Fig 1: Functional and pathway analyses of PGK1 in ovarian cancer. (A) The correlation between PGK1 and other glycolysis-related genes. (B) GO and KEGG analyses of genes interacting with PKG1. (C) The correlation between PGK1 and immune or inflammatory cell infiltration (TISIDB database). (D) The correlation between PGK1 and immune or inflammatory cell infiltration (TIMER database). (E) The correlation between PGK1 and chemokines (TISIDB database). There is no PKM2 expression data in the cBioPortal database.
Fig 2: P7C3 directly binds to PGK1. (A) Streptavidin affinity assay for P7C3-Bio binds to PGK1 in P7C3-treated glioma cells. Cells in culture were treated with 50 µM P7C3-Bio for 24 h, and then streptavidin agarose was added to incubate with the cell lysates. (B) Streptavidin affinity assay for P7C3-Bio binds to PGK1 upon cell lysates. Cell lysates were incubated with 10 µM P7C3-Bio at 37°C for 1 h, and then streptavidin agarose was added to incubate with the cell lysates. (C) Streptavidin affinity assay for P7C3-Bio binds to purified PGK1 protein. In total, 25 µg recombinant human PGK1 solution was incubated with 10 µM P7C3-Bio at 37°C for 1 h, and then streptavidin agarose was added to incubate with the cell lysates. (D) Motif analysis based on top 100 P7C3 target proteins was performed by MEME, and three motifs were identified. The motif consensus and motif locations at PGK1 are shown. (E) The P7C3 binding sites of PGK1 were identified by ESI-LC-MSMS. The P7C3 binding sites were identified by the predicted peptide mass plus P7C3 [C21H18Br2N2O with a loss of a water] and minus one H.
Fig 3: PGK1 affected EMT process and glycolysis of ovarian cancer cells. (A) PGK1 protein expression in three kinds of ovarian cancer cell lines. (B) PGK1-siRNA inhibited the proliferation of ovarian cancer cells. (C) PGK1-siRNA reduced the formation of colonies of ovarian cancer cells. (D) PGK1-siRNA reduced the migration of ovarian cancer cells. (E) PGK1-siRNA reduced the invasion of ovarian cancer cells. (F) PGK1-siRNA decreased the protein expression levels of MMP2, MMP9, and N-cadherin but increased the protein expression level of E-cadherin. (G) Chart of ECAR measurement in PGK1-silenced cells. (H) Chart of OCR measurement in PGK1-silenced cells. Error bars represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 4: P7C3 accelerates PGK1 protein degradation. (A) PGK1 mRNA level was measured by RT-PCR. P7C3 upregulates the PGK1 mRNA levels in U87MG and U118MG cells (n = 3). (B) Time-course assay of P7C3 effects on PGK1 degradation. U87MG and U118MG cells were treated by CHX (100 ug/mL) with or without P7C3 (50 µM) for 24 h, and then PGK1 protein levels were detected by western blotting. (C) Protein level curve for PGK1 based on time-course analysis of U87MG and U118MG cells (n = 3). (D) Western blotting of PGK1 protein degradation pathway. MG132 (5 µM) and CQ (20 µM) were used to treat cells for 6 h, followed by P7C3 treatment for another 18 h, and the protein level of PGK1 was detected by western blotting. (E) The statistical analysis of the quantized protein levels of PGK1 in U87MG and U118MG cells (n = 3). (F) Transmission electron microscopy assay after glioma cells were treated with P7C3 for 24 hours. Autophagosomes, autolysosomes, and phagophores are indicated by yellow, green, and red arrows (scale bar: original mage, 5 µm; Zoom image, 1 µm). (G) Western blotting for two key autophagy-associated proteins Beclin-1, and LC3A/B, after U87MG and U118MG cells were treated with 0 µM, 30 µM, and 50 µM P7C3 for 24 h. (Data are expressed as mean ± SD, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means none significance).
Fig 5: The effect of NG52 on ovarian cancer cells. (A) The effect of NG52 on PGK1 activity. (B) NG52 inhibited the proliferation of ovarian cancer cells in a dose-dependent manner. (C) Time points of NG52 anti-proliferative effects on ovarian cancer cells. (D) NG52 induced apoptosis of ovarian cancer cells. (E) NG52 reduced the migration of ovarian cancer cells. (F) NG52 reduced the invasion of ovarian cancer cells. (G) NG52 decreased the protein expression levels of MMP2 and MMP9. (H) Chart of ECAR measurement in cells treated with NG52. (I) Chart of OCR measurement in cells treated with NG52. (J) Graphical abstract. Error bars represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.
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