Journal: Circulation

Article Title: Impaired Cholesterol Metabolism and Enhanced Atherosclerosis in Clock Mutant Mice

doi: 10.1161/circulationaha.113.002885

Figure Lengend Snippet: Figure 6. Clock modulates expression of USF2 to regulate ABCA1. A, Wild-type macrophages were transfected with a pGL3 plasmid expressing luciferase under the control of ABCA1 promoter, along with a control plasmid, a plasmid expressing Clock, or lentiviruses expressing shClock. After 72 hours, cells were used to quantify luciferase activity. B, Protein levels of different transcription factors were quantified in macrophages treated with siControl or siClock. C, Wild-type macrophages were treated with different siRNAs, individually or in combination. After 48 hours, mRNA levels of ABCA1 were quantified. D, ClkΔ19/Δ19Apoe−/− and Apoe−/− macrophages were cultured for 7 days. They were used to study the binding of different transcription factors to ABCA1 promoter by chromatin immunoprecipitation. Proteins were cross-linked to DNA, sheared, and used to immunoprecipitate protein/DNA complexes using specific antibodies against indicated transcription factors. Sequences specific for ABCA1 promoter were amplified, separated on agarose gels, and visualized. E, ClkΔ19/Δ19Apoe−/− macrophages were treated with siControl or siUSF2 for 48 hours, incubated with 3H-cholesterol (5 µCi/mL) and acetylated low-density lipoprotein (50 µg/mL) for 24 hours, and injected intraperitoneally into wild-type mice. At different times, plasma was used to measure counts. Feces were collected over 48 hours, and lipids were extracted and counted. At 48 hours, liver segments were solubilized and counted. F, Wild-type bone marrow macrophages were treated with siControl or siClock for 48 hours and incubated in serum-free media for 18 hours and in media containing 50% FCS for 2 hours. Macrophages were washed and incubated in serum-free media. Three wells were harvested to measure ABCA1, USF2, and β-actin mRNA at indicated times. Comparisons were performed by 2-way ANOVA. Mean±SD; n=6 to 9. Hif indicates hypoxia-inducible factor. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Materials: siHif1α (sc-35562), siHif1β (sc-29734), siUSF1 (sc-36784), siUSF2 (sc-36785) as well as lentiviruses expressing shRNA against Clock (sc-35075-v) were from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay, Cell Culture, Binding Assay, Chromatin Immunoprecipitation, Amplification, Incubation, Injection, Clinical Proteomics

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Diurnal expression of the thrombopoietin gene is regulated by CLOCK.

doi: 10.1111/j.1538-7836.2012.04643.x

Figure Lengend Snippet: Fig. 2. CLOCK regulates thrombopoietin gene (THPO) expression in vitro. The expression of THPO was measured in two human cell lines (HepG2 and Huh7). Cells were synchronized by brief exposure to dexamethasone, and samples of cells were then collected every 4–5 h for 26–48 h (circadian time is the time in hours after cells were synchronized). (A) Rhythmic expression of the circadian gene NR1D1 verified that HepG2 cells were synchronized and expressing a circadian clock. (B) Expression of THPO was also rhythmic (P < 0.01 by one-way ANOVA for the effect of time). (C) THPO expression was rhythmic in Huh7 cells, and transfecting cells with small interfering RNA (siRNA) to CLOCK abolished this rhythm. N = 6 independent cultures per time point. (D) CLOCK mRNA levels in Huh7 cells treated with control siRNA or siRNA to CLOCK, N = 4, *P < 0.01. (E) CLOCK levels relative to glyceradehyde-3-phosphate dehydrogenase (GAPDH) levels determined from two independent western blots of lysates from Huh7 cells treated with control siRNA or siRNA to CLOCK. *P < 0.01.

Article Snippet: Small interfering RNA and serum shock Small interfering RNA (siRNA) directed against CLOCK, and non-specific control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA,USA), were introduced intoHuh7 human hepatoma cells plated in 12-well plates by the use of siRNA transfection reagent.

Techniques: Expressing, In Vitro, Small Interfering RNA, Control, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Epigallocatechin-3-gallate inhibits self-renewal ability of lung cancer stem-like cells through inhibition of CLOCK

doi: 10.3892/ijmm.2020.4758

Figure Lengend Snippet: Effect of CLOCK siRNA on CSC-like properties of lung cancer cells. Expression of CLOCK in (A) A549 and (B) H1299 sphere cells. Cells were transfected with CLOCK siRNA. Protein levels of CD133, CD44, Sox2, Nanog and Oct4 in (C) A549 and (D) H1299 sphere cells. (E) Tumor volume of A549 and H1299 sphere cells transfected with CLOCK siRNA. Flow cytometry analyses of CD133 + cell ratios in (F) A549 and (G) H1299 sphere cells. Cells were transfected with CLOCK siRNA or siRNA negative control. Data are presented as the mean ± SD from at least triplicate experiments. * P<0.05, *** P<0.001. CSC, cancer stem cell; Sox2, sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4.

Article Snippet: siRNA targeting CLOCK at a concentration of 100 nmol or a corresponding negative control (Guangzhou RiboBio, Co., Ltd.) were transfected into NSCLC/CSCs using LipofectamineTM 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions at room temperature.

Techniques: Expressing, Transfection, Flow Cytometry, Negative Control, Binding Assay