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

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