Fig 1: PHLDB2 is the key downstream modulator of miR-491-5p/miR-875-5p-NOTCH3 cascade in GC.A The top-listed genes which were regulated by NOTCH3 knockdown or miR-491-5p/miR-875-5p overexpression. B PHLDB2 is positively correlated with NOTCH3 mRNA expression in TCGA primary GC tissues (r = 0.372, P < 0.001). C PHLDB2 mRNA expression is negatively associated with miR-491-5p (r = −0.197, P < 0.001). D PHLDB2 mRNA was downregulated after NOTCH3 knockdown or miR-491-5p/miR-875-5p overexpression in AGS and MKN28 cells (**P < 0.001). E Both NOTCH3 knockdown or miR-491-5p/miR-875-5p overexpression decreased the protein expression of PHLDB2 by western blot analysis. F ChIP-qPCR experiments revealed that NOTCH3 directly interacts with the two NOTCH3-CSL binding motifs in AGS cells (**P < 0.001). G Expression of NOTCH3-ICD and PHLDB2 after treating AGS cells with RO4929097.
Fig 2: Stratifying primary GC samples as NOTCH3-PHLDB2-Akt co-activation and inactivation subgroups.A Representative images of immunohistochemistry staining for NOTCH3, PHLDB2, and Akt in primary GC samples. B NOTCH3-PHLDB2-Akt co-activation group was associated with poor outcomes both in intestinal and diffuse-type GC. C AGS and MKN28 cells with NOTCH3/PHLDB2 knockdown or miR-491-5p/miR-875-5p overexpression were more sensitive to Cisplatin or 5-FU treatment, compared with siScramble or Negative control, respectively (P < 0.05). D The summarized IC50 of Cisplatin and 5-FU on AGS and MKN28 cells with NOTCH3/PHLDB2 knockdown or miR-491-5p/miR-875-5p overexpression (*P < 0.05; **P < 0.001).
Fig 3: PHLDB2 plays an oncogenic role and its overexpression correlates with poor survival in GC.A siPHLDB2 inhibited cell proliferation in AGS and MKN28 cell lines (**P < 0.001). B PHLDB2 silencing decreased monolayer colony formation ability (**P < 0.001). C Tumor cell invasion ability was suppressed by PHLDB2 knockdown (**P < 0.001). D Western blot analysis demonstrated elevated expression of cell cycle regulator proteins, p21 and p27, together with activated apoptosis marker cleaved-PARP after silencing PHLDB2 and inactivated Akt and mTOR in GC cells. E Upregulation of PHLDB2 is correlated with poor overall survival in GC patients (TCGA cohort, P = 0.009). F Immunohistochemistry images of PHLDB2 in primary GC samples. PHLDB2 showed negative expression in normal epithelium cells, but it was expressed in the membrane and cytoplasm of the cancer cells. G PHLDB2 abundance was associated with poor disease-specific survival in Hong Kong cohort (P = 0.006). H The expression of PHLDB2 in the four molecular subtypes of GC. I NOTCH3 and PHLDB2 were overexpressed in diffuse-type GC compared with intestinal type GC (TCGA cohort; NOTCH3, P < 0.001; PHLDB2, P < 0.001).
Fig 4: Targeting NOTCH3-PHLDB2-Akt signaling cascades by small molecule inhibitors in GC.A Knockdown efficiency of shNOTCH3 was validated in MGC-803 cells (P < 0.05). B Knockdown of NOTCH3 by shRNA significantly suppressed the xenograft formation in nude mice compare with negative control (n = 5, P = 0.008). C The shNOTCH3 group formed smaller and less number of nodules than the negative control group (P < 0.005). D RO4929097 inhibited subcutaneous tumor growth in nude mice (Vehicle group n = 9, RO4929097 group n = 10, P = 0.039). E MK-2206 2HCl suppressed xenograft formation of the MGC-803 cells (n = 10, P = 0.0002). F Western blot analysis: NOTCH3-ICD and PHLDB2 expression after RO4929097 treatment; Akt-mTOR phosphorylation upon MK-2206 2HCl. Two tumor samples were applied for detecting the related protein levels. G Monolayer colony formation images of MGC-803 and SGC-7901 cells treated with RO4929097 or MK-2206 2HCl alone, or a combination of these two inhibitors. H Overall schematic presentation of miR-491-5p/miR-875-5p-NOTCH3-PHLDB2-Akt cascade in gastric carcinogenesis.
Supplier Page from Abcam for Anti-PHLDB2 antibody