Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 is aberrantly overexpressed in gastric cancer. (A) Venn diagrams illustrating the differentially expressed genes. (B) Expression levels of DEPP1 in GC and normal tissues in the TCGA-STAD cohort. Relative expression of DEPP1 in 11 pairs of GC and adjacent normal tissues measured by (C) reverse transcription-quantitative PCR and (D) western blotting. (E) Representative immunofluorescence images of DEPP1 in 11 pairs of GC and adjacent normal tissues. *P<0.05, **P<0.01 and ***P<0.001. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma; N, normal; T, tumor; Ns, not significant.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Prognostic value of DEPP1 is explored in the TCGA-STAD cohort. Kaplan-Meier survival analyses of DEPP1 in patients with GC on (A) overall survival and (B) disease-free survival. DEPP1 expression between patients with GC who are (C) alive or dead and (D) patients with or without disease progression or recurrence. (E) Univariate and (F) multivariate Cox regression analysis of DEPP1 and other clinicopathologic features. (G) Receiver operating characteristic curves of DEPP1 and conventional oncogenes EGFR and KRAS in predicting five-year overall survival of patients with GC. **P<0.01 and ***P<0.001. DEPP1, decidual protein induced by progesterone; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma; GC, gastric cancer; OS, overall survival; DFS, disease-free survival; HR, hazard ratio; CI, confidence interval.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Expressing, Biomarker Discovery

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Prognostic value of DEPP1 is validated in GSE84437 , GSE15459 and Renji cohorts. Kaplan-Meier survival analyses of DEPP1 on overall survival in (A) GSE84437 and (D) GSE15459 cohorts. (B) Univariate and (C) multivariate Cox regression analysis of DEPP1 and other clinicopathologic parameters in the GSE84437 cohort. (E) Univariate and (F) multivariate Cox regression analysis of DEPP1 and other clinicopathologic factors in the GSE15459 cohort. (G) Representative immunohistochemistry images of DEPP1 in gastric cancer and normal tissues. (H) Kaplan-Meier survival analysis of DEPP1 on overall survival in the Renji cohort. DEPP1, decidual protein induced by progesterone; OS, overall survival; DFS, disease-free survival; HR, hazard ratio; CI, confidence interval.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Immunohistochemistry

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Establishment and evaluation of a prognostic nomogram for GC. (A) Construction of a nomogram integrating DEPP1, age and TNM stage to predict the 1-, 3- and 5-year overall survival of patients with GC. (B) Calibration curves to assess the predictive accuracy of the nomogram for survival outcomes. (C) Results of decision curve analysis comparing the nomogram's performance with that of individual predictors, including DEPP1, age and TNM stage. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; TNM, tumor-node-metastasis; OS, overall survival; Pr, probability.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques:

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Relationship between DEPP1 expression and clinicopathologic factors in patients with GC. (A) DEPP1 expression levels across different Lauren phenotypes. (B) Kaplan-Meier survival analyses of DEPP1 in diffuse, intestinal and mixed subtypes of the GSE15459 cohort. (C) DEPP1 expression levels in stage I and advanced stages of GC. (D) Kaplan-Meier survival analyses of DEPP1 in stage I, II, III and IV of the TCGA-STAD cohort. (E) DEPP1 expression levels across distinct T stages. (F) The histogram displaying the distribution of T stages between the low- and high-expression groups of DEPP1. (G) The alluvial diagram depicting the association between DEPP1 expression, T stages and overall survival status. (H) Kaplan-Meier survival analyses of DEPP1 in T1, T2, T3 and T4 stages of the GSE84437 cohort. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 correlates with a stroma-rich and immunosuppressive microenvironment of GC. (A) The stromal, immune, and estimate scores in the low- and high-expression groups of DEPP1. (B) The correlation between DEPP1 expression and stromal score. (C) The distribution of various immune and stromal cells in the GC microenvironment estimated by the EPIC algorithm. (D) The heatmap showing the correlation between DEPP1 expression and immune and stromal cell populations. The infiltration levels of distinct immune and stromal cells in the low- and high-expression groups of DEPP1, as assessed by (E) EPIC, (F) TIMER and (G) quanTIseq methods, respectively. (H) A correlation heatmap illustrating the relationships between DEPP1 expression and various immune and stromal cells based on the results of the quanTIseq algorithm. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; NK, natural killer; ns, not significant.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Potential regulatory mechanisms underlying DEPP1. (A) The volcano plot depicting the differentially expressed genes between the low- and high-expression groups of DEPP1. (B) Gene Ontology and (C) Kyoto Encyclopedia of Genes and Genome enrichment analyses of the differentially expressed genes. Gene Set Enrichment Analysis demonstrates the hallmark pathways enriched in the (D) high- and (E) low-expression groups of DEPP1. (F) The box plot of the downstream hub genes of DEPP1. (G) The correlation heatmap displays the relationships among the hub genes. DEPP1, decidual protein induced by progesterone.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: High DEPP1 expression predicts poor chemotherapy sensitivity in patients with GC. (A) The association between DEPP1 expression levels and the IC 50 of selective chemotherapy drugs as shown by the heatmap. The estimated IC 50 of chemotherapy drugs in the low- and high-expression groups of DEPP1, as analyzed using data from the (B) GDSC and (C) CTRP datasets, respectively. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; IC 50 , half-maximal inhibitory concentration; GDSC, Genomics of Drug Sensitivity in Cancer; CTRP, Cancer Therapeutics Response Portal; ns, not significant.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: Expressing, Concentration Assay

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vitro . Effects of oxaliplatin and fluorouracil supplementation on DEPP1 protein levels in (A) MKN45 and (B) HGC27 cells, respectively. Validation of DEPP1 overexpression in (C) MKN45 and (D) HGC27 cells. (E) Impact of DEPP1 overexpression on MKN45 cell proliferation, assessed using EdU flow cytometry. (F) Influence of DEPP1 overexpression on MKN45 proliferation following a 24-h treatment with 10 µM oxaliplatin. (G) Effects of ectopic DEPP1 expression on oxaliplatin-induced apoptosis (20 µM) in MKN45 cells, as measured by Annexin V/PI flow cytometry. (H) Analysis of full-length and cleaved PARP protein levels following forced DEPP1 expression in (H) MKN45 and (J) HGC27 cells. *P<0.05 and ***P<0.001 DEPP1, decidual protein induced by progesterone. ns, not significant.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: In Vitro, Biomarker Discovery, Over Expression, Flow Cytometry, Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vivo . (A) Schematic representation of the subcutaneous mouse model receiving either 5% GS or 5 mg/kg oxaliplatin intraperitoneally. (B) Images and (C) weights of control and DEPP1-overexpressing tumors treated with or without oxaliplatin. (D) Representative immunohistochemistry images of DEPP1, Ki67, and cleaved caspase 3 in subcutaneous tumor tissues. DEPP1, decidual protein induced by progesterone; GS, glucose saline.

Article Snippet: Following this, sections were incubated with primary antibody against DEPP1 (1:200; cat. no. NBP3-17581; Novus Biologicals, Ltd.), Ki67 (1:2,000; cat. no. ab15580; Abcam), or cleaved caspase 3 (1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with Goat Anti-Rabbit IgG (1:600; cat. no. 33101ES60, 1:600; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 1 h. Finally, sections were subjected to a DAB Substrate Kit (cat. no. BL732A; Biosharp Life Sciences) and counterstained with hematoxylin.

Techniques: In Vivo, Control, Immunohistochemistry, Saline

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 is aberrantly overexpressed in gastric cancer. (A) Venn diagrams illustrating the differentially expressed genes. (B) Expression levels of DEPP1 in GC and normal tissues in the TCGA-STAD cohort. Relative expression of DEPP1 in 11 pairs of GC and adjacent normal tissues measured by (C) reverse transcription-quantitative PCR and (D) western blotting. (E) Representative immunofluorescence images of DEPP1 in 11 pairs of GC and adjacent normal tissues. *P<0.05, **P<0.01 and ***P<0.001. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma; N, normal; T, tumor; Ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Prognostic value of DEPP1 is explored in the TCGA-STAD cohort. Kaplan-Meier survival analyses of DEPP1 in patients with GC on (A) overall survival and (B) disease-free survival. DEPP1 expression between patients with GC who are (C) alive or dead and (D) patients with or without disease progression or recurrence. (E) Univariate and (F) multivariate Cox regression analysis of DEPP1 and other clinicopathologic features. (G) Receiver operating characteristic curves of DEPP1 and conventional oncogenes EGFR and KRAS in predicting five-year overall survival of patients with GC. **P<0.01 and ***P<0.001. DEPP1, decidual protein induced by progesterone; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma; GC, gastric cancer; OS, overall survival; DFS, disease-free survival; HR, hazard ratio; CI, confidence interval.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Expressing, Biomarker Discovery

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Prognostic value of DEPP1 is validated in GSE84437 , GSE15459 and Renji cohorts. Kaplan-Meier survival analyses of DEPP1 on overall survival in (A) GSE84437 and (D) GSE15459 cohorts. (B) Univariate and (C) multivariate Cox regression analysis of DEPP1 and other clinicopathologic parameters in the GSE84437 cohort. (E) Univariate and (F) multivariate Cox regression analysis of DEPP1 and other clinicopathologic factors in the GSE15459 cohort. (G) Representative immunohistochemistry images of DEPP1 in gastric cancer and normal tissues. (H) Kaplan-Meier survival analysis of DEPP1 on overall survival in the Renji cohort. DEPP1, decidual protein induced by progesterone; OS, overall survival; DFS, disease-free survival; HR, hazard ratio; CI, confidence interval.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Immunohistochemistry

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Establishment and evaluation of a prognostic nomogram for GC. (A) Construction of a nomogram integrating DEPP1, age and TNM stage to predict the 1-, 3- and 5-year overall survival of patients with GC. (B) Calibration curves to assess the predictive accuracy of the nomogram for survival outcomes. (C) Results of decision curve analysis comparing the nomogram's performance with that of individual predictors, including DEPP1, age and TNM stage. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; TNM, tumor-node-metastasis; OS, overall survival; Pr, probability.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques:

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Relationship between DEPP1 expression and clinicopathologic factors in patients with GC. (A) DEPP1 expression levels across different Lauren phenotypes. (B) Kaplan-Meier survival analyses of DEPP1 in diffuse, intestinal and mixed subtypes of the GSE15459 cohort. (C) DEPP1 expression levels in stage I and advanced stages of GC. (D) Kaplan-Meier survival analyses of DEPP1 in stage I, II, III and IV of the TCGA-STAD cohort. (E) DEPP1 expression levels across distinct T stages. (F) The histogram displaying the distribution of T stages between the low- and high-expression groups of DEPP1. (G) The alluvial diagram depicting the association between DEPP1 expression, T stages and overall survival status. (H) Kaplan-Meier survival analyses of DEPP1 in T1, T2, T3 and T4 stages of the GSE84437 cohort. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 correlates with a stroma-rich and immunosuppressive microenvironment of GC. (A) The stromal, immune, and estimate scores in the low- and high-expression groups of DEPP1. (B) The correlation between DEPP1 expression and stromal score. (C) The distribution of various immune and stromal cells in the GC microenvironment estimated by the EPIC algorithm. (D) The heatmap showing the correlation between DEPP1 expression and immune and stromal cell populations. The infiltration levels of distinct immune and stromal cells in the low- and high-expression groups of DEPP1, as assessed by (E) EPIC, (F) TIMER and (G) quanTIseq methods, respectively. (H) A correlation heatmap illustrating the relationships between DEPP1 expression and various immune and stromal cells based on the results of the quanTIseq algorithm. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; NK, natural killer; ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: Potential regulatory mechanisms underlying DEPP1. (A) The volcano plot depicting the differentially expressed genes between the low- and high-expression groups of DEPP1. (B) Gene Ontology and (C) Kyoto Encyclopedia of Genes and Genome enrichment analyses of the differentially expressed genes. Gene Set Enrichment Analysis demonstrates the hallmark pathways enriched in the (D) high- and (E) low-expression groups of DEPP1. (F) The box plot of the downstream hub genes of DEPP1. (G) The correlation heatmap displays the relationships among the hub genes. DEPP1, decidual protein induced by progesterone.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: High DEPP1 expression predicts poor chemotherapy sensitivity in patients with GC. (A) The association between DEPP1 expression levels and the IC 50 of selective chemotherapy drugs as shown by the heatmap. The estimated IC 50 of chemotherapy drugs in the low- and high-expression groups of DEPP1, as analyzed using data from the (B) GDSC and (C) CTRP datasets, respectively. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. DEPP1, decidual protein induced by progesterone; GC, gastric cancer; IC 50 , half-maximal inhibitory concentration; GDSC, Genomics of Drug Sensitivity in Cancer; CTRP, Cancer Therapeutics Response Portal; ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: Expressing, Concentration Assay

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vitro . Effects of oxaliplatin and fluorouracil supplementation on DEPP1 protein levels in (A) MKN45 and (B) HGC27 cells, respectively. Validation of DEPP1 overexpression in (C) MKN45 and (D) HGC27 cells. (E) Impact of DEPP1 overexpression on MKN45 cell proliferation, assessed using EdU flow cytometry. (F) Influence of DEPP1 overexpression on MKN45 proliferation following a 24-h treatment with 10 µM oxaliplatin. (G) Effects of ectopic DEPP1 expression on oxaliplatin-induced apoptosis (20 µM) in MKN45 cells, as measured by Annexin V/PI flow cytometry. (H) Analysis of full-length and cleaved PARP protein levels following forced DEPP1 expression in (H) MKN45 and (J) HGC27 cells. *P<0.05 and ***P<0.001 DEPP1, decidual protein induced by progesterone. ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Flow Cytometry, Expressing

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vivo . (A) Schematic representation of the subcutaneous mouse model receiving either 5% GS or 5 mg/kg oxaliplatin intraperitoneally. (B) Images and (C) weights of control and DEPP1-overexpressing tumors treated with or without oxaliplatin. (D) Representative immunohistochemistry images of DEPP1, Ki67, and cleaved caspase 3 in subcutaneous tumor tissues. DEPP1, decidual protein induced by progesterone; GS, glucose saline.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: In Vivo, Control, Immunohistochemistry, Saline

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF reverses a cross-tissue stress–autophagy signature and highlights DEPP1 as a shared node. (A) Venn diagram of TFDF-reversal DEGs in bone and the hippocampus (reversal defined as Model vs. Sham significant change, directionally opposed in TFDF-H vs. Model). (B) Pathway enrichment of reversal genes highlighting FOXO signaling (database and statistics in Materials and Methods). (C) Cross-filtering for Model↑ and TFDF-H↓ genes in both tissues identifies DEPP1 as a shared, treatment-reversed transcript (expression changes shown for bone and hippocampus). (D) Complementary enrichment of the reversal sets prioritizes FOXO signaling in both tissues. (E) GSEA plots demonstrating negative enrichment of FOXO programs in the Model vs. Sham groups and a positive shift in TFDF-H vs. Model (bone and hippocampus) groups.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Expressing

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF reduces DEPP1 expression and normalizes the expression of autophagy–mitochondrial markers in the hippocampus and bone of OVX–CUMS mice. (A) Representative hippocampal immunofluorescence images of NeuN (neurons, red) and DEPP1 (green) in the Sham, Model, TFDF-L, and TFDF-H groups; nuclei are stained with DAPI (blue). (B) Quantification of the DEPP1/NeuN double-positive area in the hippocampus. (C) Representative DEPP1 immunohistochemistry in trabecular bone. (D) Quantification of the DEPP1-positive area in bone. (E and F) Representative Western blots for SIRT1, FOXO3, and DEPP1 in the hippocampus (E) and bone (F). (G and H) Densitometric analysis of SIRT1, FOXO3, and DEPP1 expression in the hippocampus (G) and bone (H) normalized to that of β-actin. (I) Representative hippocampal immunofluorescence for NeuN (red) and LC3B (green). (J) Quantification of the hippocampal LC3B/NeuN double-positive area. (K) Representative LC3 immunohistochemistry in trabecular bone. (L) Quantification of the LC3-positive area in bone. (M and N) Western blots for p62, LC3B, and TOM20 in the hippocampus (M) and bone (N). (O and P) Densitometric analysis of p62, LC3B, and TOM20 expression in the hippocampus (O) and bone (P), normalized to that of β-actin.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Expressing, Immunofluorescence, Staining, Immunohistochemistry, Western Blot

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF normalizes the SIRT1–FOXO3–DEPP1 axis, rebalances excessive autophagy, and rescues mitochondrial and osteogenic functions in MC3T3-E1 cells. MC3T3-E1 cells were assigned to Control, Model (H 2 O 2 injury), TFDF, or NAC (positive antioxidant control) groups. Immunoblotting revealed that SIRT1 down-regulation, FOXO3 hyperacetylation, and DEPP1 up-regulation after H 2 O 2 were reversed by TFDF (A), as determined by densitometry (B). TMRE microscopy and quantification revealed ΔΨm loss in the Model group and restoration by TFDF (C and D). Autophagy/mitochondrial markers indicated over-autophagy under injury that were recalibrated toward baseline by TFDF (E and F). TEM revealed autophagosome accumulation and swollen mitochondria in the Model group, which were mitigated by TFDF (G). Multicolor IF (LC3B/TOM20/DEPP1) revealed increased LC3B puncta, TOM20 fragmentation, and DEPP1 elevation in the Model group, all of which improved with TFDF (H); colocalization analyses confirmed normalization of LC3B–TOM20 (mitophagy coupling) and a reduction in TOM20–DEPP1 coupling by TFDF (I and J). Osteogenic function assays demonstrated TFDF-mediated recovery of ALP activity and ARS mineral deposition (K), accompanied by increased RUNX2 expression and OCN/ALP expression, as determined by Western blotting (L and M). Data are presented as the mean ± SEM, with n indicated on the plots; the statistical tests and multiple-comparison procedures are described in Materials and Methods. Abbreviations: TFDF, total flavonoids of Drynaria fortunei ; NAC, N-acetyl-L-cysteine; TMRE, tetramethylrhodamine ethyl ester; LC3, microtubule-associated protein 1 light chain 3; OCN, osteocalcin.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Control, Western Blot, Microscopy, Activity Assay, Expressing, Comparison

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF mitigates oxidative injury in HT22 cells by restoring SIRT1–FOXO3–DEPP1 signaling and autophagy–mitochondrial homeostasis, thereby improving neuroplasticity. Cells were assigned to Control, Model (H 2 O 2 ), TFDF, or NAC (antioxidant control) groups. Western blots showing SIRT1–FOXO3–DEPP1↑ after injury and reversal by TFDF (A and B). TMRE imaging revealed ΔΨm loss in the Model group and rescue by TFDF (C and D). Autophagy/mitochondrial markers were recalibrated toward baseline by TFDF (E and F), which is consistent with TEM showing fewer autophagosomes and preserved cristae (G). IF (DAPI/LC3B/TOM20/DEPP1) demonstrated reduced LC3B puncta, increased TOM20 integrity, and decreased DEPP1 with TFDF (H), which was supported by colocalization readouts (DEPP1–TOM20 and LC3B–TOM20) (I and J). Quantification of DEPP1–TOM20 colocalization, and the mitophagy index is shown in (K). TFDF further increased the expression of BDNF and p-CREB/CREB and restored the expression of synapsin I and PSD-95 (L and M). Data are presented as the mean ± SEM; statistics and replicate numbers are provided in Materials and Methods. Abbreviations: TFDF, total flavonoids of Drynaria fortunei ; NAC, N-acetyl-L-cysteine; TMRE, tetramethylrhodamine ethyl ester.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Control, Western Blot, Imaging, Expressing

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: DEPP1 bidirectionally alters autophagy–mitochondrial coupling under oxidative injury in HT22 and MC3T3-E1 cells. DEPP1 expression was reduced by siRNA (KD) or increased by plasmid (OE); cells were exposed to H 2 O 2 to model injury. Protein and mRNA assays confirmed effective KD/OE in both lines (A and B). TMRE imaging revealed ΔΨm loss in the Model group, partial recovery in the KD+Model group, and a further decrease in the OE+Model group (C and D). TEM revealed swollen mitochondria and autophagosomes in the Model group, fewer autophagosomes after KD, and abundant autophagosomes after OE (E). Western blots demonstrated model-associated LC3-II accumulation, p62 depletion, and TOM20 reduction; KD shifted these toward control, whereas OE intensified them (F) with densitometry in (G). LC3B/TOM20 immunofluorescence revealed parallel changes in puncta burden and mitochondrial network integrity, as shown by the statistical data in (J) (H to J). Group labels: Control, Model, KD+Model, and OE+Model. Data are presented as the mean ± SEM; replicate numbers and statistics are provided in Materials and Methods.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Expressing, Plasmid Preparation, Imaging, Western Blot, Control, Immunofluorescence

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: DEPP1 knockdown with TFDF maintains and often augments pathway activity and functional rescue in injured osteoblasts and neurons. (A) Schematic of the gene–drug design and hypothesized placement of DEPP1 downstream of TFDF-responsive signaling. (B) Autophagy/mitochondrial Western blots (LC3-I/II, p62, and TOM20) in MC3T3-E1 and HT22 cells under Control, Model, siDEPP1+Model, TFDF+Model, and siDEPP1+TFDF+Model conditions. (C) Densitometry for panel (B). (D) Representative ROS fluorescence micrographs. (E) Quantification of intracellular ROS levels. (F) Osteogenic function of MC3T3-E1 cells: ALP staining (day 7) and ARS mineralization (days 14 to 21). (G) Osteogenic markers (RUNX2, OCN, and ALP) were measured by Western blotting. (H) Corresponding quantification. (I) Neuronal plasticity markers in HT22 cells (BDNF, p-CREB/CREB, Synapsin I, and PSD-95) were measured by Western blotting. (J) Quantification. In both cell types, siDEPP1 and TFDF each improved the injury phenotype, and siDEPP1+TFDF achieved comparable or greater improvement without occluding the effects of TFDF. Statistical tests and n values are provided in Materials and Methods and on the plots.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Knockdown, Activity Assay, Functional Assay, Western Blot, Control, Fluorescence, Staining

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: Docking and functional perturbation support SIRT1 as a key TFDF-responsive effector. (A) Docked pose of naringenin in the SIRT1 activator pocket with key hydrogen-bond and hydrophobic contacts indicated. (B) Radius of gyration, (C) number of protein–ligand hydrogen bonds, (D) backbone RMSF, (E) solvent-accessible surface area, and (F) ligand RMSD, all indicating rapid stabilization after ~10 ns and maintenance of a compact, well-behaved complex. (G) Free-energy landscape of the SIRT1–naringenin trajectory plotted along PC1 and PC2 (Δ G = − k B T ln P ), showing a dominant low-energy basin corresponding to the bound state. (H) SPR analysis of SIRT1–naringenin binding, showing concentration-dependent sensorgrams and a 1:1 Langmuir fit consistent with specific interaction. (I) Western blots of FOXO3 and Depp1 (HT22 and MC3T3-E1) after Sirt1 knockdown (KD) or overexpression (OE). (J) TMRE staining (ΔΨm) and (K) corresponding quantification: ΔΨm decreases with KD and increases with OE. (L) Triplex IF (LC3, TOM20, and DEPP1) showing the autophagy burden, mitochondrial network integrity, and DEPP1 levels across KD/OE conditions. (M) Quantifications of puncta burden, TOM20 continuity, and DEPP1 intensity. Docking to DEPP1 with neoeriocitrin, naringin, and naringenin produced low-affinity, nonconvergent poses (not shown); docking and molecular dynamics results for naringin, naringenin, and the positive control are provided in the Supplementary Materials. Abbreviations: R g , radius of gyration; RMSF, root mean square fluctuation; SASA, solvent-accessible surface area; RMSD, root mean square deviation.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Functional Assay, Solvent, Binding Assay, Concentration Assay, Western Blot, Knockdown, Over Expression, Staining, Produced, Positive Control

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: SIRT1 determines cellular responsiveness to TFDF in neurons and osteoblasts. Under H 2 O 2 injury, cells were treated with TFDF alone or in combination with Sirt1 knockdown (KD) or overexpression (OE). Western blots showed that TFDF decreased acetyl-FOXO3 and DEPP1 levels and that these effects were abrogated by KD and strengthened by OE in HT22 and MC3T3-E1 cells (A, densitometry in B). ROS imaging/quantification demonstrated that injury-induced oxidative stress was reduced by TFDF, partially reversed by KD, and further reduced by OE (C and D). Immunofluorescence staining for LC3B/TOM20/DEPP1 revealed a TFDF-driven improvement in autophagy–mitochondrial morphology that was attenuated by KD and potentiated by OE in both cell types (E and F). Autophagy immunoblots (LC3-II and p62) confirmed the same interaction pattern (G, densitometry in H).

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Knockdown, Over Expression, Western Blot, Imaging, Immunofluorescence, Staining

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF alleviates OVX–CUMS-associated bone–brain comorbidity by engaging SIRT1-centered stress–autophagy signaling. Left: Experimental framework: 7-week-old female mice underwent ovariectomy combined with chronic unpredictable mild stress (OVX+CUMS, 12 weeks) and received total flavonoids of Drynaria fortunei (TFDF), resulting in improved bone density and depression-like behaviors. Right: Working model: OVX+CUMS-related cellular stress elevates reactive oxygen species (ROS) and disrupts autophagy–mitochondrial homeostasis. TFDF activates SIRT1, reduces FOXO3 acetylation, suppresses stress-responsive DEPP1, and restores antioxidant capacity (e.g., catalase [CAT]), thereby lowering ROS (with N-acetyl-L-cysteine [NAC], shown as an antioxidant control) and rebalancing autophagy to support organelle quality control. These coordinated effects ultimately improve cellular function and the observed functional phenotype in bone- and brain-relevant cells.

Article Snippet: Sections were blocked with 5% BSA (or normal serum, as appropriate) for 30 min, then incubated overnight at 4 °C with primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU) and LC3 (Immunoway, PT0235R) at manufacturer-recommended dilutions.

Techniques: Control, Cell Function Assay, Functional Assay