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ABclonal Biotechnology p cdk6 ap0326
P Cdk6 Ap0326, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti%E2%80%90p%E2%80%90cdk6/Phospho-CDK6-Y13+Rabbit+pAb/pmc13449087-63-43-46
Average 94 stars, based on 2 article reviews

p cdk6 ap0326 - by Bioz Stars, 2026-10

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Article Title: MKL1/miR34a/FOXP3 axis regulates cell proliferation in gastric cancer.
Article Snippet: Funding information the innovation and entrepreneurship fund for Graduate of Wuhan University of Science and Technology, Grant/Award Numbers: JCX2017033, JCX2016024, JCX2017032; Hubei natural science foundation, Grant/ Award Number: 2017CFB537; Hubei Province health and family planning scientific research project, Grant/Award Number: WJ2017M173; Educational Commwassion of Hubei, Grant/Award Number: B2017009; National Natural Science Foundation of China, Grant/Award Numbers: 31770815, 31570764, 31501149; Science and Technology Young Training Program of the Wuhan University of Science and Technology, Grant/Award Numbers: 2017xz027, 2016xz035 Abstract Megakaryoblastic leukemia 1 (MKL1) was closely related to the pathogenesis of various human malignant cancers.. MiR34a was reported to be closely related to cancer cell proliferation.. Forkhead box protein 3 (FOXP3) was a transcription factor that played a different role in different cancer types.

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(A) Immunoblot for p-RB S780, RB, CDK4, CDK6 and histone 3 after treatment with 200 nM BSJ, palbociclib or BSJ-bump. (B) Global phosphoproteomics. Heat map depicting fold changes in peptide phosphorylation after BSJ or palbociclib treatment (200 nM, 2 h) compared to DMSO for 305 differentially phosphorylated peptides. Hits (log2 FC > 0.5 or < −0.5, adj. p-value < 0.05) and peptides phosphorylated at canonical SP/TP CDK phosphorylation motifs are annotated. (C) Heat map of DMSO-normalized fold changes in gene expression after BSJ, YKL, or palbociclib treatment (200 nM, 6h) for 993 significantly deregulated genes (log2 FC > 1.5 or < −1.5, adj. p-value < 0.05). (D) Functional network of BSJ treatment. Nodes represent GO-terms enriched among genes that are differentially expressed upon treatment, scaled by magnitude and color coded by significance of enrichment. Edges represent parent-child relationships of GO-terms. Molecular network of BSJ treatment. Hits identified via global phosphoproteomics were mapped on a protein-protein interaction network and expanded to include first order neighbors limited to ENCODE transcriptional regulators. Node shape distinguishes transcriptional regulators (TR, diamonds) from phosphoproteomics hits (round). Node color represents the number of quantified phosphopeptides. Diamonds are scaled proportional to percentage of dysregulated TR target genes upon treatment. Proteins phosphorylated at CDK consensus motif (SP/TP) are annotated by edge color. Edges represent physical interaction between proteins. See also Figure S3–4.

Journal: Cell chemical biology

Article Title: Homolog-selective degradation as a strategy to probe the function of CDK6 in AML

doi: 10.1016/j.chembiol.2018.11.006

Figure Lengend Snippet: (A) Immunoblot for p-RB S780, RB, CDK4, CDK6 and histone 3 after treatment with 200 nM BSJ, palbociclib or BSJ-bump. (B) Global phosphoproteomics. Heat map depicting fold changes in peptide phosphorylation after BSJ or palbociclib treatment (200 nM, 2 h) compared to DMSO for 305 differentially phosphorylated peptides. Hits (log2 FC > 0.5 or < −0.5, adj. p-value < 0.05) and peptides phosphorylated at canonical SP/TP CDK phosphorylation motifs are annotated. (C) Heat map of DMSO-normalized fold changes in gene expression after BSJ, YKL, or palbociclib treatment (200 nM, 6h) for 993 significantly deregulated genes (log2 FC > 1.5 or < −1.5, adj. p-value < 0.05). (D) Functional network of BSJ treatment. Nodes represent GO-terms enriched among genes that are differentially expressed upon treatment, scaled by magnitude and color coded by significance of enrichment. Edges represent parent-child relationships of GO-terms. Molecular network of BSJ treatment. Hits identified via global phosphoproteomics were mapped on a protein-protein interaction network and expanded to include first order neighbors limited to ENCODE transcriptional regulators. Node shape distinguishes transcriptional regulators (TR, diamonds) from phosphoproteomics hits (round). Node color represents the number of quantified phosphopeptides. Diamonds are scaled proportional to percentage of dysregulated TR target genes upon treatment. Proteins phosphorylated at CDK consensus motif (SP/TP) are annotated by edge color. Edges represent physical interaction between proteins. See also Figure S3–4.

Article Snippet: Following antibodies were used for detection: Name Origin Cat. No. CDK4 Cell Signaling #12790 CDK6 Santa Cruz sc-7961 Histone 3 Abcam ab1791 β-actin Sigma-Aldrich A5441 p-Rb S780 Cell Signaling #8180 Rb Cell Signaling #9309 Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG Jackson Immuno Research 115–035-003 Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG Jackson Immuno Research 111–035-003 Open in a separate window Immunoblotting Cellular Thermal Shift Assay 4×10 6 MV4–11 CRBN−/− cells were treated for 3 h with 20 µM palbociclib, BSJ-03–123 or vehicle.

Techniques: Western Blot, Expressing, Functional Assay

Journal: Cell chemical biology

Article Title: Homolog-selective degradation as a strategy to probe the function of CDK6 in AML

doi: 10.1016/j.chembiol.2018.11.006

Figure Lengend Snippet: Immunoblotting

Techniques:

Journal: Cell chemical biology

Article Title: Homolog-selective degradation as a strategy to probe the function of CDK6 in AML

doi: 10.1016/j.chembiol.2018.11.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Techniques: Recombinant, CRISPR, Expressing, RNA Sequencing Assay, Software

Fig. 1 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−ΔΔCt) 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

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: Fig. 1 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−ΔΔCt) 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, Phospho-proteomics, Comparison, Derivative Assay, Peptide Microarray, Western Blot, Activation Assay, Expressing, Cell Culture, Control, MANN-WHITNEY

Fig. 2 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

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: Fig. 2 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

Techniques: Staining, Activation Assay, Derivative Assay, Marker

Fig. 6 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−ΔΔCt) 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

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: Fig. 6 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−ΔΔCt) 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

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

Fig. 8 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−ΔΔCt) 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

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: Fig. 8 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−ΔΔCt) 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

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

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

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

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

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

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

Techniques: Staining, Activation Assay, Derivative Assay, Marker

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

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

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

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

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

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

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

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

Techniques: Activation Assay, Expressing, Activity Assay

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