Journal:

Article Title: Control of cell cycle progression by c-Jun is p53 dependent

doi:

Figure Lengend Snippet: Model for the dual functions of c-Jun in connecting mitogenic signaling to the cell cycle clock. Stimulation of cells by growth factors leads to increased expression of c-Jun, cyclin D1, p53, and p21. Cyclin D1 positively and p21 negatively influences activation of CDK4/CDK6, the first cyclin-dependent kinase to be activated as cells progress through G1. Once CDK4/CDK6 is activated and phosphorylates pRb and its related proteins, cells are committed to complete progression through the entire cell cycle in the absence of further mitogen stimulation (Harper and Elledge 1996; Sherr 1996). c-Jun contributes to mitogen-dependent activation of cyclin D1–CDK4/CDK6 in two ways. (1) It transduces mitogenic signals to induce cyclin D1 expression, although cyclin D1 can still be induced by mitogens in the absence of c-Jun (indicated by dashed arrow); (2) it attenuates accumulation of p21 through repression of p53 expression. In cells lacking p53, low levels of p21 allow efficient activation of cyclin D1–CDK4/CDK6, cyclin E–CDK2, E2F, and G1-to-S-phase progression whether or not c-Jun is present.

Article Snippet: The following primary antibodies were used (1:1000 unless otherwise indicated): CM1 (1:2000) and CM5 (Medac) for p53; M20 (Santa Cruz, sc-481) for cyclin E; HD11 (Santa Cruz, sc-246) for cyclin D1; M20 (Santa Cruz, sc-593) for cyclin D2; C16 (Santa Cruz, sc-182) for cyclin D3; M2 (Santa Cruz, sc-163) for CDK2; C22 (Santa Cruz, sc-260) for CDK4; C21 (Santa Cruz, sc-177) for CDK6; KM-1 (Santa Cruz, sc-822) for amino-terminally phosphorylated c-Jun; C19 (Santa Cruz, sc-528) for p27; C19 (Santa Cruz, sc-397) for p21; M-156 (Santa Cruz, sc-1207) for p16; PC10 (Santa Cruz, sc-056) for PCNA; A-2066 (Sigma) for actin (1:100).

Techniques: Expressing, Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA RP11-624L4.1 Is Associated with Unfavorable Prognosis and Promotes Proliferation via the CDK4/6-Cyclin D1-Rb-E2F1 Pathway in NPC

doi: 10.1016/j.omtn.2020.10.017

Figure Lengend Snippet: RP11-624L4.1 Interacts with CDK4 and Affects CDK4, CDK6, Cyclin D1, p-Rb (Ser 780), Rb, and E2F1 Protein Levels (A) RIP assays of the interaction between RP11-624L4.1 and CDK4 by CDK4 antibody in CNE1 and 5-8F cells. The level of RP11-624L4.1 was determined by quantitative real-time PCR and normalized by the input levels. (B) The mRNA expression of CDK4 was determined by qRT-PCR in sh-NC and sh-1 cells. The results are expressed as log2 (2−ΔΔCt). Data are presented as the means ± SD; N.S. p > 0.05, Student’s t test. (C) The effects of RP11-624L4.1 on the protein expression levels of CDK4/6, cyclin D1, p-Rb (Ser 780), Rb, and E2F1 were detected by western blot (from three independent experiments). GAPDH and α-Tubulin were used as loading controls. The band intensity was quantified by ImageJ. (D) Confocal RNA fluorescence ISH and immunofluorescence images are shown as the colocalization of CDK4 and RP11-624L4.1. Scale bars: 5 μm. DAPI, 4′,6-diamidino-2- phenylindole.

Article Snippet: Proteins were extracted, and a WB assay was performed according to previously described methods., , Anti-CDK4 antibodies, anti-CDK6 antibodies (Proteintech, P.R.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Western Blot, Fluorescence, Immunofluorescence

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA RP11-624L4.1 Is Associated with Unfavorable Prognosis and Promotes Proliferation via the CDK4/6-Cyclin D1-Rb-E2F1 Pathway in NPC

doi: 10.1016/j.omtn.2020.10.017

Figure Lengend Snippet: Rescue Assay Results by Overexpressing CDK4 in the Knocked Down lncRNA RP11-624L4.1 Cell sh-1 (A) The line diagram of CCK-8 cell proliferation assay. (B) Changes in proteins by western blotting in the CDK4/CDK6-Cyclin D1-Rb-E2F1 pathway. Sh-1+OE, overexpression of GV141-CDK4 in sh-1, which knocked down lncRNA RP11-624L4.1; Sh-1+vector, without overexpression of CDK4 (GV141 vector only) in sh-1, which knocked down lncRNA RP11-624L4.1.

Article Snippet: Proteins were extracted, and a WB assay was performed according to previously described methods., , Anti-CDK4 antibodies, anti-CDK6 antibodies (Proteintech, P.R.

Techniques: Rescue Assay, CCK-8 Assay, Proliferation Assay, Western Blot, Over Expression, Plasmid Preparation

Journal: Nature Communications

Article Title: Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension

doi: 10.1038/s41467-019-10135-x

Figure Lengend Snippet: Kinome profiling reveals increased activity of the CDK-Rb-E2F signaling pathway in HPASMCs from IPAH patients. a Mean value of raw data for all individual samples, such as HPASMCs from healthy individuals ( n = 5) and from IPAH patients ( n = 6), presented as a heat map of log-transformed fluorescence signals upon substrate serine/threonine phosphorylation that are used to perform an upstream kinase analysis. b Mean value of a two-group comparison for all samples indicating the global changes in serine/threonine phosphorylation pattern between both HPASMCs entities under basal media conditions. c Bar chart displaying the overall prediction of kinases with higher activity in IPAH-patient-derived samples. Normalized kinase statistics is a mathematical-based algorithm indicating the estimated relative kinase activity while the specificity score reflects the reliability and accuracy of the prediction. d Two-group comparison for the four selected CDKs only including the phosphorylation pattern of their particular peptide substrate sets on the peptide array. e Western blot analysis for CDK2, CDK4, CDK6, and CDK9 activation and subsequent Rb-E2F downstream signaling in HPASMCs obtained from healthy donors and IPAH patients exposed to basal media. f CDK1 mRNA expression normalized to GAPDH as reference gene in HPASMCs of healthy individuals ( n = 3) and IPAH patients ( n = 3). After starvation, cells were cultured in basal media for 24 h. All data are presented as mean ± SEM of the n -fold change (2 −∆∆Ct ) compared with a healthy control and analyzed statistically using a Mann–Whitney test; ** p < 0.01. Source data are provided as a Source Data file

Article Snippet: Primary antibodies against cleaved Caspase-3 (#9665, 1:500 dilution), (P-)CDK2 (#2561, #2546, 1:500–1:1000 dilution), CDK4 (#12790, 1:500–1:1000 dilution), CDK6 (#3136, 1:1000–1:2000 dilution), (P-)CDK9 (#2549 S, #2316 S, 1:1000 dilution), cyclin D1 (#2978, 1:500–1:1000 dilution), cyclin D3 (#2936, 1:1000 dilution), GAPDH (#2118, 1:2000 dilution), P-ERK (#4370, 1:1000 dilution), (P-)Rb (#8516 S, #9309 S, 1:1000–1:2000 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA), for P-CDK6 (#ab131439, 1:500 dilution), beta-actin (#ab8226, #ab8227, 1:2000 dilution), and vinculin (ab18058, 1:2000 dilution) from Abcam (Cambridge, UK), and for ERK (#sc-93, 1:2000 dilution), and PCNA (#sc-56, 1:500–1:1000) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activity Assay, Transformation Assay, Fluorescence, Derivative Assay, Peptide Microarray, Western Blot, Activation Assay, Expressing, Cell Culture, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension

doi: 10.1038/s41467-019-10135-x

Figure Lengend Snippet: Immunohistological staining of distinct CDKs and P-Rb activation in human lung specimen. Representative images of serial sections from healthy ( n = 5) and IPAH-patient derived lungs ( n = 5) analyzed for CDK2 ( a ), CDK4 ( b ), and CDK6 ( c ), with corresponding IHC for P-Rb protein and its downstream target gene and common proliferation marker PCNA (proliferation cell nuclear antigen). Cellular identity was visualized by antibodies against α-SMA (alpha-smooth muscle actin) and vWF (von-Willebrand factor). Images were taken at 400-fold magnification with a scale bar of 20 µm

Article Snippet: Primary antibodies against cleaved Caspase-3 (#9665, 1:500 dilution), (P-)CDK2 (#2561, #2546, 1:500–1:1000 dilution), CDK4 (#12790, 1:500–1:1000 dilution), CDK6 (#3136, 1:1000–1:2000 dilution), (P-)CDK9 (#2549 S, #2316 S, 1:1000 dilution), cyclin D1 (#2978, 1:500–1:1000 dilution), cyclin D3 (#2936, 1:1000 dilution), GAPDH (#2118, 1:2000 dilution), P-ERK (#4370, 1:1000 dilution), (P-)Rb (#8516 S, #9309 S, 1:1000–1:2000 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA), for P-CDK6 (#ab131439, 1:500 dilution), beta-actin (#ab8226, #ab8227, 1:2000 dilution), and vinculin (ab18058, 1:2000 dilution) from Abcam (Cambridge, UK), and for ERK (#sc-93, 1:2000 dilution), and PCNA (#sc-56, 1:500–1:1000) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Staining, Activation Assay, Derivative Assay, Marker

Journal: Nature Communications

Article Title: Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension

doi: 10.1038/s41467-019-10135-x

Figure Lengend Snippet: Ex vivo analyses of lung tissue for reversal of remodeling and in vivo drug efficacy in the MCT rat model. a The degree of muscularization of small pulmonary arteries (diameter 25–50 µm) was determined ex vivo via immunhistological staining of lung sections for vWF (brown) and α-SMA (violet) together with methylgreen for counterstaining. M: fully muscularized; P: partially muscularized; N: non-muscularized. Representative images for all three study groups are shown (healthy n = 6; MCT n = 5, MCT + Palbo n = 5). Images were taken at adequate magnification with a scale bar of 20 µm. b Medial wall thickness of vessels with a diameter of 20–50 µm was determined by Elastica-van-Gieson staining and is presented as percentage. c Proliferation index as a quantitative measure of PCNA-positive cells (purple) per vessel. Representative images (using hematoxylin/eosin as counterstain) for all three study groups are included. Images were taken at adequate magnification with a scale bar of 20 µm. Data from all individual animals (healthy n = 6; MCT n = 5, MCT + Palbo n = 5) are presented as mean ± SEM of 80–100 counted vessels and statistical analysis was performed using one-way ANOVA with Newman–Keuls post-hoc test for multiple comparisons; ** p < 0.01, *** p < 0.001 for MCT + Palbo versus MCT; § p < 0.05, §§ p < 0.01 for MCT + Palbo versus healthy; ### p < 0.001 for healthy versus MCT. d Western blot analysis for CDK2 and CDK6 activation and subsequent Rb-E2F downstream signaling in lung homogenates of representative samples from all three experimental groups (healthy, MCT, MCT + Palbo). e Analysis of CCNA2 (left) and CDK1 (right) mRNA expression normalized to GAPDH as reference gene in lung homogenates from all three experimental groups. Data from all individual animals are presented as mean ± SEM of the n -fold change (2 −∆∆Ct ) compared with a healthy control rat (healthy n = 6; MCT n = 5, MCT + Palbo n = 5). Statistical analysis was performed using one-way ANOVA with Newman–Keuls post-hoc test for multiple comparisons; *** p < 0.001 for MCT + Palbo versus MCT; § p < 0.05 for MCT + Palbo versus healthy; ### p < 0.001 for healthy versus MCT. Source data are provided as a Source Data file

Article Snippet: Primary antibodies against cleaved Caspase-3 (#9665, 1:500 dilution), (P-)CDK2 (#2561, #2546, 1:500–1:1000 dilution), CDK4 (#12790, 1:500–1:1000 dilution), CDK6 (#3136, 1:1000–1:2000 dilution), (P-)CDK9 (#2549 S, #2316 S, 1:1000 dilution), cyclin D1 (#2978, 1:500–1:1000 dilution), cyclin D3 (#2936, 1:1000 dilution), GAPDH (#2118, 1:2000 dilution), P-ERK (#4370, 1:1000 dilution), (P-)Rb (#8516 S, #9309 S, 1:1000–1:2000 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA), for P-CDK6 (#ab131439, 1:500 dilution), beta-actin (#ab8226, #ab8227, 1:2000 dilution), and vinculin (ab18058, 1:2000 dilution) from Abcam (Cambridge, UK), and for ERK (#sc-93, 1:2000 dilution), and PCNA (#sc-56, 1:500–1:1000) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Ex Vivo, In Vivo, Staining, Western Blot, Activation Assay, Expressing

Journal: Nature Communications

Article Title: Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension

doi: 10.1038/s41467-019-10135-x

Figure Lengend Snippet: Ex vivo analyses of lung tissue for reversal of remodeling and in vivo drug efficacy in the Su/Hox model. a The degree of muscularization of small pulmonary arteries (diameter 25–50 µm) was determined ex vivo via immunhistological staining of lung sections for vWF (brown) and α-SMA (violet) together with methylgreen for counterstaining. M: fully muscularized; P: partially muscularized; N: non-muscularized. Representative images for all three study groups (Nox n = 8; Su/Hox n = 8; Su/Hox + Palbo n = 7) are shown. Images were taken at adequate magnification with a scale bar of 50 µm. b The medial wall thickness, as well as the corresponding ratio of neointima/media of the depicted study groups were determined by Elastica-van-Gieson staining allowing the calculation of the vessel occlusion score ( c ). O: opened; P: partial closed; C: closed. d Proliferation index as a quantitative parameter of PCNA-positive cells (purple) per vessel. Representative images (using hematoxylin/eosin as counterstain) for all three study groups are included. Images were taken at adequate magnification with a scale bar of 50 µm. Data from all individual animals (Nox n = 8; Su/Hox n = 8; Su/Hox + Palbo n = 7) are presented as mean ± SEM of 80–100 counted vessels and statistical analysis was performed using one-way ANOVA with Newman–Keuls post-hoc test for multiple comparisons; * p < 0.05; ** p < 0.01, *** p < 0.001 for Su/Hox + Palbo versus Su/Hox; § p < 0.05; §§§ p < 0.001 for Su/Hox + Palbo versus Nox; ### p < 0.001 for Nox versus Su/Hox. e Western blot analysis for CDK2 and CDK6 activation and subsequent Rb-E2F downstream signaling in lung homogenates of representative samples from all three experimental groups (Nox, Su/Hox, Su/Hox + Palbo). f Analysis of CCNA2 (left) and CDK1 (right) mRNA expression normalized to GAPDH as reference gene in lung homogenates from all three experimental groups. Data from all individual animals are presented as mean ± SEM of the n -fold change (2 −∆∆Ct ) compared with a healthy control rat (Nox n = 8; Su/Hox n = 8; Su/Hox + Palbo n = 7). Statistical analysis was performed using one-way ANOVA with Newman–Keuls post-hoc test for multiple comparisons; *** p < 0.001 for Su/Hox + Palbo versus Su/Hox; §§ p < 0.01, §§§ p < 0.001 for Su/Hox + Palbo versus Nox; ### p < 0.001 for Nox versus Su/Hox. Source data are provided as a Source Data file

Article Snippet: Primary antibodies against cleaved Caspase-3 (#9665, 1:500 dilution), (P-)CDK2 (#2561, #2546, 1:500–1:1000 dilution), CDK4 (#12790, 1:500–1:1000 dilution), CDK6 (#3136, 1:1000–1:2000 dilution), (P-)CDK9 (#2549 S, #2316 S, 1:1000 dilution), cyclin D1 (#2978, 1:500–1:1000 dilution), cyclin D3 (#2936, 1:1000 dilution), GAPDH (#2118, 1:2000 dilution), P-ERK (#4370, 1:1000 dilution), (P-)Rb (#8516 S, #9309 S, 1:1000–1:2000 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA), for P-CDK6 (#ab131439, 1:500 dilution), beta-actin (#ab8226, #ab8227, 1:2000 dilution), and vinculin (ab18058, 1:2000 dilution) from Abcam (Cambridge, UK), and for ERK (#sc-93, 1:2000 dilution), and PCNA (#sc-56, 1:500–1:1000) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Ex Vivo, In Vivo, Staining, Western Blot, Activation Assay, Expressing

Journal: Nature Communications

Article Title: Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension

doi: 10.1038/s41467-019-10135-x

Figure Lengend Snippet: Proposed mechanism of action of palbociclib and dinaciclib in PAH. Multiple growth factors, cytokines, and mitogens induce the activation of cyclin-dependent kinases (CDKs), e.g., by increasing the expression of cyclin D1. Palbociclib specifically targets active complexes consisting of cyclin D1 and CDK4 or CDK6, whereas dinaciclib blocks the activity of the cyclin E-CDK2 complex. This leads to reduced levels of phosphorylated Rb protein and, as a consequence, transcriptional repression of E2F downstream target genes, e.g., Cyclin A2 , CDK1 , and PCNA . Thus, palbociclib (and dinaciclib) interfere with cell cycle progression from G1 into S phase, causing a reduction of proliferation of pulmonary vascular cells

Article Snippet: Primary antibodies against cleaved Caspase-3 (#9665, 1:500 dilution), (P-)CDK2 (#2561, #2546, 1:500–1:1000 dilution), CDK4 (#12790, 1:500–1:1000 dilution), CDK6 (#3136, 1:1000–1:2000 dilution), (P-)CDK9 (#2549 S, #2316 S, 1:1000 dilution), cyclin D1 (#2978, 1:500–1:1000 dilution), cyclin D3 (#2936, 1:1000 dilution), GAPDH (#2118, 1:2000 dilution), P-ERK (#4370, 1:1000 dilution), (P-)Rb (#8516 S, #9309 S, 1:1000–1:2000 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA), for P-CDK6 (#ab131439, 1:500 dilution), beta-actin (#ab8226, #ab8227, 1:2000 dilution), and vinculin (ab18058, 1:2000 dilution) from Abcam (Cambridge, UK), and for ERK (#sc-93, 1:2000 dilution), and PCNA (#sc-56, 1:500–1:1000) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Oncology reports

Article Title: Saffron carotenoids inhibit STAT3 activation and promote apoptotic progression in IL-6-stimulated liver cancer cells.

doi: 10.3892/or.2018.6232

Figure Lengend Snippet: Figure 2. Crocin inhibits DNA-binding activity of STAT3 in IL-6-stimulated liver cancer cells. (A) Hep3B and HepG2 cells (1x106/ml) were treated with the indicated concentrations of crocin for 24 h and then stimulated with IL-6 (10 ng/ml) for 60 min. In addition, nuclear STAT3 levels were analyzed using EMSA as described in Materials and methods. (B) Hep3B and HepG2 cells (1x106/ml) were treated at the indicated time-pointswith 20 µM crocin and then stimulated with IL-6 (10 ng/ml) for 60 min. In addition nuclear STAT3 levels were analyzed using EMSA. (C) Crocin inhibits IL-6-induced JAK1, JAK2, and Src kinase activation in Hep3B and HepG2 cells. These cells were treated with the indicated concentrations of crocin for 24 h and then stimulated with IL-6 (10 ng/ml) for 60 min. Whole-cell extracts were prepared and 40 µg aliquots of those extracts were resolved on 10% SDS-PAGE, electro transferred onto PVDF membranes and probed with p-JAK1, p-JAK2 and p-Src. The same blots were stripped and reprobed with JAK1, JAK2 and Src antibodies to ascertain equal protein loading.

Article Snippet: The antibodies used were as follows: p-STAT3 (1:1,000; rabbit, monoclonal, cat. no. 9145), STAT3 (1:1,000; rabbit, monoclonal, cat. no. 12640), p-JAK1 (1:1,000; rabbit, polyclonal, cat. no. 3331), p-JAK2 (1:1,000; rabbit, monoclonal, cat. no. 8082), p-Src (1:1,000; rabbit, polyclonal, cat. no. 2101), Src (1:1,000; rabbit, polyclonal, cat. no. 2108), SHP-1 (1:1,000; rabbit, monoclonal, cat. no. 3759), cleaved PARP (1:1,000; rabbit, monoclonal, cat. no. 5625), cleaved caspase-9 (1:1,000; rabbit, monoclonal, cat. no. 7237), cleaved caspase-3 (1:1,000; rabbit, polyclonal, cat. no. 9661), β-actin (1:1,000; rabbit, monoclonal, cat. no. 4970) and anti-rabbit IgG (1:5,000; rabbit, polyclonal, cat. no. 14708) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA).

Techniques: Binding Assay, Activity Assay, Activation Assay, SDS Page

Journal: Oncology reports

Article Title: Saffron carotenoids inhibit STAT3 activation and promote apoptotic progression in IL-6-stimulated liver cancer cells.

doi: 10.3892/or.2018.6232

Figure Lengend Snippet: Figure 4. Crocin induces the expression of SHP-1 protein in IL-6-stimulated Hep3B cells. (A) Pervanadate reversed crocin-mediated inhibition of STAT3 phosphorylation in IL-6-stimulated Hep3B cells. The cells were treated with pervanadate or crocin for 24 h, and then stimulated with IL-6 for 60 min. The phospho-STAT3 was detected by western blotting, and the same blots were stripped and reprobed with STAT3 antibodies. (B) Crocin induced the expression of the SHP-1 protein. Hep3B cells were treated with 0, 5, 10 and 20 µM crocin for 24 h and then stimulated with IL-6 (10 ng/ml). After whole-cell extracts were prepared, SHP-1was detected by western blotting. The same blots were stripped and reprobed with the β-actin antibody to ascertain equal protein loading. (C and D) Hep3B cells were transfected with either SHP-1 siRNA or scrambled siRNA (50 nM). After 24 h, the cells were treated with or without 20 µM crocin for 24 h and then stimulated with IL-6 (10 ng/ml) for 60 min. Then, whole-cell extracts were subjected to western blot analysis for SHP-1, p-STAT3, p-JAK1, p-JAK2 and p-Src. The same blots were stripped and reprobed with β-actin, STAT3, JAK1, JAK2 and Src antibodies to ascertain equal protein loading.

Article Snippet: The antibodies used were as follows: p-STAT3 (1:1,000; rabbit, monoclonal, cat. no. 9145), STAT3 (1:1,000; rabbit, monoclonal, cat. no. 12640), p-JAK1 (1:1,000; rabbit, polyclonal, cat. no. 3331), p-JAK2 (1:1,000; rabbit, monoclonal, cat. no. 8082), p-Src (1:1,000; rabbit, polyclonal, cat. no. 2101), Src (1:1,000; rabbit, polyclonal, cat. no. 2108), SHP-1 (1:1,000; rabbit, monoclonal, cat. no. 3759), cleaved PARP (1:1,000; rabbit, monoclonal, cat. no. 5625), cleaved caspase-9 (1:1,000; rabbit, monoclonal, cat. no. 7237), cleaved caspase-3 (1:1,000; rabbit, polyclonal, cat. no. 9661), β-actin (1:1,000; rabbit, monoclonal, cat. no. 4970) and anti-rabbit IgG (1:5,000; rabbit, polyclonal, cat. no. 14708) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA).

Techniques: Expressing, Inhibition, Phospho-proteomics, Western Blot, Transfection