Oasis Trim Keto UPDATE 2020 Activity Via SET In Acute Myeloid Leukemia!

January 8 2020

Oasis Trim Keto and MOLM-13 cell lines were treated with the p38 inhibitors SB203580 (2.5 µM) and PH797804 (250 nM) for 24 h. Protein expression for p-HSP27/HSP27 (p38 substrate) and SET was analyzed by western blot. B Measurement of PP2A activity after p38 inhibition by immunoprecipitation and phosphatase assay. C Silencing of p38α and p38β with specific siRNA (50 nM for 48 h), and analysis of total protein by western blot. D Measurement of PP2A activity by immunoprecipitation and phosphatase assay. E Western blot analysis of total protein in AML cell lines and AML patient samples, compared to peripheral blood mononuclear cells (PB-MC). The results are corrected by the specific loading control and are expressed as fold-change of the control, which are assigned a value of 1 and are mean values. Experiments were performed in triplicate four times. *p < 0.05.

The p38 family has four members: p38α, p38β, p38γ, and p38δ. As p38α and p38β are the main targets of SB203580 and PH797804 at the tested concentrations27,28, we focused on these two kinases. Knockdown of p38α or p38β by specific siRNAs showed that downregulation of p38β, but not p38α, significantly decreased SET protein levels and increased PP2A activity (Fig. 1c, d).

To explore the clinical relevance of this finding, we assessed p38α and p38β expression in AML. The p38β protein was highly expressed in 5 out of 7 AML cell lines (71%), and in 23 out of 27 AML patient samples (85%); whereas the p38α protein was almost equally Oasis Trim Keto expressed in PB and AML specimens (Fig. 1e, Supplementary Fig. S2). Correlation analysis indicated a positive co-expression between SET and p38β protein levels, which was statistically significant (R2 = 0.376 p-value 0.0014). However, no correlation was found between p38α and SET (R2 = 0.004 p-value 0.7694). Quantitative analysis confirmed that p38α was expressed at similar levels in PB and AML cell lines (20–30 ng/100 µg total protein). However, p38β was expressed at lower levels than p38α in HL60 and MOLM-13 cells (2–3 ng/100 µg total protein), but it was undetectable in PBMC (Supplementary Fig. S3). Taken together, these results suggest that p38β is overexpressed in AML and can regulate PP2A activity via SET.

p38β binds to and stabilizes SET in AML cells

We next focus on dissecting the mechanisms through which p38β regulates the SET protein. Co-immunoprecipitation experiments indicated that SET bound to p38β in both HL60 and MOLM-13 cells, and to a lesser extent, to p38α in HL60 cells (Fig. 2a). Immunofluorescence analysis confirmed high expression and cytoplasmic colocalization between p38β and SET, which disappeared after silencing p38β, whereas p38α silencing had no effect (Fig. 2b).

Fig. 2: p38β co-localizes with SET in AML cells.

a Immunoprecipitation of SET, p38α and p38β with specific antibodies in HL60 and MOLM-13 cells. Normal goat Ig was used as negative control b Knockdown of p38α and p38β with siRNA (50 nM for 48 h), using scramble siRNA as control, in HL60 and MOLM-13 cells. Immunofluorescence analysis of either p38α or p38β (red) and SET (green). Nuclei were stained with DAPI. Immunofluorescences were visualized by confocal microscopy. Quantification table of colocalization fluorescence and green fluorescence (SET) in nucleus and cytoplasm. Quantification analysis showed ~60% of SET-p38β colocalization in both cell lines, and only 12 and 29% of SET-p38α colocalization in MOLM-13 and HL60, respectively. Oasis Trim Keto Immunofluorescence analysis of p38β (red) and SET (green) in peripheral blood mononuclear cells (PB-MC) and the primary AML patient samples AML-23 and AML-24. Nuclei were stained with DAPI. Quantification table of colocalization fluorescence. Immunofluorescences were visualized by confocal microscopy. The results are expressed as mean values ± SEM. Experiments were performed in triplicate four times. *p < 0.05, **p < 0.01. Scale bar represents 5 µm.

We hypothesized that p38β might phosphorylate SET. Surprisingly, in vitro kinase assays showed no direct SET phosphorylation either by p38β or p38α (Supplementary Fig. S4a). Importantly, co-immunoprecipitation in HL60 cells treated with p38 inhibitors showed that SET-p38 interaction did not require kinase activation (Supplementary Fig. S4b). For these reasons, we postulated that p38β could regulate SET stability in a kinase-independent manner. Treatment of cells with cycloheximide demonstrated that SET is stable up to 48 h (Supplementary Fig. S5a). Treatment of p38β-silenced cells with cycloheximide resulted in a significant decrease in SET (Supplementary Fig. S5b), suggesting that p38β-SET interaction is critical for SET stability. Besides, immunofluorescence analysis in samples from AML patients that overexpress SET and p38β, such as AML-23 or AML-25, demonstrated that both proteins tend to associate and colocalized in the cytoplasm. In contrast, samples from patients with no SET or p38β overexpression, such as AML-24, showed minimal colocalization (Fig. 2c, Supplementary Fig. S6a, b). These results support the biological importance of SET-p38β binding in AML, and suggest that p38β contributes to cytoplasmic SET stability. Data from our group previously reported that SET protein stability is enhanced through its binding to SETBP125. Here, we show that SETBP1 and p38β colocalized along with SET in the cytoplasm (Fig. 3a, Supplementary Fig. S7). Furthermore, we found SET-PP2Ac and PP2Ac-p38β colocalization and interaction in AML cells (Fig. 3b). Taken together, these results suggest that p38β acts as a SET stabilizing protein, together with SETBP1, allowing SET to inhibit PP2A in the cytoplasm.

a Immunofluorescence analysis of SET (green) and SETBP1 (red), and SETBP1 (green) and p38β (red), in HL60 and MOLM-13 cells. Nuclei were stained with DAPI. Quantification of colocalization fluorescence. (b) Immunofluorescence analysis of SET (green) and PP2Ac (red) and PP2Ac (green) and p38β (red), in HL60 and MOLM-13 cells. Nuclei were stained with DAPI. Quantification of colocalization fluorescence. Immunofluorescences were visualized by confocal microscopy. The results are expressed as mean values ± SEM. Experiments were performed in triplicate four times. *p < 0.05, **p < 0.01. Scale bar represents 5 µm.

p38β regulates CK2-mediated phosphorylation of SET and facilitates its translocation to the cytoplasm

SET is mainly localized in the nucleus29, but AML cells overexpressing SET showed strong cytoplasmic half-moon-shape localization (Fig. 2b, c). It has been reported in Alzheimer’s disease models that CK2 phosphorylates Ser9 on SET, leading to its cytoplasmic translocation and inhibition of PP2A, resulting in tau phosphorylation30,31. CK2 is overexpressed in Oasis Trim Keto most hematological tumors, including AML32, and it is a target of p38 signaling33. This data prompted us to postulate the potential role of p38β in regulating CK2 and, consequently, the phosphorylation of SET in AML. Western blot showed that overexpression of CK2 is a recurrent event in both AML cell lines and patient samples (Fig. 4a, Supplementary Fig. S2). First, we confirmed that CK2 phosphorylation is indeed regulated by p38 in AML cells, as it is decreased after p38α or p38β knockdown (Fig. 4b). Next, we investigated whether p38β silencing or CK2 inhibition using CX-4945 affects SET phosphorylation, by using Phos-tagTM SDS-PAGE. Inhibition of CK2 and silencing of p38β, but not p38α, substantially decreased the phosphorylated forms of SET in AML cells (Fig. 4c, Supplementary Fig. S8a). Interestingly, while both p38α and p38β can potentially regulate CK2 phosphorylation, only the inhibition of p38β affected SET phosphorylation and SET interaction with CK2.

https://bit.ly/2Ft3mXs

https://pillsfect.com/oasis-trim-keto/

http://fitnessketohealth.over-blog.com/2020/01/slimphoria-keto

http://ketofitpill.over-blog.com/2020/01/keto-blaze-extreme

http://ketofitpill.over-blog.com/2020/01/slimphoria-keto

http://fitnessketohealthtone.over-blog.com/2020/01/keto-blaze-extreme

http://fitnessketohealthtone.over-blog.com/2020/01/slimphoria-keto

http://fitnessketohealthtone.over-blog.com/2020/01/oasis-trim-keto

http://healthfitnessdiet.over-blog.com/2020/01/slimphoria-keto

http://healthfitnessdiet.over-blog.com/2020/01/oasis-trim-keto

http://healthfitnessdiet.over-blog.com/2020/01/keto-blaze-extreme

http://ketopimsreviews.over-blog.com/2020/01/slimphoria-keto