cdk4 6 inhibitor iv  (Santa Cruz Biotechnology)

Journal: Cancer research

Article Title: Inactivation of the Dlc1 gene cooperates with down-regulation of p15 INK4b and p16 Ink4a leading to neoplastic transformation and poor prognosis in human cancer

doi: 10.1158/0008-5472.CAN-12-2368

Figure Lengend Snippet: (A) p15 and p16 expression become undetectable in later passage MEFs, leading to up-regulation of Cdk4/6 and phospho-cyclin D1. Cell extracts from MEF-Vt and MEF-Cre cells at early (~6o) or later (~25 o) passages were analyzed by immunobloting with anti-p15, p16, Cdk4, Cdk6, phospho-Cyclin D1 (T286), and anti-β-actin antibodies. (B and C) siRNA knock-down of p15 or p16 in early passage of MEF-Cre cells (A) and A549 cells (C, right) results in colony growth in soft agar. Cell extracts from siRNA transfected cells were analyzed to verify reduced expression of p15 and p16. Cells were grown in soft agar for 4 weeks. Unlike NSCLC H1703 cells, which contain detectable DLC1 protein, DLC1 expression is undetectable in NSCLC A549 cells (left). (D) Genotyping of isolated colonies from MEF-Cre mass culture grown in soft agar. Individual colonies isolated from agar colonies, designated anchorage-independent growth (AIG) clones, were analyzed by RT-PCR using Dlc1 exon 4-specific primers (top) or GAPDH primers (bottom). (E) Inverse relationship between p16 expression and CdK6 level in MEFs. The later passage MEF-Vt, MEF-Cre and derived AIG clones were analyzed for expression of p16 and Cdk6. β-actin blot serves as a loading control. (F) The presence of p16 in AIG-2 line grew poorly in soft agar. The indicted MEFs were grown in soft agar, and the quantization of colony number (top) and representative images (bottom) are shown.

Article Snippet: In some experiments, the ROCK inhibitor Y27362 (5 nM), JNK inhibitor VIII (10 nM), or CDK4/6 inhibitor IV (10 nM) (all from EMD Millipore) was added to the agar plates every 5–6 days, to test the contribution of the respective kinase to cell growth in agar.

Techniques: Expressing, Western Blot, Transfection, Isolation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Cancer research

Article Title: Inactivation of the Dlc1 gene cooperates with down-regulation of p15 INK4b and p16 Ink4a leading to neoplastic transformation and poor prognosis in human cancer

doi: 10.1158/0008-5472.CAN-12-2368

Figure Lengend Snippet: (A) Re-expression of p15 or p16 in MEF-Cre cells reduces RhoGTP, Cdc42GTP, Cdk4/6, pCyclin D1, and growth in soft agar. MEF-Cre AIG-5 cells stably expressing GFP, p15, or p16 were analyzed for various biochemical properties (top) and agar growth (bottom). (B) siRNA knock-down of Cdk4/6 can inhibit phosphorylation of Cyc D1 and cell migration. Reduced Cdk4 and Cdk6 expression was verified by immunoblotting. (C) Inhibition of Cdk4/6 activity in MEF-Cre cells reduces phosphorylation of Cdk4/6 substrates. pCyclin D1 (T286) and pRB (S780) levels are validated by immunoblots. (D) Validation of ROCK and Jnk pathway-specific inhibitors in signaling. Top panels: The inhibitory effect of Y27632 on transfected rat ROKα-HA activity in MEF-Cre cells was assayed by anti-HA immunoprecipitation followed by Phospho-Ser/Thr immunoblotting. The membrane was re-probed by anti-HA to show the loading control (bottom). Bottom panels: MEF-Cre cells treated with JNK inhibitor VIII were assayed by phospho-c-jun (S78) and c-jun antibody blots. (E) Partial reversion of soft agar growth of MEF-Cre cells treated with the pathway-specific inhibitors in (C) and (D). Cells were grown in agar for 5 weeks. Quantitation of colony numbers (top) and representative images of agar colonies (bottom) are shown.

Article Snippet: In some experiments, the ROCK inhibitor Y27362 (5 nM), JNK inhibitor VIII (10 nM), or CDK4/6 inhibitor IV (10 nM) (all from EMD Millipore) was added to the agar plates every 5–6 days, to test the contribution of the respective kinase to cell growth in agar.

Techniques: Expressing, Stable Transfection, Migration, Western Blot, Inhibition, Activity Assay, Transfection, Immunoprecipitation, Quantitation Assay

Journal: Cancer research

Article Title: Inactivation of the Dlc1 gene cooperates with down-regulation of p15 INK4b and p16 Ink4a leading to neoplastic transformation and poor prognosis in human cancer

doi: 10.1158/0008-5472.CAN-12-2368

Figure Lengend Snippet: The reverse expression pattern of DLC1 and CDK4/6 in human cancers

Article Snippet: In some experiments, the ROCK inhibitor Y27362 (5 nM), JNK inhibitor VIII (10 nM), or CDK4/6 inhibitor IV (10 nM) (all from EMD Millipore) was added to the agar plates every 5–6 days, to test the contribution of the respective kinase to cell growth in agar.

Techniques: Expressing

Journal: Cancer research

Article Title: Inactivation of the Dlc1 gene cooperates with down-regulation of p15 INK4b and p16 Ink4a leading to neoplastic transformation and poor prognosis in human cancer

doi: 10.1158/0008-5472.CAN-12-2368

Figure Lengend Snippet: (A-C) Kaplan-Meier analysis of DLC1, p15, p16, CDK4, and CDK6 gene expression in NSCLC (A and B) and colon cancer (C). Low vs. high expression of each gene by itself does not have prognostic value in these cohorts (p>0.05), but there is prognostic significance when low DLC1 is combined with low p15 (B and C), with low p16 (A and B), with high CDK4 (A, “DL4H”), and with high CDK6 (C, “DL6H”). The p value for each comparison is shown.

Article Snippet: In some experiments, the ROCK inhibitor Y27362 (5 nM), JNK inhibitor VIII (10 nM), or CDK4/6 inhibitor IV (10 nM) (all from EMD Millipore) was added to the agar plates every 5–6 days, to test the contribution of the respective kinase to cell growth in agar.

Techniques: Expressing