Fig 1: The protein staining of the fetal membrane cells for the 22 markers evaluated by immunohistochemistry. AEC: amnion epithelial cells, AMSC: amnion mesenchymal stromal cells, CMSC: chorion mesenchymal stromal cells, CTC: chorion trophoblast cells, DSC: decidual stromal cells. Proteins significantly expressed in AEC: AHNAK2, CK5, CK7, CK17, CNR1, DPYSL3, EMP1, FERMT2, FLT1, GPX8, MUC16, PRLR, PVRL4, RXFP1, UCHL1, UPK1B, and VIM; proteins significantly expressed in AMSC: AHNAK2, DPYSL3, EMP1, FLT1, GPX8, PRLR, RXFP1, UCHL1, and VIM; proteins significantly expressed in CMSC: AHNAK2, DPYSL3, EMP1, FLT1, GPX8, PDLIM4, PRLR, THY1, UCHL1, and VIM; proteins significantly expressed in CTC: CK7, CNR1, FLT1, GPX8, PRTG, PVRL4, UCHL1, and UPK1B; proteins significantly expressed in DSC: AHNAK2, DPYSL3, FERMT2, FLT1, GPX8, PDLIM4, PRTG, THY1, UCHL1, and VIM.
Fig 2: PDLIM4 promotes malignant progression of GC cells in vitro and in vivo.a RT-qPCR analysis demonstrated the efficiency of PDLIM4 KD at the mRNA level in MKN45 and HGC27 cells. b Western blotting analysis corroborated the knockdown efficiency of PDLIM4 at the protein level in MKN45 and HGC27 cells. c-d Clonal formation assays were conducted in MKN45 and HGC27 cells with PDLIM4 KD. e Cell viability assays indicated the effects of PDLIM4 depletion on MKN45 and HGC27 cells. f-g Apoptotic responses in MKN45 and HGC27 cells following PDLIM4 depletion were assessed. h The expression levels of apoptosis-related protein and cell cycle proteins were evaluated in MKN45 and HGC27 cells with PDLIM4 KD. i Representative images of tumors harvested from mice injected with shPDLIM4 or shControl MKN45 cells are presented. j Tumor growth was monitored in vivo, measuring tumor volume over time. k The weight and volume of harvested tumors were recorded (n = 6). l The mRNA expression levels of PDLIM4 in each tumor group were quantified. Data were presented as means±SD. *P<0.05; **P<0.01; ***P<0.001.
Fig 3: PDLIM4 interacts with HSP70.a Integration of mass spectrometry data with the BioGRID database. b Kaplan-Meier survival analysis of OS was performed using HSP70 expression levels. Patients were split into two groups, HSP70High and HSP70low, based on the median IHC staining score (6 for HSP70). c Kaplan-Meier survival curves for OS in patients categorized by the IHC staining scores (5.33 for PDLIM4 and 6 for HSP70) are shown. Patients were grouped into three categories based on the expression levels of PDLIM4 and HSP70: PDLIM4High + HSP70High, PDLIM4Low + HSP70Low and others. d Examination of the interaction between exogenously expressed SFB-tagged PDLIM4 and HSP70 in HEK-293T cells using a co-immunoprecipitation assay. e Investigation of the interaction between exogenously expressed SFB-tagged PDLIM4 and HSP70 in MKN45 cells via a co-immunoprecipitation assay. f Immunofluorescence analysis of PDLIM4 and HSP70 expression in HGC27 cells, Scale bar: 20 μm. g The interaction between PDLIM4 and HSP70 in HGC27 cells was detected by PLA, Scale bar: 20μm. h Based on previous studies, PDLIM4 is categorized into distinct structural domains. i-j Analysis of the interaction between various exogenous SFB-tagged PDLIM4 constructs (330AA, 1-88AA, 1-260AA, 80-260AA, 80-330AA, 250-330AA) and HSP70 in HEK-293T cells through a co-immunoprecipitation (CO-IP) assay.
Fig 4: PDLIM4 positive regulates HSP70’s Protein level through ubiquitination. a Representative immunohistochemistry staining images of PDLIM4 and HSP70 across various grades of GC tissues and adjacent normal tissues, scale bars: 50μm. b IHC scores for HSP70 in 40 pairs of samples of GC tissues and adjacent normal tissues. c Analysis of the protein expression levels of PDLIM4 and HSP70 in GC tissues using Spearman 's correlation. d Evaluation of the mRNA expression level of HSP70 in GC cells following PDLIM4 KD. e Assessment of the protein expression level of HSP70 in GC cells with PDLIM4 KD. f Western blotting analysis of HSP70 in MKN45 and HGC27 cells with PDLIM4 KD after treatment with MG132 (20 μM). g Determination of the HSP70 degradation half-life in MKN45 and HGC27 cells with PDLIM4 KD in the presence of cycloheximide (CHX, 200 μg/mL). h Analysis of HSP70 ubiquitination levels in cells with PDLIM4 knockdown or overexpression. Data were presented as means±SD. ***P<0.001; ns, no statistical difference.
Fig 5: DLIM4 promotes GC cells malignant progression by regulating the HSP70-mediated MAPK signaling pathway.a Diagrammatic representation of the in vivo and image of tumors. b Growth of tumor volume in vivo. c-d Weight and volume of the harvested tumors (n = 7). e-f KEGG enrichment analysis of differentially expressed genes between shControl and shPDLIM4#1/2 MKN45 cells. g Western blotting was used to analyze the total and phosphorylation protein levels of P38, JNK, and ERK on MKN45 and HGC27 cells with PDLIM4 KD. h Western blotting was used to analyze the total and phosphorylation protein levels of P38, JNK, and ERK on PDLIM4 KD MKN45 and HGC27 cells with HSP70 overexpression. Data were presented as means±SD. *P<0.05; **P<0.01; ***P<0.001.
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