Fig 1: PHLDA1/2 are highly expressed in cancers harboring ERK activating oncogenes.a Oncogenic ERK signaling induces simultaneous PHLDA1/2 expression. HEK293 cells were stimulated with EGF, TPA, or insulin with or without trametinib for the indicated times. The cell lysates were probed for PHLDA1/2 expression, and for the phosphorylation states and protein levels of ERK and AKT by immunoblotting. b HEK293 cells were treated with various concentration of TPA for 6 h, and the cell lysates were analyzed as in (a). The intensity of the PHLDA1/2 bands was quantified (bottom graphs). c, d PHLDA1/2 mRNA and protein expression levels in HEK293 cells stably expressing NRasG12V or BRafV600E were assessed using qRT-PCR (c) and by immunoblotting (d), respectively. Where indicated, the cells were pretreated with the MEK inhibitor, trametinib. e Immunoblot analysis of PHLDA1/2 expression in various cancer cell lines harboring the indicated oncogenes. Where indicated, cells were pretreated with the EGFR inhibitor (canertinib), the BRaf inhibitor (vemurafenib), or trametinib. b, c Data are mean ± SEM from three independent experiments. P-values were assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a Source Data file.
Fig 2: PHLDA1/2 proteins are highly expressed in human clinical cancers.a, b Representative immunohistochemical staining for PHLDA1 (a) and PHLDA2 (b) in tumor and corresponding normal tissues of lung, pancreas, and colon. Right graphs show the staining profiles of PHLDA1/2 in normal (N) and tumor (T) specimens. Scale bars, 60 µm. P-values were assessed using the Mann–Whitney U test. c, d Analyses of PHLDA1-3 mRNA expression in normal vs. various cancer tissues (c), and in normal vs. colon cancer tissues harboring mutations in the indicated oncogenes (d) based on the TCGA and GEO databases. Each horizontal bar represents the mean. Significance was determined using two-tailed Student t-test (c) or using one-way ANOVA followed by Tukey’s multiple comparisons test (d). ns, not significant. Numbers in parentheses indicate sample sizes. Source data are provided as a Source Data file.
Fig 3: miR-526b-5p inhibition resulted in enhanced the viability, migration, invasion, and proliferation of osteosarcoma (OS) cells in a pleckstrin homology-like domain family A member 1 (PHLDA1)-dependent way. (A) The transfection efficiency of sh-PHLDA1, miR-526b-5p inhibitor, and sh-PHLDA1 + miR-526b-5p inhibitor in HOS and U2OS cells was determined at messenger RNA (mRNA) level. (B) The transfection efficiency of sh-PHLDA1, miR-526b-5p inhibitor, and sh-PHLDA1 + miR-526b-5p inhibitor in HOS and U2OS cells was determined at protein level. (C) The cell viability of OS cells in the five groups was analyzed by CCK8 assay. (D) The migration ability of OS cells in the five groups was analyzed by wound healing assay. The migration rate of every group was interpreted. Magnification: 40×. (E) The invasion ability of OS cells in the five groups was analyzed by Transwell assay. The invading cell number was counted in every selected field. Magnification: 100×. (F) Cell proliferation was determined using colony formation assay. The colony number in every group was analyzed. Con, control; NC, negative control. *p < 0.05 vs. control. #p < 0.05 vs. sh-PHLDA1 group.
Fig 4: MiR-526b-5p directly targeted pleckstrin homology-like domain family A member 1 (PHLDA1) in osteosarcoma (OS) cells. (A) Potential binding sites between miR-526b-5p and PHLDA1 mRNA 3' untranslated region (UTR). (B) Luciferase reporter assay in OS cells cotransfected with miR-526b-5p mimic and PHLDA1-Wt or PHLDA1-Mut reporter plasmids. *p < 0.05 compared with Wt + mimic group. (C) Protein expression of PHLDA1 was detected by Western blot analysis. *p < 0.05 compared with control group. #p < 0.05 vs. sh-circ0085539 group. (D) Quantitative reverse transcription PCR (qRT-PCR) showed that PHLDA1 expression was significantly upregulated in OS tissues. Normal: adjacent tissues. *p < 0.05 compared with normal group. (E) Correlation analysis showed that miR-526b-5p expression was inversely associated with PHLDA1 expression in OS tissues.
Fig 5: PHLDA1 Levels Regulate Sensitivity to Trastuzumab and Lapatinib Treatment(A) Western blot analysis of PHLDA1 levels in MCF7/HER2-18 cells cultured with 1 µM trastuzumab or IgG control for 72 hr.(B) MCF7/HER2-18 cell number following 3-day treatment with 1 µM trastuzumab preceded by 48-hr siRNA knockdown of PHLDA1 or scrambled control.(C) In situ hybridization for PHLDA1 expression in MCF7/HER2-18 xenograft tumors. Four-week-old tumors from mice treated with an IgG control showed strong PHLDA1 mRNA expression (brown), whereas treatment with trastuzumab resulted in significantly weaker staining, as shown in graph on right. Sections were counterstained with hematoxylin, and dotted boxes represent zoomed-in areas. Data are presented as mean ± SEM from at least eight mice for each condition. *p = 0.05, **p = 0.01, compared with IgG controls.(D) Western blot showing PHLDA1 levels in parental and lapatinib-resistant SKBR3 and HCC1954 cells treated with 2 µM lapatinib or DMSO control for 48 hr.(E and G). Upper: H&E staining of SKBR3LapR (E) and HCC1954LapR cells (G) containing a doxycycline-inducible PHLDA1 expression construct. Cells were grown in mini-organotypic cultures for 7 days with or without 2 µM lapatinib and 1 µg/mL doxycycline. Lower: Ki67 staining with nuclei counterstained by DAPI. Right: quantitation of cell number and Ki67-positive nuclei. Data are presented as mean ± SEM. Images are representative of at least three independent experiments. H&E image scale bar, 100 µm; Ki67 image scale bar, 50 µm. ***p = 0.001. H&E images are automatically spliced composites.(F and H) Western blot showing PHLDA1 levels in parental and resistant SKBR3 cells (F) and HCC1954 cells (H) following treatment with doxycycline.
Supplier Page from Abcam for Anti-PHLDA1 antibody [EPR6674]