP1 expression. SNAI1 increases SP1 expression through miR-128. The SNAI1/miR-128/SP1 axis promotes cell invasion, cell migration, cell proliferation and cell cycle via increasing MMP-2, MMP-9, PLAU and CCNE1 expression and reducing CDKN1A expression.

    Article Snippet: Primary antibodies were as follows: SNAI1 (1∶200, RnD systems), SP1 (1∶1000, Cell Signalling), CDKN1a (1∶1000, Cell Signalling), CCNE1 (1∶1000, Cell Signalling), MMP2 (1∶1000, Cell Signalling), MMP9 (1∶1000, Cell Signalling), PLAU (1∶150, Abcam), GAPDH (1∶5000, Bioworld) and β-actin (1∶5000, Bioworld).

    Techniques: Expressing, Migration

    Rescue of pathological features of LVNC iPSC-CMs a–b , Percentage of EdU + control and LVNC iPSC-CMs (a) or scramble control and TBX20 knock-down ESC-CMs (b) at 2 weeks after induction of cardiac differentiation with or without treatment of TGFβ receptor-1 inhibitors (SD208 or RepSox) for 2 continuous days. n=7 independent experiments. c , Adenoviral mediated overexpression of dominant negative form of TGFβ receptor-2 (TGFBRIIDN) significantly restored proliferative potential in LVNC iPSC-CMs compared to control (adenoviral mediated GFP overexpression; GFP-Ad). n=8 independent experiments. d , Knockdown efficiency of CDKN1A protein (upper) and mRNA (lower) in iPSC-CMs by siRNA. n=6 independent experiments. e , Immunostaining for nuclear (blue), TNNT2 (green), and EdU (red) in control and LVNC iPSC-CMs with CDKN1A or scramble siRNA knockdown. f , Percentage of EdU + iPSC-CMs at 2 weeks after induction of cardiac differentiation with CDKN1A or scramble siRNA knockdown. n=6 independent experiments. g , The efficiency of cardiac differentiation of LVNC and mutation corrected LVNC iPSC lines before glucose deprivation as validated by FACS for TNNT2. n=5 independent experiments per each group. h , mRNA expression of cardiac transcription factors in differentiating LVNC and mutation corrected LVNC iPSCs at day 6 and day 9 after induction of cardiac differentiation. LVNC n=6 independent experiments; LVNC corrected n=5 independent experiments. i , Immunostaining for nuclear (blue), TNNT2 (green), and EdU (red) in control, LVNC, and TBX20 mutation corrected (LVNC corrected) iPSC-CMs. j , Percentage of EdU + iPSC-CMs at 2 weeks in control, LVNC, and LVNC corrected group. n=6 independent experiments. k , Reversible CDKN1A, TGFB1, and PRDM16 mRNA expression abnormality in LVNC corrected iPSC-CMs compared to LVNC iPSC-CMs. n=6 independent experiments. CON, unrelated controls. Scale bars, 100 μm. *p

    Journal: Nature cell biology

    Article Title: iPSC-derived cardiomyocytes reveal abnormal TGFβ signaling in left ventricular non-compaction cardiomyopathy

    doi: 10.1038/ncb3411

    Figure Lengend Snippet: Rescue of pathological features of LVNC iPSC-CMs a–b , Percentage of EdU + control and LVNC iPSC-CMs (a) or scramble control and TBX20 knock-down ESC-CMs (b) at 2 weeks after induction of cardiac differentiation with or without treatment of TGFβ receptor-1 inhibitors (SD208 or RepSox) for 2 continuous days. n=7 independent experiments. c , Adenoviral mediated overexpression of dominant negative form of TGFβ receptor-2 (TGFBRIIDN) significantly restored proliferative potential in LVNC iPSC-CMs compared to control (adenoviral mediated GFP overexpression; GFP-Ad). n=8 independent experiments. d , Knockdown efficiency of CDKN1A protein (upper) and mRNA (lower) in iPSC-CMs by siRNA. n=6 independent experiments. e , Immunostaining for nuclear (blue), TNNT2 (green), and EdU (red) in control and LVNC iPSC-CMs with CDKN1A or scramble siRNA knockdown. f , Percentage of EdU + iPSC-CMs at 2 weeks after induction of cardiac differentiation with CDKN1A or scramble siRNA knockdown. n=6 independent experiments. g , The efficiency of cardiac differentiation of LVNC and mutation corrected LVNC iPSC lines before glucose deprivation as validated by FACS for TNNT2. n=5 independent experiments per each group. h , mRNA expression of cardiac transcription factors in differentiating LVNC and mutation corrected LVNC iPSCs at day 6 and day 9 after induction of cardiac differentiation. LVNC n=6 independent experiments; LVNC corrected n=5 independent experiments. i , Immunostaining for nuclear (blue), TNNT2 (green), and EdU (red) in control, LVNC, and TBX20 mutation corrected (LVNC corrected) iPSC-CMs. j , Percentage of EdU + iPSC-CMs at 2 weeks in control, LVNC, and LVNC corrected group. n=6 independent experiments. k , Reversible CDKN1A, TGFB1, and PRDM16 mRNA expression abnormality in LVNC corrected iPSC-CMs compared to LVNC iPSC-CMs. n=6 independent experiments. CON, unrelated controls. Scale bars, 100 μm. *p

    Article Snippet: Primary antibodies used were biotinylated SMAD2/3 (Cell Signaling), phospho-SMAD2/3 (Cell Signaling), CDKN1A (Cell Signaling), TBX20 (Sigma Aldrich), and HRP conjugated α–Tubulin (Cell Signaling).

    Techniques: Over Expression, Dominant Negative Mutation, Immunostaining, Mutagenesis, FACS, Expressing

    Upregulation of TGFβ signaling in LVNC phenotype a , Upstream regulator analysis of signaling pathway comparing LVNC and control iPSC-CMs at 2 weeks after induction of cardiac differentiation. The p -value measures whether there is a statistically significant overlap between the dataset genes and the genes that are regulated by a transcription regulator. b , Heat map showing upregulation of TGFβ signaling pathway in LVNC (III-2, 3, 4; mean of four samples) and mild DCM (II-2; mean of two samples) compared to control iPSC-CMs (unrelated controls; mean of two samples). Mean=0, variance=1. c , Western blot of total and phospho-SMAD2/3 in control and patient-specific iPSC-CMs (upper) and densitometry analysis, normalized against α-tubulin (lower). d , Western blot of CDKN1A protein in control and patient-specific iPSC-CMs (upper) and densitometry analysis, normalized against α-tubulin (lower). e , Immunostaining of nuclear (blue), alpha-sarcomeric actin (green), and phospho-SMAD2 (red) in LV of control donor heart tissue vs. explanted heart of proband #1. f , Allele-specific mRNA expression analysis by mRNA-sequencing (upper panel) and digital droplet PCR (lower panel) showed a higher ratio of TBX20 mutant allele expression in LVNC iPSC-CMs compared to mild DCM iPSC-CMs. n=6 independent experiments. g , The effect of TGFβ isoform treatments on the percentage of EdU + cardiomyocytes in control iPSC-CMs with or without growth factors. PBS treated control; n = 4 independent experiments, TGFβ treated samples; n = 3 independent experiments. CON, unrelated controls. * p

    Journal: Nature cell biology

    Article Title: iPSC-derived cardiomyocytes reveal abnormal TGFβ signaling in left ventricular non-compaction cardiomyopathy

    doi: 10.1038/ncb3411

    Figure Lengend Snippet: Upregulation of TGFβ signaling in LVNC phenotype a , Upstream regulator analysis of signaling pathway comparing LVNC and control iPSC-CMs at 2 weeks after induction of cardiac differentiation. The p -value measures whether there is a statistically significant overlap between the dataset genes and the genes that are regulated by a transcription regulator. b , Heat map showing upregulation of TGFβ signaling pathway in LVNC (III-2, 3, 4; mean of four samples) and mild DCM (II-2; mean of two samples) compared to control iPSC-CMs (unrelated controls; mean of two samples). Mean=0, variance=1. c , Western blot of total and phospho-SMAD2/3 in control and patient-specific iPSC-CMs (upper) and densitometry analysis, normalized against α-tubulin (lower). d , Western blot of CDKN1A protein in control and patient-specific iPSC-CMs (upper) and densitometry analysis, normalized against α-tubulin (lower). e , Immunostaining of nuclear (blue), alpha-sarcomeric actin (green), and phospho-SMAD2 (red) in LV of control donor heart tissue vs. explanted heart of proband #1. f , Allele-specific mRNA expression analysis by mRNA-sequencing (upper panel) and digital droplet PCR (lower panel) showed a higher ratio of TBX20 mutant allele expression in LVNC iPSC-CMs compared to mild DCM iPSC-CMs. n=6 independent experiments. g , The effect of TGFβ isoform treatments on the percentage of EdU + cardiomyocytes in control iPSC-CMs with or without growth factors. PBS treated control; n = 4 independent experiments, TGFβ treated samples; n = 3 independent experiments. CON, unrelated controls. * p

    Article Snippet: Primary antibodies used were biotinylated SMAD2/3 (Cell Signaling), phospho-SMAD2/3 (Cell Signaling), CDKN1A (Cell Signaling), TBX20 (Sigma Aldrich), and HRP conjugated α–Tubulin (Cell Signaling).

    Techniques: Western Blot, Immunostaining, Expressing, Sequencing, Polymerase Chain Reaction, Mutagenesis

    RT-PCR and Western blot validation of microarrays. (A) RT-PCR analysis of selected genes. Induction of dnTCF1E WT and dnTCF1E mut decreased AXIN2 and cMYC mRNA expression. Induction of dnTCF1E WT but not dnTCF1E mut caused a decrease in SP5 mRNA and an increase in CDKN1A (p21) mRNA levels. (B) Stable cell lines were induced with doxycycline, and lysates were collected over the course of 9 h. Induction of dnTCF1E WT with 0.0005 μg/ml doxycycline caused an increase in p21, a decrease in SP5, and no change in cMYC protein levels. Induction of dnTCF1E mut with 1 μg/ml doxycycline caused little or no change in p21, SP5, and cMYC protein levels. Western blot analysis shows that induced protein levels of dnTCF1E WT and dnTCF1E mut were equivalent. (C) dnTCF1E WT -expressing stable cells were transfected with EVR2 (mock) and increasing amounts of FLAG-SP5 expression plasmid (500 ng, 1 μg, and 2 μg). After 24 h, mock- and SP5-transfected cells were induced with doxycycline, and lysates were collected after an additional 9 h to allow induction of dnTCF1E WT . (D) SP5 binds near the p21 transcription start site. ChIP was performed with mock- or FLAG-SP5-transfected DLD1 cells. FLAG-SP5 was immunoprecipitated from cellular lysates with IgG-agarose or FLAG-agarose beads. Quantitative PCR was performed with the indicated primer set. FLAG-SP5 was enriched near the GC-rich p21 transcription start site but not at a downstream intragenic region.

    Journal: Molecular and Cellular Biology

    Article Title: A WNT/p21 Circuit Directed by the C-Clamp, a Sequence-Specific DNA Binding Domain in TCFs

    doi: 10.1128/MCB.06769-11

    Figure Lengend Snippet: RT-PCR and Western blot validation of microarrays. (A) RT-PCR analysis of selected genes. Induction of dnTCF1E WT and dnTCF1E mut decreased AXIN2 and cMYC mRNA expression. Induction of dnTCF1E WT but not dnTCF1E mut caused a decrease in SP5 mRNA and an increase in CDKN1A (p21) mRNA levels. (B) Stable cell lines were induced with doxycycline, and lysates were collected over the course of 9 h. Induction of dnTCF1E WT with 0.0005 μg/ml doxycycline caused an increase in p21, a decrease in SP5, and no change in cMYC protein levels. Induction of dnTCF1E mut with 1 μg/ml doxycycline caused little or no change in p21, SP5, and cMYC protein levels. Western blot analysis shows that induced protein levels of dnTCF1E WT and dnTCF1E mut were equivalent. (C) dnTCF1E WT -expressing stable cells were transfected with EVR2 (mock) and increasing amounts of FLAG-SP5 expression plasmid (500 ng, 1 μg, and 2 μg). After 24 h, mock- and SP5-transfected cells were induced with doxycycline, and lysates were collected after an additional 9 h to allow induction of dnTCF1E WT . (D) SP5 binds near the p21 transcription start site. ChIP was performed with mock- or FLAG-SP5-transfected DLD1 cells. FLAG-SP5 was immunoprecipitated from cellular lysates with IgG-agarose or FLAG-agarose beads. Quantitative PCR was performed with the indicated primer set. FLAG-SP5 was enriched near the GC-rich p21 transcription start site but not at a downstream intragenic region.

    Article Snippet: Blots were probed with 1:2,000 anti-Flag antibody (Sigma), 1:1,000 p21 antibody (Cell Signaling), 1:500 SP5 antibody (Abcam), 1:1,000 cMYC antibody (Cell Signaling), and 1:2,000 lamin AC antibody (Cell Signaling).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Mechanism of XWL-1-48 inhibits growth of breast cancer cells. XWL-1-48, a novel orally topoisomeraseII inhibitor, exerts potent in vitro and in vivo anti-tumor activity against breast cancer. Treatment with XWL-1-48 significantly inhibited TopoII activity, triggered DNA damage response, activated ATM/p53/p21 pathways, arrested cell cycle at S phase, and induced mitochondria-mediated apoptosis. Meanwhile, XWL-1-48 strongly blocked PI3K/Akt/Mdm2 pathway, prompted degradation of Mdm2, and suppressed breast cancer cell survival

    Journal: Cell Communication and Signaling : CCS

    Article Title: DNA damage and apoptosis induced by a potent orally podophyllotoxin derivative in breast cancer

    doi: 10.1186/s12964-018-0263-9

    Figure Lengend Snippet: Mechanism of XWL-1-48 inhibits growth of breast cancer cells. XWL-1-48, a novel orally topoisomeraseII inhibitor, exerts potent in vitro and in vivo anti-tumor activity against breast cancer. Treatment with XWL-1-48 significantly inhibited TopoII activity, triggered DNA damage response, activated ATM/p53/p21 pathways, arrested cell cycle at S phase, and induced mitochondria-mediated apoptosis. Meanwhile, XWL-1-48 strongly blocked PI3K/Akt/Mdm2 pathway, prompted degradation of Mdm2, and suppressed breast cancer cell survival

    Article Snippet: The membrane was immunoblotted with anti-γ - H2AX, anti-p21, anti-p-ATM, anti-ATM, anti-p-Mdm2, anti-Mdm2, anti-p-p53, anti-AKT, anti-β-actin (Cell Signaling Technology, USA), anti-p53, anti-Bax and anti-Bcl-2 (Santa Cruz, USA) antibodies in 5% milk TBST, at 4 °C overnight.

    Techniques: In Vitro, In Vivo, Activity Assay

    Effect of XWL-1-48 on ROS production and DNA damage, a , b After the cells treated with desired concentrations of XWL-1-48, GL331 and H 2 O 2 for 24 h, ROS production was determined by flow cytometry. Columns represent the mean ± SD values obtained from three individual experiments; c MCF-7 cells were pre-treated with or without GSH (5 mM) for 1 h and then with XWL-1-48 (10 μM) for 24 h. The cell viability was determined by the MTT assay; d MCF-7 cells were treated with various concentration of XWL-1-48 or GL331 for 24 h, induction of γ-H2AX was determined by western blot; e MCF-7 cells were incubated with XWL-1-48 (10 μM) for indicated time point, γ-H2AX expression was measured; f Immunofluorescent staining of γ-H2AX in MCF-7 cells. Cells were incubated in 6-well plate overnight and then exposed to XWL-1-48 (1 μM) for 24 h. Cells were incubated with γ-H2AX antibody followed by incubation of secondary anti-rabbit IgG-FITC antibody (green). The nucleus was stained with PI (red). Images were captured using fluorescence microscope. Scale bar, 25 μm. g XWL-1-48 induced DNA damage by triggering ATM-related signaling pathways in MCF-7 cells. Expression levels of ATM, p-ATM, p-p53(ser15), p53 and p21 were analyzed. h , i MCF-7 cells were incubated with XWL-1-48 (10 μM) for the indicated time-point, the expression level of p-p53(ser15), p53 and p21 were determined by immunoblot. Data were shown as mean ± SD of three independent experiments. * p

    Journal: Cell Communication and Signaling : CCS

    Article Title: DNA damage and apoptosis induced by a potent orally podophyllotoxin derivative in breast cancer

    doi: 10.1186/s12964-018-0263-9

    Figure Lengend Snippet: Effect of XWL-1-48 on ROS production and DNA damage, a , b After the cells treated with desired concentrations of XWL-1-48, GL331 and H 2 O 2 for 24 h, ROS production was determined by flow cytometry. Columns represent the mean ± SD values obtained from three individual experiments; c MCF-7 cells were pre-treated with or without GSH (5 mM) for 1 h and then with XWL-1-48 (10 μM) for 24 h. The cell viability was determined by the MTT assay; d MCF-7 cells were treated with various concentration of XWL-1-48 or GL331 for 24 h, induction of γ-H2AX was determined by western blot; e MCF-7 cells were incubated with XWL-1-48 (10 μM) for indicated time point, γ-H2AX expression was measured; f Immunofluorescent staining of γ-H2AX in MCF-7 cells. Cells were incubated in 6-well plate overnight and then exposed to XWL-1-48 (1 μM) for 24 h. Cells were incubated with γ-H2AX antibody followed by incubation of secondary anti-rabbit IgG-FITC antibody (green). The nucleus was stained with PI (red). Images were captured using fluorescence microscope. Scale bar, 25 μm. g XWL-1-48 induced DNA damage by triggering ATM-related signaling pathways in MCF-7 cells. Expression levels of ATM, p-ATM, p-p53(ser15), p53 and p21 were analyzed. h , i MCF-7 cells were incubated with XWL-1-48 (10 μM) for the indicated time-point, the expression level of p-p53(ser15), p53 and p21 were determined by immunoblot. Data were shown as mean ± SD of three independent experiments. * p

    Article Snippet: The membrane was immunoblotted with anti-γ - H2AX, anti-p21, anti-p-ATM, anti-ATM, anti-p-Mdm2, anti-Mdm2, anti-p-p53, anti-AKT, anti-β-actin (Cell Signaling Technology, USA), anti-p53, anti-Bax and anti-Bcl-2 (Santa Cruz, USA) antibodies in 5% milk TBST, at 4 °C overnight.

    Techniques: Flow Cytometry, Cytometry, MTT Assay, Concentration Assay, Western Blot, Incubation, Expressing, Staining, Fluorescence, Microscopy

    Observed phenotype following CDK2AP1 knockdown is p53 dependent. Panel A: Primary HDFs were transduced with EV or CDK2AP1-shRNA or CDK2AP1-shRNA and a p53 specific shRNA and were analyzed by qPCR for mRNA levels of p53 , p21 , BAX and PUMA . Knockdown of p53 with CDK2AP1 prevented the increase in p21, BAX and PUMA. Panel B: The knockdown of p53 with CDK2AP1 increased the percentage of cells in the S and reduced the cells in G1 when compared to CDK2AP1 only knockdown cells (p-value

    Journal: PLoS ONE

    Article Title: Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

    doi: 10.1371/journal.pone.0120782

    Figure Lengend Snippet: Observed phenotype following CDK2AP1 knockdown is p53 dependent. Panel A: Primary HDFs were transduced with EV or CDK2AP1-shRNA or CDK2AP1-shRNA and a p53 specific shRNA and were analyzed by qPCR for mRNA levels of p53 , p21 , BAX and PUMA . Knockdown of p53 with CDK2AP1 prevented the increase in p21, BAX and PUMA. Panel B: The knockdown of p53 with CDK2AP1 increased the percentage of cells in the S and reduced the cells in G1 when compared to CDK2AP1 only knockdown cells (p-value

    Article Snippet: Cell lysates were prepared from wild type and CDK2AP1 knockdown primary human fibroblasts and analyzed for p53, p21 and β-tubulin expression by Western blot using specific antibodies. p53 and p21 antibodies were obtained from Cell Signaling, MA, p14ARF antibodies were obtained from Santa Cruz Technologies, while β-tubulin antibody was obtained from Developmental Studies Hybridoma Bank, IA.

    Techniques: Transduction, shRNA, Real-time Polymerase Chain Reaction, Significance Assay

    Knockdown of CDK2AP1 increases the expression of p53, p21 and the Apoptotic Genes BAX and PUMA . Panel A: HDFs transduced with EV, CDK2AP1-shRNA1 or CDK2AP1-shRNA2 were harvested and analyzed for the mRNA expression of p53 , p21 , BAX and PUMA . Knockdown of CDK2AP1 in HDFs increased the expression of these genes. Panel B: Whole cell lysates were extracted from wild and knockdown HDFs and analyzed for p21 and p53 levels by Western Blot. Panel C: Whole cell lysates were extracted from wild and knockdown HDFs and analyzed for p14 ARF by Western Blot.

    Journal: PLoS ONE

    Article Title: Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

    doi: 10.1371/journal.pone.0120782

    Figure Lengend Snippet: Knockdown of CDK2AP1 increases the expression of p53, p21 and the Apoptotic Genes BAX and PUMA . Panel A: HDFs transduced with EV, CDK2AP1-shRNA1 or CDK2AP1-shRNA2 were harvested and analyzed for the mRNA expression of p53 , p21 , BAX and PUMA . Knockdown of CDK2AP1 in HDFs increased the expression of these genes. Panel B: Whole cell lysates were extracted from wild and knockdown HDFs and analyzed for p21 and p53 levels by Western Blot. Panel C: Whole cell lysates were extracted from wild and knockdown HDFs and analyzed for p14 ARF by Western Blot.

    Article Snippet: Cell lysates were prepared from wild type and CDK2AP1 knockdown primary human fibroblasts and analyzed for p53, p21 and β-tubulin expression by Western blot using specific antibodies. p53 and p21 antibodies were obtained from Cell Signaling, MA, p14ARF antibodies were obtained from Santa Cruz Technologies, while β-tubulin antibody was obtained from Developmental Studies Hybridoma Bank, IA.

    Techniques: Expressing, Transduction, Western Blot

    CDKN1A mutations in bladder cancer

    Journal: Molecular cancer therapeutics

    Article Title: Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target

    doi: 10.1158/1535-7163.MCT-14-0622-T

    Figure Lengend Snippet: CDKN1A mutations in bladder cancer

    Article Snippet: Antibodies against CDKN1A, TP53, PARP-1, P-γH2Ax, p-Chk1-317, p-Chk-345 were purchased from Cell Signaling Tech. (New Bedford, MA).

    Techniques:

    p21 expression is critical to the response to combination treatment in p21 deficient bladder cancer cell lines

    Journal: Molecular cancer therapeutics

    Article Title: Combined CDKN1A/TP53 mutation in bladder cancer is a therapeutic target

    doi: 10.1158/1535-7163.MCT-14-0622-T

    Figure Lengend Snippet: p21 expression is critical to the response to combination treatment in p21 deficient bladder cancer cell lines

    Article Snippet: Antibodies against CDKN1A, TP53, PARP-1, P-γH2Ax, p-Chk1-317, p-Chk-345 were purchased from Cell Signaling Tech. (New Bedford, MA).

    Techniques: Expressing

    Stress hormones induce ATR, Chk1, and P21 in TNBC cells. ( A ) MDA-MB-231 cells were incubated with Cort or NE in the presence or absence of receptor antagonists, RU-486 and propranolol for 2 and 6 h and cell lysates were prepared and resolved by SDS-PAGE and analysed for phospho ATR (ser 428) and phospho Chk1 (ser 345) by western blotting ( n =4 and n =3). Total protein levels of ATR and Chk1 were included as controls ( n =2). Density of western blot bands was analysed using Image J software ( http://rsbweb.nih.gov/ij/ ). Densities of phospho ATR and Chk-1 phosphorylation in stress hormone-treated proteins bands were normalised to density of actin ( B – D ). Theoretical molecular masses in kilodaltons are shown. Stress hormones increased the expression of ATR, CHK1, and p21 proteins in MDA-MB-231 cells. * P > 0.05 and ** P > 0.005.

    Journal: British Journal of Cancer

    Article Title: Stress hormones reduce the efficacy of paclitaxel in triple negative breast cancer through induction of DNA damage

    doi: 10.1038/bjc.2015.133

    Figure Lengend Snippet: Stress hormones induce ATR, Chk1, and P21 in TNBC cells. ( A ) MDA-MB-231 cells were incubated with Cort or NE in the presence or absence of receptor antagonists, RU-486 and propranolol for 2 and 6 h and cell lysates were prepared and resolved by SDS-PAGE and analysed for phospho ATR (ser 428) and phospho Chk1 (ser 345) by western blotting ( n =4 and n =3). Total protein levels of ATR and Chk1 were included as controls ( n =2). Density of western blot bands was analysed using Image J software ( http://rsbweb.nih.gov/ij/ ). Densities of phospho ATR and Chk-1 phosphorylation in stress hormone-treated proteins bands were normalised to density of actin ( B – D ). Theoretical molecular masses in kilodaltons are shown. Stress hormones increased the expression of ATR, CHK1, and p21 proteins in MDA-MB-231 cells. * P > 0.05 and ** P > 0.005.

    Article Snippet: Blots were blocked with 5% BSA in 0.1% TBS-T followed by probing with the following primary antibodies p21; Cell Signaling, Phospho-ATR (Ser428), ATR Phospho-Chk1 (Ser345) (133D3) Rabbit mAb, (DNA Damage Antibody Sampler Kit #9947), ATR, Cell Signaling (2390) Rabbit mAb, Chk-1 goat mAB (Abcam AB2845, Cambridge, MA, USA), Cell Signaling and phospho histone γH2AX; Millipore, Temecula, CA, USA), and then incubated with corresponding HRP-conjugated secondary antibodies.

    Techniques: Multiple Displacement Amplification, Incubation, SDS Page, Western Blot, Software, Expressing

    P21 knockdown prevents G1 arrest in MDA-MB-231 cells. MDA-MB 231 cells were treated with stress hormones for 24 h after knockdown of p21 by siRNA. ( A ) Flow cytometry showing that there was a higher percentage of cells in the G1 phase (grey bar) of the cell cycle in cells treated with control siRNA and stress hormones. There was no significant difference in cells treated with p21 siRNA and stress hormones compared with unstim ( n =3). ( B ) Western blot showing p21 knockdown in MDA-MB-231 cells. Stress hormones had no effect on cell cycle regulation ( C ) and cell viability ( D ) in the p21 null MDA-MB 436 cell lines. Stress hormones do not induce phospho ATR or Chk-1 ( E ). Outcomes were compared using analysis of variance followed by planned paired comparisons. ** P > 0.01.

    Journal: British Journal of Cancer

    Article Title: Stress hormones reduce the efficacy of paclitaxel in triple negative breast cancer through induction of DNA damage

    doi: 10.1038/bjc.2015.133

    Figure Lengend Snippet: P21 knockdown prevents G1 arrest in MDA-MB-231 cells. MDA-MB 231 cells were treated with stress hormones for 24 h after knockdown of p21 by siRNA. ( A ) Flow cytometry showing that there was a higher percentage of cells in the G1 phase (grey bar) of the cell cycle in cells treated with control siRNA and stress hormones. There was no significant difference in cells treated with p21 siRNA and stress hormones compared with unstim ( n =3). ( B ) Western blot showing p21 knockdown in MDA-MB-231 cells. Stress hormones had no effect on cell cycle regulation ( C ) and cell viability ( D ) in the p21 null MDA-MB 436 cell lines. Stress hormones do not induce phospho ATR or Chk-1 ( E ). Outcomes were compared using analysis of variance followed by planned paired comparisons. ** P > 0.01.

    Article Snippet: Blots were blocked with 5% BSA in 0.1% TBS-T followed by probing with the following primary antibodies p21; Cell Signaling, Phospho-ATR (Ser428), ATR Phospho-Chk1 (Ser345) (133D3) Rabbit mAb, (DNA Damage Antibody Sampler Kit #9947), ATR, Cell Signaling (2390) Rabbit mAb, Chk-1 goat mAB (Abcam AB2845, Cambridge, MA, USA), Cell Signaling and phospho histone γH2AX; Millipore, Temecula, CA, USA), and then incubated with corresponding HRP-conjugated secondary antibodies.

    Techniques: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Western Blot

    Proposed diagram of a stress hormone effect on an induction of DNA damage through ATR, Chk-1, and p21.

    Journal: British Journal of Cancer

    Article Title: Stress hormones reduce the efficacy of paclitaxel in triple negative breast cancer through induction of DNA damage

    doi: 10.1038/bjc.2015.133

    Figure Lengend Snippet: Proposed diagram of a stress hormone effect on an induction of DNA damage through ATR, Chk-1, and p21.

    Article Snippet: Blots were blocked with 5% BSA in 0.1% TBS-T followed by probing with the following primary antibodies p21; Cell Signaling, Phospho-ATR (Ser428), ATR Phospho-Chk1 (Ser345) (133D3) Rabbit mAb, (DNA Damage Antibody Sampler Kit #9947), ATR, Cell Signaling (2390) Rabbit mAb, Chk-1 goat mAB (Abcam AB2845, Cambridge, MA, USA), Cell Signaling and phospho histone γH2AX; Millipore, Temecula, CA, USA), and then incubated with corresponding HRP-conjugated secondary antibodies.

    Techniques:

    A-B. p21 but not p27 is involved in TNF-α induced inhibition of proliferation . (A) LN-18 cells were transfected with p21 siRNA oligonucleotides (100 nM) or (B) p27 antisense (As) oligonucleotides (200 nM) for 6 hr and cultured as monolayers or spheroids for 18 hr. Cells were treated with TNF-α (10 ng/ml) for 24 hr, and tritiated thymidine incorporation assay was done as described in Fig. 1. * p

    Journal: Molecular Cancer

    Article Title: Differential expression and role of p21cip/waf1 and p27kip1 in TNF-?-induced inhibition of proliferation in human glioma cells

    doi: 10.1186/1476-4598-6-42

    Figure Lengend Snippet: A-B. p21 but not p27 is involved in TNF-α induced inhibition of proliferation . (A) LN-18 cells were transfected with p21 siRNA oligonucleotides (100 nM) or (B) p27 antisense (As) oligonucleotides (200 nM) for 6 hr and cultured as monolayers or spheroids for 18 hr. Cells were treated with TNF-α (10 ng/ml) for 24 hr, and tritiated thymidine incorporation assay was done as described in Fig. 1. * p

    Article Snippet: Transfection with p21 siRNA Cells were cultured for 24 hr and transfected with p21 siRNA oligonucleotides (50 nM) according to the manufacturer's instructions (Cell Signaling Technology, U.S.A).

    Techniques: Inhibition, Transfection, Cell Culture, Thymidine Incorporation Assay

    A-B. TNF-α-induced p21 does not have antiapoptotic function . (A) LN-18 cells were grown on coverslips and transfected with p21 siRNA oligonucleotides (100 nM) for 24 hr. Control and transfected cells were treated with TNF-α (10 ng/ml) for 3 hr. Cells were stained with p21 antibody followed by secondary antibody labeled with Cy3. DAPI was used for nuclear staining (blue fluorescence), merged images show nuclear localization of p21 (Magnification 63×). (B) Effect of TNF-α on viability in p21 siRNA transfected cells done by MTT assay. The graph represents mean +/- SD of 3 experiments done in duplicates.

    Journal: Molecular Cancer

    Article Title: Differential expression and role of p21cip/waf1 and p27kip1 in TNF-?-induced inhibition of proliferation in human glioma cells

    doi: 10.1186/1476-4598-6-42

    Figure Lengend Snippet: A-B. TNF-α-induced p21 does not have antiapoptotic function . (A) LN-18 cells were grown on coverslips and transfected with p21 siRNA oligonucleotides (100 nM) for 24 hr. Control and transfected cells were treated with TNF-α (10 ng/ml) for 3 hr. Cells were stained with p21 antibody followed by secondary antibody labeled with Cy3. DAPI was used for nuclear staining (blue fluorescence), merged images show nuclear localization of p21 (Magnification 63×). (B) Effect of TNF-α on viability in p21 siRNA transfected cells done by MTT assay. The graph represents mean +/- SD of 3 experiments done in duplicates.

    Article Snippet: Transfection with p21 siRNA Cells were cultured for 24 hr and transfected with p21 siRNA oligonucleotides (50 nM) according to the manufacturer's instructions (Cell Signaling Technology, U.S.A).

    Techniques: Transfection, Staining, Labeling, Fluorescence, MTT Assay

    Elimination of PJ34 activated p21 expression attenuates growth arrest. (A) Western blot for p21 and tubulin loading control in MCF7 and MCF7:PARP1KD cells untreated (Cont), transfected with either a non-specific siRNA (siRC), or siRNAs against p21 (siR1, siR2) for 18 hours followed by treatment with (+) or without (−) 50 µM PJ34 for 24 hours. (B) Normalized mitotic index plotted from parallel cultures treated as in (9 (A)). A single representative triplicate experiment is shown for Control or siRNA transfected cultures with (+) or without (−) 50 µM PJ34. Error bars, SEM (ns = not significant, ***p

    Journal: DNA repair

    Article Title: The PARP inhibitor PJ34 causes a PARP1-independent, p21 dependent mitotic arrest

    doi: 10.1016/j.dnarep.2011.07.006

    Figure Lengend Snippet: Elimination of PJ34 activated p21 expression attenuates growth arrest. (A) Western blot for p21 and tubulin loading control in MCF7 and MCF7:PARP1KD cells untreated (Cont), transfected with either a non-specific siRNA (siRC), or siRNAs against p21 (siR1, siR2) for 18 hours followed by treatment with (+) or without (−) 50 µM PJ34 for 24 hours. (B) Normalized mitotic index plotted from parallel cultures treated as in (9 (A)). A single representative triplicate experiment is shown for Control or siRNA transfected cultures with (+) or without (−) 50 µM PJ34. Error bars, SEM (ns = not significant, ***p

    Article Snippet: First, cells were transfected with either a non-specific control or two different p21 siRNAs (100 nM; Cell Signaling) using Lipofectamine 2000 (Invitrogen) and incubated for 16 hours.

    Techniques: Expressing, Western Blot, Transfection

    P21 deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control siRNA or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi)

    Journal: EBioMedicine

    Article Title: MDM2 Antagonists Counteract Drug-Induced DNA Damage

    doi: 10.1016/j.ebiom.2017.09.016

    Figure Lengend Snippet: P21 deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control siRNA or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi)

    Article Snippet: P21 siRNA (Cell Signaling, #6456) was mixed with in vivo JetPei (Polyplus-transfection SA, Illkirch, France) in accordance with manufacturer's recommendations and injected into the tumor.

    Techniques: Transfection

    Validation of top gene hits in ESCC cell lines. A. Cell proliferation assay of ESCC cells overexpressed CDKN1A, ELAVL2 or TSPAN4 under the treatment of PTX and DDP. B. Annexin V staining of ESCC cells overexpressed CDKN1A, ELAVL2 or TSPAN4 under the treatment of PTX and DDP. C. Western blot analysis of CDKN1A, ELAVL2, TSPAN4, Bax, P53 and GAPDH in ESCC cells overexpressed CDKN1A or ELAVL2. * P

    Journal: American Journal of Cancer Research

    Article Title: Genome-scale CRISPR activation screening identifies a role of ELAVL2-CDKN1A axis in paclitaxel resistance in esophageal squamous cell carcinoma

    doi:

    Figure Lengend Snippet: Validation of top gene hits in ESCC cell lines. A. Cell proliferation assay of ESCC cells overexpressed CDKN1A, ELAVL2 or TSPAN4 under the treatment of PTX and DDP. B. Annexin V staining of ESCC cells overexpressed CDKN1A, ELAVL2 or TSPAN4 under the treatment of PTX and DDP. C. Western blot analysis of CDKN1A, ELAVL2, TSPAN4, Bax, P53 and GAPDH in ESCC cells overexpressed CDKN1A or ELAVL2. * P

    Article Snippet: The membranes were blocked with Tris-buffered saline (pH 7.5) containing 0.2% Tween-20 and 5% nonfat milk at room temperature for 1 h. Primary antibodies used were as follows: rabbit anti-CDKN1A (Cell Signaling Technology, 1:1000 dilution), rabbit anti-TSPAN4 (Abcam, 1:5000 dilution), rabbit anti-ELAVL2 (Proteintech, 1:5000 dilution), rabbit anti-JUNB (Proteintech, 1:1000 dilution), rabbit anti-PAAF1 (Proteintech, 1:2000 dilution), rabbit anti-Bax (Proteintech, 1:1000 dilution), rabbit anti-p53 (Proteintech, 1:2000 dilution) and rabbit anti-GAPDH (Cell Signaling Technology, 1:5000 dilution).

    Techniques: Proliferation Assay, Staining, Western Blot

    CDK inhibitors are upregulated in breast cancer cells following treatment with HDAC inhibitors (A) CDK inhibitor genes CDKN1C, CDKN1A, CDKN2B, and CDKN2D were significantly (p value

    Journal: The pharmacogenomics journal

    Article Title: A genomic approach to predict synergistic combinations for breast cancer treatment

    doi: 10.1038/tpj.2011.48

    Figure Lengend Snippet: CDK inhibitors are upregulated in breast cancer cells following treatment with HDAC inhibitors (A) CDK inhibitor genes CDKN1C, CDKN1A, CDKN2B, and CDKN2D were significantly (p value

    Article Snippet: Total CDKN1C and CDKN1A expression was detected by primary antibody to CDKN1C (Cell Signaling), and mouse p21(Amersham).. GAPDH (AbCam) was used as loading control.

    Techniques: Significance Assay

    Figure 6. Amodiaquine treatment modulates cell cycle regulators (CDKN1A, RB1 [Ser780; Ser807/811], CCND1, E2F1) causing inhibition of proliferation and S phase cell cycle arrest. ( A ) Immunoblot detection of AQ-induced (≤ 20 µM;

    Journal: Autophagy

    Article Title: The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death

    doi: 10.4161/auto.26506

    Figure Lengend Snippet: Figure 6. Amodiaquine treatment modulates cell cycle regulators (CDKN1A, RB1 [Ser780; Ser807/811], CCND1, E2F1) causing inhibition of proliferation and S phase cell cycle arrest. ( A ) Immunoblot detection of AQ-induced (≤ 20 µM;

    Article Snippet: Membranes were incubated with primary antibodies in 5% milk-TBST overnight at 4 °C as follows: rabbit anti-phospho-RB1(Ser780) polyclonal (Cell Signaling Technology, 9307); rabbit anti-phospho-RB1(Ser807/811) polyclonal (Cell Signaling Technology, 9308); mouse anti-RB1 monoclonal (Cell Signaling Technology, 9309); mouse anti-CDKN1A monoclonal (Cell Signaling Technology, 2946); rabbit anti-LAMP1 monoclonal (Cell Signaling Technology, 3243); rabbit anti-SNCA polyclonal (Cell Signaling Technology, 4179); rabbit anti-RPL13A polyclonal (Cell Signaling Technology, 2765); rabbit anti-MYC monoclonal (Cell Signaling Technology, 5605P); mouse anti-TP53 monoclonal (Santa Cruz Biotechnology, sc-126); mouse anti-E2F1 monoclonal (Santa Cruz Biotechnology, sc-251); mouse anti-BECN1 monoclonal (Santa Cruz Biotechnology, sc-48341); anti-CCND1 polyclonal (Santa Cruz Biotechnology, sc-718); mouse anti-SQSTM1/p62 monoclonal (Santa Cruz Biotechnology, sc-48402); rabbit anti-LC3 polyclonal (Novus Biologics, 100-2331); mouse anti-HSPA1A/Hsp70 monoclonal (Enzo Life Sciences, SPA-810-F); anti-HSP90AA1/Hsp90 monoclonal (Enzo Life Sciences, SPA-836-D); mouse anti-amodiaquine monoclonal (Thermo Scientific, 320-04-02).

    Techniques: Inhibition

    Extracellular AGR2 enhances RhoA, CDC42 expression and stimulates FAK phosphorylation in NIH3T3 cells. (a) Western blot analysis of RhoA expression in NIH3T3 cells stimulated for 24 h with AGR2 coupled with or without bFGF. The concentration of AGR2 is 500 ng/ml. The concentration of bFGF is 1 ng/ml. (b) NIH3T3 cells were treated by 500 ng/ml AGR2 for the indicated time. RhoA, cyclin D1, P21, ERK1/2, pERK1/2, CDC42, Rac1, RhoB, and RhoC were detected by western blots. β-actin served as loading control. (c) Significant reduction in RhoA expression, cyclin D1 expression, and FAK phosphorylation was observed in the NIH3T3 cells pretreated with 20 nM/ml FGFR1 inhibitor PD173074 and 10 nM/ml VEGFR inhibitor axitinib before AGR2 treatment. β-actin served as loading control.

    Journal: Cell Adhesion & Migration

    Article Title: Paracrine signalling of AGR2 stimulates RhoA function in fibroblasts and modulates cell elongation and migration

    doi: 10.1080/19336918.2019.1685928

    Figure Lengend Snippet: Extracellular AGR2 enhances RhoA, CDC42 expression and stimulates FAK phosphorylation in NIH3T3 cells. (a) Western blot analysis of RhoA expression in NIH3T3 cells stimulated for 24 h with AGR2 coupled with or without bFGF. The concentration of AGR2 is 500 ng/ml. The concentration of bFGF is 1 ng/ml. (b) NIH3T3 cells were treated by 500 ng/ml AGR2 for the indicated time. RhoA, cyclin D1, P21, ERK1/2, pERK1/2, CDC42, Rac1, RhoB, and RhoC were detected by western blots. β-actin served as loading control. (c) Significant reduction in RhoA expression, cyclin D1 expression, and FAK phosphorylation was observed in the NIH3T3 cells pretreated with 20 nM/ml FGFR1 inhibitor PD173074 and 10 nM/ml VEGFR inhibitor axitinib before AGR2 treatment. β-actin served as loading control.

    Article Snippet: Immunoblotting was performed as described previously with RhoA, p-FAK, Cyclin D1, p-ERK, ERK, P21 primary antibody (Cell signalling technology Inc., Denver, MA, USA), FAK, RhoB, RhoC, Rac1 Primary antibody (ABclonal, Woburn, MA, USA), CDC42 primary antibody (Abcam, Cambridge, MA, USA) and β-actin purchased from Abgent [ ].

    Techniques: Expressing, Western Blot, Concentration Assay