Fig 1: PLAAT4 suppresses malignant behaviors in HGSOC cells. (A) Representative images of PLAAT4 protein staining by IHC in normal fallopian tube and HGSOC tissues. (B) Kaplan-Meier analysis of overall survival in 41 HGSOC patients stratified by PLAAT4 expression. (C,D) Western blot analysis of AKT and p-AKT in total cell lysates after PLAAT4 was overexpressed in SKOV3 and COV504 cells. (E) Assessment of the colony-forming ability of SKOV3 cells via colony formation assays. (F) Assessment of the colony-forming ability of COV504 via colony formation assays. (G) Assessment of proliferation in SKOV3 cells via EdU incorporation assays (scale bar = 1,000 µm). (H) Assessment of COV504 proliferation via EdU assays (scale bar = 1,000 µm). (I) Assessment of the invasiveness of SKOV3 cells via transwell assays (scale bar = 200 µm). (J) Assessment of the invasiveness of COV504 cells via transwell assays (scale bar = 200 µm). (K) Assessment of migration in SKOV3 cells via wound-healing assays (scale bar = 200 µm). (L) Assessment of COV504 cell migration in wound-healing assays (scale bar = 200 µm). (M,N) Western blot analysis of cyclin D1, vimentin, E-cadherin, and MMP1 in total cell lysates after overexpression of PLAAT4 in SKOV3 and COV504 cells.
Fig 2: BCL6 induces HGSOC cell malignant behaviors via the tumor suppressor protein PLAAT4. (A,B) Colony formation of BCL6-overexpressing SKOV3 and COV504 cells, with or without PLAAT4 re-expression, was measured via colony formation assays. (C,D) Proliferation of BCL6-overexpressing SKOV3 and COV504 cells, with or without PLAAT4 re-expression, was measured via EdU assays. (E–H) The migration and invasion of BCL6-overexpressing SKOV3 and COV504 cells, with or without PLAAT4 re-expression, were analyzed via transwell and wound healing assays. (I,J) Western blot analysis of BCL6, PLAAT4, cyclin D1, vimentin, E-cadherin, and MMP1 in BCL6-overexpressing SKOV3 and COV504 cells with or without PLAAT4 re-expression. The data are presented as the means ± SDs of n = 3 independent biological experiments; two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.
Fig 3: BCL6 activates the PI3K‒AKT signaling pathway by negatively regulating PLAAT4. (A) Genomic distribution of CUT&Tag peaks in SKOV3 cells. (B) KEGG analysis of the peak-related genes in (A). (C) Venn diagram of overlapping genes between sets of genes downregulated through RNA-seq and the target gene set identified by CUT&Tag. (D) qRT‒PCR revealed that SKOV3 and COV504 cells overexpressing BCL6 presented decreased PLAAT4 mRNA expression. (E,F) Western blotting revealed that SKOV3 and COV504 cells overexpressing BCL6 presented decreased PLAAT4 protein expression, whereas BCL6 knockout resulted in the opposite trend. (G) BCL6 peak enrichment in the promoter region of the PLAAT4 gene according to CUT&Tag analysis. (H) Sequence logo of a potential BCL6-binding site in CUT&Tag. (I) A diagram of mutant sites in the PLAAT4 sequence. (J) Dual-luciferase reporter assay with the promoter of PLAAT4 and the mutated promoter of PLAAT4 in BCL6-overexpressing SKOV3 cells. (K,L) Immunoblot analysis of AKT, p-AKT, BCL6, and PLAAT4 in BCL6-overexpressing SKOV3 and COV504 cells transfected with or without PLAAT4. The data are presented as the means ± SDs of n = 3 independent biological experiments; two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.
Fig 4: BCL6 promotes HGSOC growth and metastasis via PLAAT4 in vivo. (A,B) BCL6-overexpressing SKOV3 cells, with or without PLAAT4 re-expression, were subcutaneously injected into nude mice. Images (A) and weights (B) of the excised xenografts recovered at 21 days. Data represent mean ± SD, n = 6 mice per group. (C) Representative IHC staining of BCL6, PLAAT4 and p-AKT in tumors derived from nude mice. (D) Bioluminescence imaging at 28 days after intraperitoneal injection using IVIS Imaging System. (E,F) HGSOC tissues were analyzed in parallel for the expression of BCL6 and PLAAT4 via immunohistochemical (IHC) staining. Representative staining of cases with low and high expression of BCL6, respectively (E,F) and quantification of IHC staining as immunoreactive scores (IRS) (G). *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.
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