casein kinase i inhibitor  (Santa Cruz Biotechnology)

Journal: Cell & Bioscience

Article Title: Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression

doi: 10.1186/s13578-024-01249-x

Figure Lengend Snippet: Casein kinase 1 inhibition with D4476 increases ENO1 nuclear accumulation in MCF10Ca1h tumour cells only. (A) MCF10 TNBC cell lines that were treated with 125 µM casein kinase 1 inhibitor D4476 or DMSO for 3 h, then fixed and stained with anti-ENO1 antibodies and imaged by CLSM. White star indicates complete nuclear accumulation of ENO1 observed in 10% of D4476 treated MCF10Ca1h cells. Images represent single typical cells from a series of 4 independent biological replicate experiments. (B) Images such as those in (A) were analysed to determine Fn/c ratio as previous. Results represent mean ± SEM ( n > 50) of a single typical experiment from a series of 4 independent biological replicate experiments. **** p < 0.0001 compared to DMSO treated cell line, all other comparisons were nonsignificant. Data was not corrected for multiple comparisons

Article Snippet: D4476 casein kinase I inhibitor was purchased from Abcam.

Techniques: Inhibition, Staining

Journal: Cell & Bioscience

Article Title: Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression

doi: 10.1186/s13578-024-01249-x

Figure Lengend Snippet: Inhibited phosphorylation of miniTurbo-ENO1 with CK1 inhibitor D4476 reduces ENO1 interaction with cytoskeletal proteins and increases interaction with DNA-metabolism proteins. MCF10Ca1h tumour cells were transfected with V5-miniTurbo or V5-miniTurbo-ENO1, then biotinylated by addition of exogenous biotin for 20 min to induce proximity labelling of ENO1 interacting proteins. (A) CLSM images of MCF10Ca1h cells transfected with V5-miniTurbo (left) or V5-miniTurbo-ENO1 (right), then left untreated (UT) or treated with 50 µM biotin, cells were then fixed and stained with anti-V5 antibodies (red), or FITC-streptavidin (green) and nuclei were counterstained with DAPI (blue). Images represent single typical cells from a series of 3 independent biological replicate experiments. (B) Western blot of MCF10Ca1h cell lysates expressing miniTurbo-ENO1 (mT-ENO1) that were either untreated (UT) or treated with 50 µM biotin to induce proximity labelling of other proteins interacting with mT-ENO1. (C) Volcano plot of proteins that showed differences in label free quantitation (LFQ) of expression between MCF10Ca1h cells expressing MiniTurbo-ENO1 that were D4476 treated or DMSO treated, identified by LC-MS. Results represent difference in mean LFQ intensity (D4476 treated – DMSO treated) from 3 replicates, plotted against -log transformed p-values. Red stars indicate statistically significant differences in LFQ intensity between samples with gene name listed. (D) Graphical representation of protein classes present in significantly reduced miniTurbo-ENO1 with D4476 treatment interactor list. Results represent percentage of genes mapped to each protein class listed from n = 10 genes. Highest percentage group was coloured red. (E) Graphical representation of protein classes present in significantly enriched miniTurbo-ENO1 with D4476 treatment interactor list. Results represent percentage of genes mapped to each protein class listed from n = 9 genes. Highest percentage group was coloured red. Protein classes were assigned using Panther Gene Ontology (GO) analysis online tool. LFQ intensity differences and p-values are reported for all differing proteins in Supplementary Table 1

Article Snippet: D4476 casein kinase I inhibitor was purchased from Abcam.

Techniques: Transfection, Staining, Western Blot, Expressing, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy, Transformation Assay

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: Early phosphorylation of p53 in S20 induced by Resv is necessary for p53-stability in MCF-7 R cells. ( A ) MCF-7 R cells were treated with CDDP (6 μM) with or without Resv (100 μM) and ( B ) MCF-7 cells were treated with Resv (100 μM); both cell cultures were treated for 6 h with specific p53-pS20 site kinase inhibitors: CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM). Total and phospho-p53 contents are assessed by Western blot using antibodies directed against total p53 (DO-1) or against the specific phosphorylated residue on S20, as indicated. ( C ) MCF-7 and MCF-7 R cells were treated with a DMSO–ethanol vehicle as control or CDDP (6 μM) with resveratrol (100 μM) and cultured in combination with CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM) inhibitors for 48 h and were double-stained with Annexin V and propidium iodide (PI) followed by flow cytometry analysis to determine apoptotic cells. The viable cells are located in the lower left quadrant (double negative with Annexin V–/PI–). Apoptotic cells (Annexin V+/PI–) appear in the lower right (early apoptosis) and upper right (late apoptosis) quadrant of data plots. Data are presented as a percentage of the cell population. ( D ) The combined results of three independent cytometry analyses depicting the mean levels of total apoptotic cells are shown. Results are presented as the means ± SD. *** p < 0.001 by one-way ANOVA followed by Turkey’s Multiple Comparison test.

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Residue, Control, Cell Culture, Staining, Flow Cytometry, Cytometry, Comparison

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: In the MCF-7 resistant cell variant (MCF-7 R ), Resv attenuates phosphorylation in S15 and S46 of p53 by dephosphorylation and deactivation of ATM. However, it activates kinases CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20 (which is required to activate p53 in order to upregulate BAX and PUMA genes) and modifies the ratio between BCL-2/BAX expression. The BAX protein was increased while BCL-2 protein was decreased, restoring apoptosis and overcoming chemoresistance. On the other hand, the overexpression of BCL-2 in MCF-7 R cells after CDDP treatment maintains the chemoresistance and blocks apoptosis despite the phosphorylation of p53 in S15 and S46 and the upregulation of NOXA and PUMA .

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Variant Assay, Phospho-proteomics, De-Phosphorylation Assay, Expressing, Over Expression