Journal: Molecular Cancer

Article Title: Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORβ

doi: 10.1186/s12943-017-0590-2

Figure Lengend Snippet: RORβ inhibits tumorigenesis and the self-renewal of CCICs. a RORB expression in colorectal cancer cells. RORB mRNA and protein expression levels were detected in colorectal cancer cells by Taqman RT-qPCR and western blotting, respectively. RORB mRNA was normalized with GAPDH . b RORβ expression in primary colorectal cancer tissues. RORβ expression in human primary colorectal cancer tissues was detected by IHC staining with antibodies against RORβ. Normal rabbit IgG was used as a negative control. c RORB mRNA in primary colorectal cancer tissues. RORB mRNA was measured by Taqman RT-qPCR in 14 patients with colorectal cancer. The results showed that the levels of RORB in colorectal cancer cells were significantly lower than those in matched adjacent tissues. * p < 0.05 (ANOVA). d Tumorigenicity of RORβ-overexpressing cells. RORβ- overexpressing SW620 cells (1 × 10 6 ) as well as their control cells infected with blank lentivirus were injected into naked Balb/c mice, respectively (n = 5). Tumor formation was quantified within 4 weeks. * p < 0.05 (ANOVA). The results showed that RORβ inhibited tumor growth. e Quantification of colospheres in RORβ-overexpressing cells. Colospheres were counted in RORβ-overexpressing P1 and control cells infected with blank lentivirus at the 5th day under low-adhesion and serum-free condition. The number of colospheres significantly decreased after RORβ overexpression compared with the controls. * p < 0.05 (ANOVA). f Determination of the percentage of CD44 + CD24+ cells after overexpression of RORβ. The percentage of CD44 + CD24+ cells were analyzed by FCM in RORβ-overexpressing HT29, P1 and SW620 cells, with cells infected with blank lentivirus as controls. The results showed that RORβ reduced the percentage of CD44 + CD24+ cells compared with the control cells, all p < 0.05 (ANOVA). g RORβ expression in RORB- knockdown cells. Cells were infected with lentivirus encoding RORB shRNA for 72 h and subsequently screened with 5 μg/mL Puromycin for 7 days. The surviving cell clone was picked out with limiting dilution analyses. RORβ was detected by western blotting in these RORB- knockdown clones, with cells infected with scrambled shRNA lentivirus as controls. h Quantification of colospheres in RORB- knockdown cells. Colospheres from the above RORB -knockdown colorectal cancer cell clones were counted at day 5 under serum-free conditions. The number of colospheres was significantly higher in RORB -knockdown cells than in the control cells. * p < 0.05 (ANOVA)

Article Snippet: Primary antibodies against the following target proteins were used in this study: NRIP2, HBP1 (1:1,000; Novus, USA), cyclin D1, c-Myc, RARα, RORβ (1:1000–2000; Epitomics, CA, USA), and GAPDH (1:5000; KangChen Biotech, Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Negative Control, Infection, Injection, Over Expression, shRNA, Clone Assay

Journal:

Article Title: GalR1, but not GalR2 or GalR3, levels are regulated by galanin signaling in the locus coeruleus through a cyclic AMP-dependent mechanism

doi: 10.1111/j.1471-4159.2005.03105.x

Figure Lengend Snippet: GalR1 is upregulated in a cyclic AMP-dependent manner in the LC-like cell line, Cath.a. A) Cath.a cells were stimulated for 5 min with 5 μM forskolin to induce cyclic AMP production and PKA activation. Activation of the cyclic AMP/PKA signaling pathway was analyzed by western analysis. Representative western blots are shown for CREB (Ser133) phosphorylation, total CREB and GAPDH (loading control). An increase in CREB (Ser133) phosphorylation indicates forskolin-induced activation of the cyclic AMP/PKA pathway. B) Cath.a cells were stimulated with 5 μM forskolin for 7h to determine cyclic AMP-dependent changes in galanin receptor protein expression. Representative western blots are shown for GalR1, GalR2, GalR3 and GAPDH. C) Galanin receptor protein levels were quantified using NIH Image software and significance was determined by ANOVA. * p < 0.001.

Article Snippet: Blots were washed 3 times for 5 min each, then incubated for 30 min at room temp in anti-GAPDH monoclonal primary antibody (Advanced ImmunoChemical Inc., Long Beach, CA) diluted to 1:2000 in 5% BSA/TBS.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control, Expressing, Software

Journal: Genes

Article Title: Prolactin-Responsive Circular RNA circHIPK3 Promotes Proliferation of Mammary Epithelial Cells from Dairy Cow

doi: 10.3390/genes11030336

Figure Lengend Snippet: CircHIPK3 knockdown represses the proliferation of HC11 cells. ( A ) Relative cell viability of HC11 measured by CCK-8; ( B ) CDK1 protein expression; ( C ) Cyclin A2 protein expression. Mean ± SEM of three samples from three independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The membrane was blocked for 1 h in PBS in 10% skim milk, and then incubated overnight with primary antibodies against CDK1, Cyclin A2 (HuaBio, Cambridge, MA, USA), and GAPDH (AbSci, Baltimore, MD, USA).

Techniques: Knockdown, CCK-8 Assay, Expressing