Fig 1: PSMG2 knockdown activated autophagy in TNBC cells(A and B) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were treated with or without AZD6244 (1 µM) for 24 h, and the relative mRNA or ribosome-bound mRNA expression level of PDPK1 was detected by qRT-PCR. Error bars represent the mean ± SD, n = 3 technical replicates.(C) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, the autophagy-related proteins were detected by western blot in BT549 and MB468 cells. Loading control, ß-actin.(D and E) BT549 and MB468 cells were treated with or without MG132 (2 µM) for 24 h or bortezomib (100 nM) for 36 h, and then the autophagy-related proteins were detected by western blot. Loading control, ß-actin.(F) BT549 cells, transfected with vector (pLKO.1) or PSMG2 shRNA (shPSMG2), were imaged by transmission electron microscopy. Two magnified views of the electron photomicrograph show a characteristic autophagosome. N, nucleus; ER, endoplasmic reticulum; M, mitochondria; A, autophagosome. The scale bar represents 2 µm and 1 µm, respectively.(G) After PSMG2 knockdown, ER stress-related proteins were detected by western blot in BT549 and MB468 cells. Loading control, ß-actin.
Fig 2: PDPK1 was degraded by autophagy in TNBC cells(A) BT549 and MB468 cells were treated with different concentrations of autophagy activator (rapamycin) for 24 h, and then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, ß-actin.(B and C) Clonogenic assays of BT549 and MB468 cells treated with different concentrations of AZD6244 and LiCl (6 mM) for 10–14 days.(D) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were treated with CQ (40 µM) for 8 or 12 h, and then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, ß-actin.(E) After treatment with or without MG132 (2 µM) for 24 h, BT549 and MB468 cells were treated with CQ (40 µM) for 8 or 12 h, then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, ß-actin.(F) Confocal microscopy images show the merged subcellular co-localization images of PDPK1 and LAMP1 of BT549 and MB468 cells. BT549 and MB468 cells treated with MG132 (2 µM) or rapamycin (1 µM) for 24 h or transfected with PSMG2 shRNA (shPSMG2) and treated with or without CQ (40 µM) for 12 h. Nucleus and LAMP1 were stained with DAPI (blue) and Alexa Fluor 594-conjugated wheat germ agglutinin (red), respectively. PDPK1 was stained with Alexa Fluor 488 combined with wheat germ agglutinin (green). White arrows indicated the co-localization between PDPK1 and LAMP1. The scale bar represents 40 µm.(G) Clonogenic assays of BT549 and MB468 cells treated with or without different concentrations of AZD6244 and CQ (0.5 µM) for 10–14 days after PSMG2 shRNA (shPSMG2) transfection.(H and I) The results of the clonogenic assays (G) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); *p < 0.05, ***p < 0.001, ****p < 0.0001; one-way ANOVA.
Fig 3: PSMG2 knockdown downregulated PDPK1/AKT signaling(A) The scatterplot shows the correlation between proteasome and all 50 hallmark gene sets in the two single-cell transcriptome datasets (GEO: GSE75688 and GEO: GSE11838). The x axis and y axis represent the correlation coefficients of proteasome and hallmark gene sets in the GEO: GSE11838 and GEO: GSE75688 datasets, respectively. The size and color of the points represent the −log10 p value in the GEO: GSE11838 and GEO: GSE75688 datasets, respectively. The points of the top 5 signal pathways in the comprehensive correlation are marked with different colored outer circles.(B) The scatterplot shows the high correlation between mTOR signaling and proteasome in two independent single-cell transcriptomic datasets (GEO: GSE75688 and GEO: GSE11838). The pink triangles represent the GEO: GSE75688 dataset, and the blue dots represent the GEO: GSE11838 dataset.(C) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were treated with AZD6244 (1 μM) for 2 or 24 h, and then the phosphorylation of ERK in the MAPK pathway and the phosphorylation and total protein of AKT and PDPK1 in the upstream mTOR pathway were detected by western blot. Loading control, β-actin.(D) BT549 and MB468 cells were treated with or without MG132 (2 μM) for 24 h and the related proteins were detected by western blot. Loading control, β-actin.(E) After transfection with vector (pLKO.1) or PDPK1 shRNA (shPDPK1), BT549 and MB468 cells were treated with AZD6244 (1 μM) for 2 or 24 h, and then the phosphorylation of ERK and AKT and total protein levels of PDPK1 were detected by western blot. Loading control, β-actin.(F) Clonogenic assays of PDPK1-knockdown cells (BT549 and MB468) treated with or without AZD6244 (1 μM) for 10–14 days.(G) The results of the clonogenic assays (F) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗∗∗p < 0.001; one-way ANOVA.(H) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were transfected with vector (cPPT) or the overexpression vector for PDPK1 (PDPK1), then the phosphorylation of PDPK1 and AKT was analyzed by western blot. Loading control, β-actin.(I) Under PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were transfected with vector (cPPT) or the vector for PDPK1 expression (cPPT-PDPK1) and treated with or without AZD6244 (0.1 or 0.5 μM) for 10–14 days.(J and K) The results of the clonogenic assays (I) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗p < 0.01, ∗∗∗∗p < 0.0001; one-way ANOVA.
Fig 4: SDC2 facilitates the membrane translocation of PDK1 by interacting with its PH domain in GC cells. A & B. Association of PDK1 and FGF2 with SDC2 (LC/MS analysis, Co-IP assay). C. Interaction of SDC2 and PDK1 in GC cells (Co-IP assays). D & E. Interaction of SDC2 and the PH domain of PDK1 (Co-IP assays, using HEK293T cells; molecular docking performed by Discovery StudioTM). F. Colocalization of SDC2 and PDK1 in AGS and HGC-27 cells (Double immunofluorescent staining). G. Effect of SDC2 on membrane translocation of PDK1 (Western blotting of lipid raft). H, I, & J. Dependence of the oncogenic function of SDC2 on the PDK1-AKT signaling pathway in GC cells (CCK8 assay, Transwell assay, and Western blotting. Scale bar: =100 µm). K. A schematic diagram showing the proposed mechanism.
Fig 5: Working model: SDC2 promotes gastric cancer progression through co-option of PDK1 and modulating the FGF2-AKT signaling axis. USP14 deubiquitinates and stabilizes SDC2, while IU1 treatment decreases the abundance of SDC2 through targeting USP14.
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