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. 2018 Feb 28;22(9):2469–2481. doi: 10.1016/j.celrep.2018.02.028

Figure 3.

Figure 3

PHLDA1 Negatively Regulates Akt and Is Downregulated in FGFR Inhibitor-Resistant Endometrial Cancer Cell Lines

(A) Top ten downregulated genes in MFE-296PDR cells (left) and MFE-296AZDR cells (right) compared to parental controls, identified by microarray analysis.

(B–D) Western blot showing downregulation of PHLDA1 levels in parental MFE-296 (B) and AN3CA (C) cells following treatment with 1 μM AZD4547 for 24 hr and persistent downregulation of PHLDA1 in MFE-296AZDR and AN3CAAZDR cells following removal of 1 μM AZD4547 for 24 hr. PHLDA1 levels in Ishikawa cells (D) were unaffected by FGFR inhibitor treatment.

(E) Left: western blot showing reduced p-Akt (pSer473) in HCC1954 cells following transfection with GFP-PHLDA1. Right: quantitation of p-Akt (Ser473), normalized to total Akt and GAPDH. Data are presented as mean fold change ±SEM in p-Akt (Ser473) ∗∗∗p ≤ 0.001.

(F) MFE-296 cells were transfected with constructs encoding GFP-PHLDA1, GFP-mtPHLDA1, or GFP-PH-Akt for 48 hr prior to fixation. Nuclei were labeled with DAPI, and F-actin was visualized using Alexa Fluor 546 Phalloidin (red). Scale bar, 50 μm.

(G) Domain organization of PHLDA1.

PH domain, pleckstrin homology domain; QQ, polyglutamine tract; P-Q, proline-glutamine rich tract; P-H, proline-histidine rich tract. Residues deleted in mtPHLDA1 are indicated in red.