Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: CuCl 2 Enhances TMZ Cytotoxicity on Glioblastoma Cells and Inhibits Proliferation and Invasion. ( A ) Flow cytometry analysis of apoptosis in U251 (upper row) and T98G (lower row) cells treated with different concentrations of CuCl 2 (0–6 μM). ( B ) Quantification of PI-positive cells in U251 and T98G cells after CuCl 2 (0–6 μM) treatment, showing that CuCl 2 alone has low toxicity ( n = 3). ( C ) Dose–response curves of U251 and T98G cell viability after treatment with different concentrations of TMZ (0–200 μM) ( n = 3). ( D ) Cell viability of U251 and T98G cells treated with TMZ (25 μM) alone or in combination with different concentrations of CuCl 2 (0–6 μM) ( n = 3). ( E ) Immunofluorescence staining of U251 cells after different treatments. Green represents DLAT, red represents the mitochondrial marker TOM20, and yellow indicates colocalization of both (63×). ( F , G ) Representative images and quantitative analysis of clonogenic assays for U251 and T98G cells under different treatments ( n = 3). ( H , I ) Representative images and quantification of Transwell invasion assays showing that the invasion ability significantly decreased in the combined treatment group ( n = 3) (20×). ( J ) CS1 fluorescence probe assay to detect Cu + levels in U251 and T98G cells, showing significant Cu + accumulation in the combined treatment group (100×). ( K , M ) Western blot and grayscale analysis showing the expression of copper transport and cuproptosis-related proteins (ATP7A, FDX1, Lipoylated-DLAT, SLC31A1, ATP7B) in T98G cells after treatment with TMZ (0, 25 μM) and CuCl 2 (0–6 μM) ( n = 3). ( L , N ) Western blot and grayscale analysis showing the expression of the same proteins in U251 cells treated with different concentrations of TMZ (0, 12.5, 25 μM) ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Marker, Fluorescence, Western Blot, Expressing

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: TMZ+CuCl 2 Combined Treatment Inhibits Tumor Growth in Mice and Alters Copper Homeostasis and EMT-Related Protein Expression. ( A ) Representative images of subcutaneous xenograft tumors in nude mice after different treatments. The tumor volume in the control group is larger, while the combined treatment group shows significantly smaller tumors. ( B ) Dynamic changes in tumor volume for each group, showing a significant inhibition of tumor growth in the combined treatment group ( n = 5). ( C ) Statistical analysis of tumor weight at the end of the experiment, with the combined treatment group showing significantly lower tumor weight compared to the control group ( n = 5). ( D ) Representative images of immunohistochemical staining of xenograft tumors showing the expression levels of proliferation-related protein PCNA and copper death-related proteins FDX1 and ATP7A. In the combined treatment group, the proportion of positive cells for PCNA, FDX1, and ATP7A is reduced (20×). ( E ) Western blot analysis of copper transport and copper deposition-related proteins (ATP7A, FDX1, Lipoylated-DLAT, SLC31A1, ATP7B) and proliferation/emt-related proteins (PCNA, vimentin, E-cadherin) in the control group, only TMZ group, only CuCl 2 group and combination treatment group. ( F ) Grayscale analysis of protein expression showing significant downregulation of ATP7A, FDX1, PCNA, and Vimentin, and upregulation of E-cadherin in the combined treatment group ( n = 3) (** p < 0.01, **** p < 0.0001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Expressing, Control, Inhibition, Immunohistochemical staining, Staining, Western Blot

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: TRIM14 Overexpression Reduces Cu + Accumulation and Weakens TMZ+CuCl 2 Antitumor Effects. ( A , B ) Western blot validation of TRIM14 overexpression efficiency ( n = 3). ( C , D ) Clonogenic assay: TRIM14 overexpression partially restores the inhibition of cell proliferation by TMZ+CuCl 2 ( n = 3). ( E , F ) Transwell invasion assay: TRIM14 overexpression significantly enhances cell invasion ability and weakens the inhibitory effect of TMZ+CuCl 2 ( n = 3) (10×). ( G ) CS1 fluorescence probe detection of Cu + levels, showing significant Cu + accumulation in the combined treatment group, while fluorescence signals are significantly reduced in the TRIM14 overexpression group (100×). ( H , I ) Western blot analysis of copper homeostasis and cuproptosis-related proteins, showing that TRIM14 overexpression reverses the downregulation of ATP7A, FDX1, and Lipoylated-DLAT, and the upregulation of SLC31A1, while restoring ATP7B expression ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Over Expression, Western Blot, Biomarker Discovery, Clonogenic Assay, Inhibition, Transwell Invasion Assay, Fluorescence, Expressing

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: TRIM14 knockdown promotes TMZ+CuCl 2 -induced copper ion accumulation and cuproptosis. ( A , B ) Western blot validation of TRIM14 knockdown efficiency, showing a significant reduction in TRIM14 expression in the sh-TRIM14 group ( n = 3). ( C – F ) Western blot analysis of copper homeostasis- and cuproptosis-related proteins. TRIM14 knockdown resulted in downregulation of ATP7A, while ATP7B and SLC31A1 showed no significant changes. Under TMZ+CuCl 2 treatment, FDX1 expression was decreased, whereas lipoylated-DLAT and lipoylated-DLST were upregulated, indicating enhanced mitochondrial lipoylated protein aggregation and amplified cuproptosis ( n = 3). ( G ) CS1 fluorescence probe detection of intracellular Cu + levels, showing that TMZ+CuCl 2 treatment induced Cu + accumulation, which was further elevated in the TRIM14 knockdown group (100×) (** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Knockdown, Western Blot, Biomarker Discovery, Expressing, Amplification, Fluorescence

Journal: Biomedicines

Article Title: TRIM14 Regulation of Copper Homeostasis and Cuproptosis: A New Strategy to Overcome Chemoresistance in Glioblastoma

doi: 10.3390/biomedicines13123085

Figure Lengend Snippet: Low Expression of ATP7B in Glioma Patients. ( A ) ATP7B expression in GBM samples. ( B ) ATP7B expression distribution based on patient race. ( C ) ATP7B expression distribution based on patient gender. ( D ) ATP7A expression in GBM based on patient age. ( E ) ATP7A expression in GBM based on patient TP53 mutation status. ( F – H ) Kaplan-Meier (KM) curves showing prognosis of patients with high or low ATP7B expression in the TCGA-GBM, CGGA, and TCGA-LGG databases (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) were blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies were applied according to the manufacturer’s instructions and incubated at 4 °C for 12 h. Primary antibodies included ATP7A (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), ATP7B (ProteinTech Group, Chicago, IL, USA, 1:1000), TRIM14 (ProteinTech Group, Chicago, IL, USA, 1:500), β-actin (ProteinTech Group, Chicago, IL, USA, 1:20,000), FDX1 (ProteinTech Group, Chicago, IL, USA, 1:1000), PCNA (ProteinTech Group, Chicago, IL, USA, 1:5000), Vimentin (ProteinTech Group, Chicago, IL, USA, 1:20,000), E-cadherin (ProteinTech Group, Chicago, IL, USA, 1:20,000), p100 (ProteinTech Group, Chicago, IL, USA, 1:5000), p52 (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), SLC31A1 (ProteinTech Group, Chicago, IL, USA, 1:5000), Lip-DLAT (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Lip-DLST (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), and others.

Techniques: Expressing, Mutagenesis