Journal: bioRxiv

Article Title: Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

doi: 10.1101/550954

Figure Lengend Snippet: Modulation of CS::FLUC rhythms in N. salina by a CK1ε/δ inhibitor. (a) In vivo bioluminescence rhythms of N. salina CS::FLUC expressing lines. The average ± SEM from rhythmic traces is shown (n =3-8, two independent lines). Cultures were entrained under cycles of 12 h light/12 h dark (200 μmol m −2 s −1 ) and released to constant light (10 μmol m −2 s −1 ) at time 24 h. Cells were treated with PF-670462 at the indicated concentrations at time 12 h. Traces from one representative experiment are shown. Dark grey shading indicates dark period, light grey shading indicates subjective dark. b Time of first peak of CS::FLUC luminescence (n=8-16) from two independent experiments. * Indicate a significant difference with the vehicle control (one-way ANOVA with Dunnett’s post hoc test, α=0.05).

Article Snippet: The casein kinase inhibitor PF-670462 (Cayman Chemicals) was resuspended in water at 2 mg ml −1 .

Techniques: In Vivo, Expressing

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A-B) Bars present relative mRNA expression of CSNK1G isotypes. After treatment with 2 μM D4476 for 24 h, (A) MCF-7 and (B) MDA-MB-231 silenced with CSNK1G2 siRNA were analyzed for the mRNA expression of CSNK1G isotype genes such as CSNK1G1 , CSNK1G2 , and CSNK1G3 . (C-D) Bars denote relative percentage of % survival of (C) ER + or (D) ER - breast cancer cells treated with D4476 alone or in combination with TAM for 24 h. MCF-7 and MDA-MB-231 cells were treated with 1 to 10 μM of D4476. For combination treatment, 1 μM TAM was also used. Cellular toxicity was measured using the MTT assay (mean (SD), n = 5; ** P < 0.01 vs . each represented counterpart). (E-G) Western blotting analysis from the breast cells treated with vehicle or 2 μM D4476 together with 1 μM TAM for 24 h were performed. Immunoblots for (E) ERα and phospho-ERα (at Ser 118 or Ser 167 ) in MCF-7 cells and for PI3K/AKT/mTOR/S6K signaling-associated proteins in (F) MCF-7 and (G) MDA-MB-231 cells. NC siRNA or CSNK1G2 siRNA-transfected cells were treated with 1 μM of TAM together with vehicle or with 2 μM of D4476 for 24 h. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis (mean ± SEM, n = 4).

Article Snippet: D4476 (#74014) was purchased from Stem Cell Technologies Inc. (Cambridge, MA, USA).

Techniques: Expressing, MTT Assay, Western Blot, Transfection

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A) Cytotoxicity assay of either GFP-C1 or GFP-ERα-transfected ER - breast cancer cells. Bar denote relative percentage of % survival of transfected MDA-MB-231 cells after treatment with vehicle, 1 μM TAM, 2 μM D4476, and 1 μM TAM plus 2 μM D4476 for 24h. Cellular toxicity was measured using the MTT assay (mean (SD), n = 5; * P < 0.05, ** P < 0.01 vs . each represented counterpart). (B) Western blotting analysis from GFP-ERα-transfected ER - breast cells treated with vehicle or 2 μM D4476 together with 1 μM TAM for 24 h were performed. Immunoblots for PI3K/AKT/mTOR/S6K signaling-associated proteins in ER - MDA-MB-231 cells. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis (mean ± SEM, n = 4).

Article Snippet: D4476 (#74014) was purchased from Stem Cell Technologies Inc. (Cambridge, MA, USA).

Techniques: Cytotoxicity Assay, Transfection, MTT Assay, Western Blot, Expressing

Journal: PLoS Genetics

Article Title: Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds

doi: 10.1371/journal.pgen.1007850

Figure Lengend Snippet: (A) Lysates from NIH/3T3 stable cell lines expressing the dog, Myc-tagged wild-type (Wt) or mutant variant (Mut) DVL2, which is 23 aa shorter than wild-type exogenous DVL2, were analyzed by western blotting using an anti-c-Myc antibody. To assess the ability of the wild-type and mutant proteins to respond to WNT stimulation, cells were treated with WNT5A or WNT3A for 6 hours. Both treatments resulted in increased gel mobility shifts of the wild-type DVL2 protein, indicative of increased phosphorylation; this effect was reduced on the mutant DVL2 protein. (B) To confirm that the DVL2 gel mobility shifts observed in (A) were due to phosphorylation, cell lysates were subjected to mock treatment (30 min incubation at 37 C), or calf intestinal phosphatase (CIP) treatment (30 min incubation at 37 C in the presence of CIP) before separation by SDS-PAGE. The DVL2 gel mobility shifts above wild-type and mutant proteins were lost after CIP treatment, confirming that they are caused by phosphorylation. (C) To test whether the DVL2 gel mobility shifts observed in (A) were driven by casein kinase 1 (CK1), cells were treated with D4476, a CK1 inhibitor, for 1 hour prior to and concurrently during the Wnt stimulation for 6 hours. The DVL2 gel mobility shifts were lost after D4476 treatment, further indicating that they are caused by CK1-dependent phosphorylation. α-tubulin was used for loading controls. Cell lysates were normalized by BCA assays for total protein.

Article Snippet: For casein kinase 1 inhibitor treatment, cells were pre-treated with D4476 (APEXBIO catalog #A3342, 100nM final concentration) for 1h prior to WNT5A or WNT3A treatment.

Techniques: Stable Transfection, Expressing, Mutagenesis, Variant Assay, Western Blot, Phospho-proteomics, Incubation, SDS Page