Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: EGF stimulation enhances the expression of EGFR, EPS8, SCAMP3, and AR-V7 in androgen-sensitive and enzalutamide-resistant prostate cancer cells. The effects of epidermal growth factor (EGF) on the expression of EGFR, EPS8, SCAMP3, and AR-V7 were examined in LNCap and LNCap-Enz cells. (A) Representative western blot analysis of LNCap cells treated with or without EGF (100 ng/ml) for 24 h, showing the protein expression levels of EGFR, EPS8, SCAMP3, AR-V7, and β-actin. (B) Densitometric quantification of EGFR, EPS8, SCAMP3, and AR-V7 protein expression in LNCap cells, normalized to β-actin and expressed as a percentage of control. EGF treatment significantly increased the expression of all four proteins (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Cell viability assay showing that EGF treatment modestly increased LNCap cell viability compared to control conditions. (D) Representative western blot analysis of LNCap-Enz cells treated with or without EGF (100 ng/ml), demonstrating up-regulation of EGFR, EPS8, SCAMP3, and AR-V7 protein levels. (E) Quantitative analysis of protein expression in LNCap-Enz cells, showing significant increases in EGFR, EPS8, SCAMP3, and AR-V7 levels following EGF stimulation (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (F) Cell viability assay revealing a slight increase in LNCap-Enz cell viability after EGF treatment. Data are presented as mean±standard deviation (SD) from three independent experiments.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Expressing, Western Blot, Control, Viability Assay, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: EGF stimulation enhances the interaction between EGFR, EPS8, and SCAMP3 in androgen-sensitive and enzalutamide-resistant prostate cancer cells. The physical interactions between EGFR, EPS8, SCAMP3, and AR-V7 were assessed by co-immunoprecipitation (co-IP) assays in LNCap and LNCap-Enz cells with or without EGF stimulation (100 ng/ml, 24 h). (A) Representative western blot following immunoprecipitation with anti-EGFR antibody in LNCap cells, showing enhanced association of EPS8, SCAMP3, and AR-V7 with EGFR after EGF treatment. (B) Densitometric quantification of EPS8, SCAMP3, and AR-V7 protein levels from panel A, normalized to control and presented as percentage [mean±standard deviation (SD), *p<0.05, one-way ANOVA followed by Tukey’s post hoc test]. (C) Representative western blot following immunoprecipitation with anti-SCAMP3 antibody in LNCap cells, demonstrating increased binding of EPS8, EGFR, and AR-V7 with SCAMP3 after EGF stimulation. (D) Quantitative analysis of protein expression from panel C, showing significant up-regulation of EPS8 and EGFR but down-regulation of AR-V7 in EGF-treated samples (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (E) Co-IP results using anti-EGFR antibody in LNCap-Enz cells, revealing enhanced interaction of EGFR with EPS8, SCAMP3, and AR-V7 following EGF treatment. (F) Densitometric analysis of protein expression from panel E, indicating significant increases in all three proteins (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (G) Western blot analysis following immunoprecipitation with anti-SCAMP3 antibody in LNCap-Enz cells, showing enhanced association of EPS8 and EGFR but decreased AR-V7 binding with SCAMP3 after EGF stimulation. (H) Quantitative assessment of protein levels from panel G, demonstrating significant up-regulation of EPS8 and EGFR but down-regulation of AR-V7 in the presence of EGF (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). IB: Immunoblotting; IP: immunoprecipitation.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Control, Standard Deviation, Binding Assay, Expressing

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: SCAMP3 knockdown reduces EGFR and EPS8 signaling in prostate cancer cells. The effect of SCAMP3 silencing on EGFR/EPS8 signaling was investigated in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells treated with EGF (100 ng/ml) under three conditions: without shRNA transfection, with shRNA-control, or with shRNA-SCAMP3. Blots show protein expression of EGFR, SCAMP3, EPS8, STAT3, AKT, ERK, and β-actin as loading control. (B) Densitometric analysis of western blot results from LNCap cells, showing relative expression levels of each protein compared to control. Data are presented as percentage of control mean±standard deviation (SD). Asterisks indicate significant differences compared to control (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative Western blot analysis of LNCap-Enz cells with the same treatment conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells, demonstrating the effect of SCAMP3 silencing on EGFR/EPS8 signaling components.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Knockdown, Western Blot, shRNA, Transfection, Control, Expressing, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: SCAMP3 overexpression enhances EGFR and EPS8 signaling pathways in prostate cancer cells. The effect of SCAMP3 overexpression on EGFR/EPS8 signaling was examined in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells under three conditions: treatment with EGF (100 ng/ml) alone, transfection with pcDNA-control vector plus EGF, or transfection with pcDNA-SCAMP3 plus EGF. Immunoblotting results show protein expression levels of EGFR, SCAMP3, EPS8, STAT3, AKT, ERK, and β-actin as loading control. (B) Densitometric analysis of protein expression in LNCap cells normalized to β-actin and expressed as a percentage of control. Bar graphs represent mean±standard deviation (SD) from three independent experiments. Asterisks indicate statistically significant differences compared to control conditions (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative western blot analysis of LNCap-Enz cells under the same three experimental conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells, showing relative expression levels of each protein as a percentage of control.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Over Expression, Protein-Protein interactions, Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: EPS8 regulates the EGFR and SCAMP3 signaling pathway in prostate cancer cells. The effect of EPS8 overexpression on EGFR and SCAMP3 signaling was investigated in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells treated with EGF (100 ng/ml) alone, or transfected with pcDNA-control plus EGF, or transfected with pcDNA-EPS8 plus EGF. Protein expression levels of EGFR, SCAMP3, EPS8, and downstream signaling molecules STAT3, AKT, and ERK were detected. β-actin served as a loading control. (B) Densitometric analysis of protein expression in LNCap cells from three independent experiments. Data are presented as percentage of control [mean±standard deviation (SD)]. Asterisks indicate statistically significant differences compared to control (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative western blot analysis of LNCap-Enz cells under the same experimental conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells showing relative protein levels normalized to β-actin.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Over Expression, Western Blot, Transfection, Control, Expressing, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: EPS8 knockdown down-regulates EGFR and SCAMP3 signaling pathways in prostate cancer cells. The effect of EPS8 silencing on EGFR/SCAMP3 signaling was investigated in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells treated with EGF (100 ng/ml) alone, or with EGF plus shRNA-control, or with EGF plus shRNA-EPS8. The blots show protein expression of EGFR, SCAMP3, EPS8, STAT3, AKT, ERK, and β-actin as loading control. (B) Densitometric analysis of protein expression in LNCap cells normalized to β-actin and expressed as percentage of control. Data are presented as mean±standard deviation (SD) from three independent experiments. Asterisks indicate statistically significant differences compared to control (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative western blot analysis of LNCap-Enz cells under the same experimental conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells showing relative expression levels of each protein.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Knockdown, Protein-Protein interactions, Western Blot, shRNA, Control, Expressing, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: EPS8 and SCAMP3 enhance the migratory ability of LNCaP and LNCaP-Enz cells. (A, C) Representative phase-contrast images from wound healing assays in LNCaP (A) and LNCaP-Enz (C) cells transfected with pcDNA-control, pcDNA-EPS8, or pcDNA-SCAMP3. Images were acquired at 0 and 12 hours after ibidi insert removal. (B, D) Quantification of migration area (%) in LNCaP (B) and LNCaP-Enz (D) cells at 12 hours. Data represent the mean±standard deviation (SD) from three independent experiments. *p<0.05 vs. control group using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Transfection, Control, Migration, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SCAMP3 and EPS8 Cooperatively Regulate EGFR Signaling to Promote Enzalutamide Resistance and Metastatic Potential in Prostate Cancer

doi: 10.21873/cgp.20549

Figure Lengend Snippet: Schematic model illustrating the cooperative regulation of EGFR signaling by SCAMP3 and EPS8 in enzalutamide-resistant prostate cancer cells. Upon EGF stimulation, EGFR forms a complex with EPS8 and SCAMP3, promoting downstream activation of STAT3, AKT, and ERK signaling pathways. This effect is more prominent in enzalutamide-resistant LNCap (LNCap–Enz-R) cells compared to parental LNCap cells. Genetic inhibition using shRNA targeting SCAMP3 or EPS8 suppresses this pathway, whereas overexpression via pcDNA transfection enhances EGFR signaling activity. These findings suggest that SCAMP3 and EPS8 act synergistically to modulate EGFR-mediated resistance signaling in advanced prostate cancer.

Article Snippet: To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used.

Techniques: Activation Assay, Protein-Protein interactions, Inhibition, shRNA, Over Expression, Transfection, Activity Assay

Journal: Biomedical Journal

Article Title: IRE1α-mediated endoplasmic reticulum stress response regulates oxidative damage in CYP4V2 deficient human retinal pigment epithelial cells

doi: 10.1016/j.bj.2025.100875

Figure Lengend Snippet: CYP4V2 -knockdown induced unfold protein response (UPR) and ER stress in ARPE-19 cells. ( A ) The differentially expressed proteins (DEPs) identified through proteomics analysis were related to ER stress, and their connections were demonstrated using the STRING database, ( B ) The expression of mRNA associated with UPR response including HSPA5 , PDIA4 , PDIA6 , and ERO1A (n = 4), ( C ) Western blot analysis of CYP4V2 protein expression, ( D ) The expression of protein markers of ER stress including GRP78/BiP, ERO1-Lα, PDI, PERK, and IRE1α (n = 4), ( E ) Immunofluorescence imaging of transcription factor XBP1s in IRE1α pathway (n = 6), ( F ) The effect of n-3 PUFA alpha linolenic acid (αLA) on GRP/78/BiP protein expression (n = 4). (CB: Coomassie blue staining, veh.: vehicle, scale bar: 20 μm, significance: ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001).

Article Snippet: Scramble control (SR30004) and the siRNA mixture A specific for human CYP4V2 (SR326149) were purchased from OriGene (Rockville, MD, USA). siRNA mixture B specific for human CYP4V2 (ON-TARGETplus siRNA) was purchased from Horizon (Cambridge, UK).

Techniques: Knockdown, Expressing, Western Blot, Immunofluorescence, Imaging, Staining

Journal: Biomedical Journal

Article Title: IRE1α-mediated endoplasmic reticulum stress response regulates oxidative damage in CYP4V2 deficient human retinal pigment epithelial cells

doi: 10.1016/j.bj.2025.100875

Figure Lengend Snippet: IRE1α signaling pathway regulated ERO1-Lα, which was involved in ROS production and mitochondrial function in CYP4V2-deficient RPE. ( A ) EN460, an ERO1-Lα inhibitor, regulated ERO1A mRNA (n = 4), ( B ) H2DCFDA staining of intracellular ROS (n = 6, EN460 15 μM, 48 h), ( C ) The effect of inhibitors for major ER stress signaling pathways on the expression of ERO1-Lα protein expression, (n = 4, 72h of treatment with 4μ8c 20 μM, GSK: GSK2656157 1 μM, Ceap: Ceapin-A7 10 μM) and ( D ) the ER-specific antioxidant genes, PDRX4, GPX7, GPX8 mRNA expression (n = 4), ( E ) Mitochondrial protein NDUFS1, ATP5A1, and SOD2 expression (n = 4), ( F ) NADH-cytochrome c reductase (NCCR) activity (n = 4), ( G ) TMRE staining mitochondrial membrane potential (n = 6, F: 4μ8c 20 μM, K: Kira8 100 nM 48h), ( H ) ATP concentration 72h after treatment of inhibitors (n = 5, F: 4μ8c 20 μM, K: Kira8 100 nM, T: TUDCA 150 μM), (I) IRE1α inhibition (Kira8 100 nM, 72h) and NDUFS1 protein expression (n = 4). (CB: Coomassie blue staining, veh.: vehicle, scale bar: 20 μm, significance: ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001).

Article Snippet: Scramble control (SR30004) and the siRNA mixture A specific for human CYP4V2 (SR326149) were purchased from OriGene (Rockville, MD, USA). siRNA mixture B specific for human CYP4V2 (ON-TARGETplus siRNA) was purchased from Horizon (Cambridge, UK).

Techniques: Staining, Protein-Protein interactions, Expressing, Activity Assay, Membrane, Concentration Assay, Inhibition