ZFIN Image: He et al., 2020, Figure 3

Image

Figure Caption

Figure 3 <italic><styled-content toggle='no' style='fixed-case'>FLT</styled-content>3</italic>/<styled-content toggle='no' style='fixed-case'>ITD</styled-content> upregulated <italic><styled-content toggle='no' style='fixed-case'>FST</styled-content></italic> through phosphorylation of <styled-content toggle='no' style='fixed-case'>CREB</styled-content>

A

In silico analysis (DECipherment of DNA Elements, SABiosciences) and schematic model of transcription factor binding sites on human FST promoter. CBP: CREB‐binding protein; CRE: cAMP‐response element; TSS: transcription start site.

B, C

The direct binding of p‐CREB to human FST promoter was detected by ChIP‐PCR (B) and ChIP‐qPCR (C). c‐Fos was used as positive control of p‐CREB target gene. Normal IgG was used as negative control of ChIP.

D

Dual‐luciferase assay demonstrating the direct binding of p‐CREB on human FST promoter. pRL‐CMV, Renilla luciferase vector; pGL‐CRE− and pGL‐CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE−) or wild type (CRE+); p‐GFPSpark, GFP‐expressing vector; p‐CREB^Y134F, CREB^Y134F‐GFP‐expressing vector.

E

FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3‐parental (P in short) and Ba/F3‐FLT3/ITD (ITD in short) cells.

F–H

Phospho‐flow analysis of p‐CREB in Ba/F3‐parental, Ba/F3‐FLT3/ITD, and Ba/F3‐FLT3/ITD cells treated with FLT3 inhibitor quizartinib (Qui in short). Isotype antibody was used as control to calculate the mean fluorescence intensity (MFI) ratio (F, G). The transcription and expression of Fst were detected by RT–qPCR after quizartinib treatment (10 nM) in Ba/F3‐FLT3/ITD cells for 1 day (H).

I–K

The expression of FST and phosphorylation of CREB were detected by Western blotting (I and K) and phospho‐flow analysis (J) in MOLM‐13 (I) and Ba/F3‐FLT3/ITD (K) cells treated with quizartinib and BRD7389 for 1 day, respectively.

L

RSK expression and FST expression were detected by Western blotting after p90RSK knockout by CRISPR/Cas9 in MOLM‐13 cells.

M

The phosphorylation of CREB and FST expression was detected by Western blotting in Ba/F3‐FLT3/ITD cells treated with CREB inhibitor 666‐15 for 1 day. ^: non‐specific staining of p‐ATF1 protein due to the conserved motif.

N

CREB expression and FST expression were detected by Western blotting after CREB knockout by CRISPR/Cas9 in MOLM‐13 cells.

O

The growth of Ba/F3‐parental (with IL‐3), Ba/F3‐FLT3/ITD (without IL‐3), and Ba/F3‐FLT3/ITD (with IL‐3) cells was measured after 3 days treatment of CREB inhibitor 666‐15 in vitro.

P

The rescue effect of CREB inhibitor 666‐15 on FLT3/ITD‐induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.

Data information: In (C, D, G, H, J, and O), the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P < 0.01 and ***P < 0.001 (Student's t‐test).Source data are available online for this figure.

Figure Data

Phenotype details

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ EMBO Mol. Med.