Journal: Cancer Management and Research

Article Title:

siPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway

doi: 10.2147/cmar.s193805

Figure Lengend Snippet: Figure 1 The mRNA expression profile of PRDX2 knockdown in HCT116 cells and DNM3 is a downstream protein of PRDX2. (A–C) PRDX2 siRNA was conducted and introduced into HCT116 cells. The mRNA expression profile was determined by RNA-Sequencing. The volcano plot (A) and heatmap (B) of different RNA expression profile, and the number of differently expressed genes (C). (D) The protein expression of PRDX2 and DNM3 was detected by Western blot in HT29, SW480, HCT116 and SW1116 cells. (E) The effects of PRDX2 knockdown or PRDX2 overexpression on DNM3 expression were tested by Western blot. The intensities of protein bands were quantified by Image J software. GAPDH was used as an internal control. *P<0.05. All experiments were performed in triplicate. Abbreviations: DNM, Dynamin; DEGs, differently expressed genes; PRDX2-KD, cells transfected with siRNA-PRDX2 (PRDX2 knockdown); PRDX2-OV, cells transfected with pcDNA3.1-PRDX2 (PRDX2 overexpression); NC, negative control.

Article Snippet: The primary antibodies against PRDX2 (Cat# 60202-1-Ig, 1:1,000), DNM3 (Cat# 14737-1-AP, 1:1,000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bcl2 (Cat# 12789-1-AP, 1:1,000), Bax (Cat# 60267-1-Ig, 1:1,000), Caspase-3-P17 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5,000), p-AKT (Cat# 66444-1-Ig, 1:2,000), p70S6K (Cat# 66638-1-Ig, 1:3,000) and Cyclin D1 (Cat# 60186-1-Ig, 1:10,000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Expressing, Knockdown, RNA Sequencing, RNA Expression, Western Blot, Over Expression, Software, Control, Transfection, Negative Control

Journal: Cancer Management and Research

Article Title:

siPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway

doi: 10.2147/cmar.s193805

Figure Lengend Snippet: Figure 2 Dynamin 3 (DNM3) was down-regulated in colon cancer. (A) The expression of DNM3 in colon cancer and paired para-carcinoma tissues was investigated by Western blot. (B) Quantitative analysis of Western blot results. (C) Immunohistochemistry analysis was performed to detect the expression of DNM3 protein. The intensities of protein bands were quantified by Image J software. GAPDH was used as an internal control. *P<0.05.

Article Snippet: The primary antibodies against PRDX2 (Cat# 60202-1-Ig, 1:1,000), DNM3 (Cat# 14737-1-AP, 1:1,000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bcl2 (Cat# 12789-1-AP, 1:1,000), Bax (Cat# 60267-1-Ig, 1:1,000), Caspase-3-P17 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5,000), p-AKT (Cat# 66444-1-Ig, 1:2,000), p70S6K (Cat# 66638-1-Ig, 1:3,000) and Cyclin D1 (Cat# 60186-1-Ig, 1:10,000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Immunohistochemistry, Software, Control

Journal: Cancer Management and Research

Article Title:

siPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway

doi: 10.2147/cmar.s193805

Figure Lengend Snippet: Figure 3 Dynamin 3 (DNM3) overexpression decreases cell viability in colon cancer cells. (A) DNM3 cDNA was cloned into pcDNA3.1 plasmid and introduced into HCT116 and HT29 cells, and the empty pcDNA3.1 plasmid was used as negative control (NC). The overexpression of DNM3 was validated by Western blot. The intensities of protein bands were quantified by Image J software. GAPDH was used as an internal control. (B) The effects of DNM3 overexpression on cell proliferation of HCT116 and HT29 cells were investigated by CKK-8 assay. (C) The cologenic ability was tested in DNM3 overexpressing HCT116 and HT29 cells. *P<0.05. All experiments were performed in triplicate.

Article Snippet: The primary antibodies against PRDX2 (Cat# 60202-1-Ig, 1:1,000), DNM3 (Cat# 14737-1-AP, 1:1,000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bcl2 (Cat# 12789-1-AP, 1:1,000), Bax (Cat# 60267-1-Ig, 1:1,000), Caspase-3-P17 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5,000), p-AKT (Cat# 66444-1-Ig, 1:2,000), p70S6K (Cat# 66638-1-Ig, 1:3,000) and Cyclin D1 (Cat# 60186-1-Ig, 1:10,000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Over Expression, Clone Assay, Plasmid Preparation, Negative Control, Western Blot, Software, Control

Journal: Cancer Management and Research

Article Title:

siPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway

doi: 10.2147/cmar.s193805

Figure Lengend Snippet: Figure 4 Dynamin 3 (DNM3) overexpression inhibits the migration and invasion of colon cancer cells. Cell invasion (A) and migration (B) were investigated by transwell assays in DNM3 overexpressing HCT116 and HT29 cells. (C) The proteolytic activities of secreted MMP9 were investigated by gelatin zymography assay. (D) Quantitative analysis of gelatin zymography assay results. *P<0.05. All experiments were performed in triplicate. Abbreviation: NC, negative control.

Article Snippet: The primary antibodies against PRDX2 (Cat# 60202-1-Ig, 1:1,000), DNM3 (Cat# 14737-1-AP, 1:1,000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bcl2 (Cat# 12789-1-AP, 1:1,000), Bax (Cat# 60267-1-Ig, 1:1,000), Caspase-3-P17 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5,000), p-AKT (Cat# 66444-1-Ig, 1:2,000), p70S6K (Cat# 66638-1-Ig, 1:3,000) and Cyclin D1 (Cat# 60186-1-Ig, 1:10,000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Over Expression, Migration, Zymography Assay, Negative Control

Journal: Cancer Management and Research

Article Title:

siPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway

doi: 10.2147/cmar.s193805

Figure Lengend Snippet: Figure 5 Dynamin 3 (DNM3) overexpression induces apoptosis in colon cancer cells. (A) Cell apoptosis was investigated by Annexin V/PI staining and flow cytometry. (B) and (C) Photomicrographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue) in HCT116 (B) and HT29 (C) cells. PI-positive cells were counted in approximately 200 cells from three random microscopic fields for each sample and are expressed as percentages of the total cells. *P<0.05. All experiments were performed in triplicate.

Article Snippet: The primary antibodies against PRDX2 (Cat# 60202-1-Ig, 1:1,000), DNM3 (Cat# 14737-1-AP, 1:1,000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bcl2 (Cat# 12789-1-AP, 1:1,000), Bax (Cat# 60267-1-Ig, 1:1,000), Caspase-3-P17 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5,000), p-AKT (Cat# 66444-1-Ig, 1:2,000), p70S6K (Cat# 66638-1-Ig, 1:3,000) and Cyclin D1 (Cat# 60186-1-Ig, 1:10,000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Over Expression, Staining, Cytometry

Journal: Cancer Management and Research

Article Title:

siPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway

doi: 10.2147/cmar.s193805

Figure Lengend Snippet: Figure 6 Dynamin 3 (DNM3) overexpression leads to activation of mitochondria apoptosis pathway and down-regulation of AKT signaling pathway. (A) The expression of mitochondria apoptosis pathway members Bcl2, Bax and Caspase-3-P17 was investigated by Western blot. (B) The expression of AKT signaling pathway members AKT, p-AKT, p70S6K and Cyclin D1 was investigated by Western blot. The intensities of protein bands were quantified by Image J software. GAPDH was used as an internal control. *P<0.05. All experiments were performed in triplicate. Supplementary data. Abbreviation: NC, negative control.

Article Snippet: The primary antibodies against PRDX2 (Cat# 60202-1-Ig, 1:1,000), DNM3 (Cat# 14737-1-AP, 1:1,000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bcl2 (Cat# 12789-1-AP, 1:1,000), Bax (Cat# 60267-1-Ig, 1:1,000), Caspase-3-P17 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5,000), p-AKT (Cat# 66444-1-Ig, 1:2,000), p70S6K (Cat# 66638-1-Ig, 1:3,000) and Cyclin D1 (Cat# 60186-1-Ig, 1:10,000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Over Expression, Activation Assay, Expressing, Western Blot, Software, Control, Negative Control

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 is downregulated after myocardial injury A, RNA transcriptome sequencing( GSE114695 ) was used to reveal the expression profile of USPs (ubiquitin-specific proteases) in myocardial infarction (MI)-induced myocardial injury. RNA transcriptome sequencing was performed on control (n = 3) and 1 d after MI (n = 3) mice heart samples, respectively. We use log2 of the fold change as the source of data for the x axis and −log10 of the P as the source of data for the y axis. Log2 FC >1 and P < 0.05 indicate statistically significant differences. Red and blue points represent the upregulated USPs and the downregulated USPs compared with control group. Meanwhile, black points represent the USPs with no statistical difference compared with the control group. B, Real-time qPCR analysis of the mRNA expression of partial USPs in heart tissues 1 day after MI. C and D, Representative immunoblotting images and quantification showing the protein expression levels of USP13 in the infarct border zone of mouse heart tissues at various time post-myocardial infarction (MI) (n = 7 for each group). E, Uniform manifold approximation and projection (UMAP) plot showing 11 annotated cell types based on the top enriched marker genes from the single-nucleus RNA sequencing (snRNA-seq) data of healthy human hearts. F, Feature plot depicting USP13 scaled expression across all cell clusters in the dataset shown in panel E. Purple dots represent cells expressing USP13 at up to 95 % of the maximum expression level. The top 5 % of high-expressing cells were filtered to avoid any unknown biases. G, Violin plot displaying USP13 scaled expression across all cell types in the heart snRNA-seq data, as shown in panel F. H and I, Representative immunoblotting images and quantification showing the expression levels of USP13 in neonatal mouse ventricular myocytes (NMCMs) and non-NMCMs (n = 4 for each group). J and K, Representative immunoblotting images and quantification showing the expression levels of USP13 in NMCMs undergoing 12 and 24 h of oxygen-glucose deprivation (OGD), (n = 4 for each group). Data were expressed as mean ± SEM. Data in D was analyzed by one-way ANOVA, data in B and I were analyzed by unpaired t -test, data in K were analyzed by Kruskal-Walli's test. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Sequencing, Expressing, Ubiquitin Proteomics, Control, Western Blot, Marker, RNA Sequencing

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 deficiency exacerbated cardiac injuries and following remodeling process following MI A, Schematic illustration of experimental design. B and C, the serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) 1 day after MI were detected by commercial kits (n = 6 for each group). D, Representative M-mode echocardiograms obtained from AAV9-Ctrl and AAV9-shUSP13 mice 28d after MI or sham surgery. E and F echocardiographic measurements of left ventricular ejection fraction (EF), fractional shortening (FS) (n = 6 for each group). G and H, Masson trichrome staining of serial sections of mice heart 28 d after MI and the quantified size of scar size (the blue collagen region) (scale bar, 1 mm). Average scar sizes calculated by ImageJ Pro Plus (n = 6 for each group). Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Staining

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 directly interacts with ALDOA A, Schematic illustration for USP13 substrate screening. B, Proteins with high confidence that may interact with USP13 from MS analysis. C, Coimmunoprecipitation (CoIP) of USP13 and ALDOA in NMCMs. D, Representative western blotting for ALDOA in the infarct border zone of mouse heart tissues at various time post-MI. E, CoIP of USP13 and ALDOA in adult mouse heart. F and G, CoIP with anti-Flag or anti-Ha antibodies in 293T cells transfected with Flag-USP13 and Ha-ALDOA. H, Colocalization of Flag-USP13 (green) and Ha-ALDOA (red) in 293T cells. Scale bar = 10 μm. I, Predicted docking module of USP13 and ALDOA visualized by PYMOL. Purple represents USP13, cyan represents ALDOA, yellow is hydrogen bond, and red is the binding site corresponding to USP13. J, Schematic illustration of the USP13 domain deletion construct used in K. K, CoIP with anti-Ha antibody in 293T cells transfected with Flag-USP13-WT, Flag-USP13-Mut-1, Flag-USP13- Mut-2 and Ha-ALDOA.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Western Blot, Transfection, Binding Assay, Construct

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 inhibits MI-induced ferroptosis A and B, Volcano plot and gene set enrichment analysis (GSEA) for differentially expressed genes (DEGs) between nc and si in NMCMs after Oxygen-glucose deprivation (OGD) 24 h. C and D, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). E, The mRNA expression levels of PTGS2 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). F, The serum levels of malondialdehyde (MDA) 1 day after MI were detected by commercial kits (n = 6 for each group). G and H, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in NMCMs after 24 h of OGD (n = 3 for each group). I and J, Representative fluorescent images and quantification of C11-BODIPY probe (n = 6 for each group), scale bar = 100 μm. The green fluorescence indicated oxidized lipid and the red fluorescence indicated non-oxidized lipid. The green and red ratios were quantitatively analyzed in each group. Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Fluorescence

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 restrains ferroptosis post-MI by stabilizing ALDOA A, USP13 and ALDOA protein expression level in the heart tissues collected from control patients and patients with myocardial infarction (n = 3). B and C, Representative immunoblotting images and quantification showing the protein expression levels of ALDOA and USP13 in mouse heart tissues at 1 day following MI (n = 6 for each group). D and E, Representative immunoblotting images and quantification showing the protein expression levels of ALDOA and USP13 in NMCMs after 24 h of OGD (n = 3 for each group). F and G, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in NMCMs after 24 h of OGD (n = 3 for each group). H, The mRNA expression levels of PTGS2 in NMCMs after 24 h of OGD (n = 3 for each group). I and J, the levels of LDH and MDA in H9C2 cells after 8 h of OGD were detected by commercial kits (n = 6 for each group). K and L, Representative fluorescent images and quantification of C11-BODIPY probe (n = 6 for each group), scale bar = 100 μm. The green fluorescence indicated oxidized lipid and the red fluorescence indicated non-oxidized lipid. The green and red ratios were quantitatively analyzed in each group. Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Expressing, Control, Western Blot, Fluorescence

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 modulates the stability of ALDOA through deubiquitination A, Representative western blotting for Flag-USP13 and Ha-ALDOA in 293T cells transfected with Flag-USP13 with various concentrations and Ha-ALDOA. B, The mRNA expression levels of USP13 and ALDOA of A. C, Representative western blotting for Ha-ALDOA in 293T cells transfected with Flag-USP13 and Ha-ALDOA, incubated with the lysosome inhibitor CQ or the proteasome inhibitor MG132 for 6 h. D and E, Representative immunoblotting images and quantification showing the protein expression levels of Ha-ALDOA in 293T cells transfected with Flag-USP13 and Ha-ALDOA and then subjected to CHX for the indicated times (n = 3 for each group). F and G, Representative immunoblotting images and quantification showing the protein expression levels of ALDOA in 293T cells transfected with Si-Ctrl and Si-USP13 and then subjected to CHX for the indicated times (n = 3 for each group). H, Immunoprecipitation with anti-Ha antibody in 293T cells transfected with Flag-USP13, Ha-ALDOA and Myc-Ub and then subjected to MG132 (10 μM). I, Immunoprecipitation with anti-Ha antibody in 293T cells transfected with Flag-USP13, Ha-ALDOA, Myc-Ub, Myc-Ub-K48, and Myc-Ub-K63 and then subjected to MG132 (10 μM). J, K, Immunoprecipitation with anti- Flag or anti-Ha antibodies in 293T cells transfected with Flag-USP13, Ha-ALDOA WT , Ha-ALDOA K13R , Ha-ALDOA K87R and Ha-ALDOA K139R and then subjected to MG132 (10 μM). K, Sequence comparison of ALDOA at K13 among HUMAN, BOVIN, RABIT, MOUSE, RAT. Data were expressed as mean ± SEM. All the data were analyzed by one-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Western Blot, Transfection, Expressing, Incubation, Immunoprecipitation, Sequencing, Comparison

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 overexpression attenuates cardiac injuries after MI A, Schematic illustration of experimental design. B and C, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). D, The mRNA expression levels of PTGS2 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). E, F and G, The serum levels of MDA, LDH and CK-MB 1 day after MI were detected by commercial kits (n = 6 for each group). H, Representative M-mode echocardiograms obtained from AAV9-Ctrl and AAV9-USP13 oe mice 28d after MI or sham surgery. I and J echocardiographic measurements of left ventricular ejection fraction (EF), fractional shortening (FS) (n = 6 for each group). K and L, Masson trichrome staining of serial sections of mice heart 28 d after MI and the quantified size of scar size (the blue collagen region) (scale bar, 1 mm). Average scar sizes calculated by ImageJ Pro Plus (n = 6 for each group). Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Over Expression, Western Blot, Expressing, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Expression of bone morphogenetic protein signaling pathway players in the jejunum and colon of adult rats

doi: 10.4081/ejh.2025.4174

Figure Lengend Snippet: Immunohistochemical localization of ACVR1 ( a,b,c ) and BMPR2 ( d,e,f ) receptors in rat jejunum. e ) The arrows show strong immunostaining in the lamina propria; the asterisk designates the longitudinal muscularis layer. To determine the localization of BMP receptors, we performed immunohistochemical staining on jejunum sections from 6 different rats. Sections were examined by light microscopy (Leica DM 2000) using 20x and 40x objectives. Representative images are shown. Scale bars: 50 μm.

Article Snippet: BMPR2 , 1:100 , ProteinTech - 19087-1 - AP.

Techniques: Immunohistochemical staining, Immunostaining, Staining, Light Microscopy

Journal: European Journal of Histochemistry : EJH

Article Title: Expression of bone morphogenetic protein signaling pathway players in the jejunum and colon of adult rats

doi: 10.4081/ejh.2025.4174

Figure Lengend Snippet: Immunohistochemical localization of ACVR1 ( a,b,c ) and BMPR2 ( d,e,f ) receptors in rat colon. b ) The asterisk shows surface colonocytes. To determine the localization of BMP receptors, we performed immunohistochemical staining on colon sections from 6 different rats. Sections were examined by light microscopy (Leica DM 2000) using 20x and 40x objectives. Representative images are shown. Scale bars: 50 μm.

Article Snippet: BMPR2 , 1:100 , ProteinTech - 19087-1 - AP.

Techniques: Immunohistochemical staining, Staining, Light Microscopy

Journal: Cancer Research

Article Title: N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

doi: 10.1158/0008-5472.can-05-1688

Figure Lengend Snippet: Figure 2. Modulation of AD198-induced apoptosis by PLS3. A, overexpression of PLS3 enhanced AD198-induced apoptosis. HeLa cells at 90% confluence were transfected with pcDNA control vector or PLS3-expression vector for 24 hours. The expression of PLS3 was shown by Western blot. Cells were then incubated with AD198 at 0, 1, 5, and 10 Amol/L. Cells were harvested at 16 hours after AD198 exposure and the apoptotic population was determined by staining with propidium iodide. The sub-G1 population was marked by M1 gate and used to represent the apoptotic population. B, down-regulation of PLS3 by siRNA. HeLa-PLS3 cells were transfected with siRNA against PLS3 or random control siRNA at 5, 10, 15, and 20 nmol/L. Whole cell lysates were harvested at 48 hours for Western blotting. Down-regulation of PLS3 abolished AD198-induced apoptosis. HeLa cells at 50% confluence were transfected with 20 nmol/L siRNA against PLS3 or a random control siRNA for 48 hours. Cells were then treated with AD198 at 0, 1, 5, and 10 Amol/L for 16 hours followed by staining with propidium iodide. The apoptotic sub-G1 population is marked as M1 gate and the percentages are indicated. Columns, mean from three independent experiments; bars, SD.

Article Snippet: The polyclonal antibody to PLS3 was raised in rabbits against fulllength recombinant PLS3 (Proteintech Group, Inc., Chicago, IL).

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Expressing, Western Blot, Incubation, Staining

Journal: Cancer Research

Article Title: N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

doi: 10.1158/0008-5472.can-05-1688

Figure Lengend Snippet: Figure 3. AD198 induces PLS3 phosphorylation at threonine. A, HeLa cells were transfected with the His-tagged PLS3 expression vector and treated with AD198 for 0, 2, 4, and 6 hours. Whole cell lysates were incubated with Ni affinity beads to pull down His-tagged PLS3. The pulldown samples were analyzed by Western blotting with antibodies against phosphothreonine and PLS3. B, HeLa cells were transfected with the His-tagged PLS3 vector combined with the pcDNA empty vector, PKC-y, or kinase-defective PKC-y (PKC-dKD) vectors. Cells were then incubated with or without AD198 (5 Amol/L) for 2 hours. Whole cell lysates were incubated with Ni beads to pull down His-tagged PLS3, and analyzed with the antibodies against phosphothreonine and PLS3. C, down-regulation of PKC-y suppresses AD198-induced PLS3 phosphorylation. HeLa cells were transfected with various combinations of pcDNA, pKD-PKC-y-v3, and pCMV-His-PLS3 as indicated. Cells were treated with or without AD198 for 16 hours. Whole cell lysates were probed with PKC-y antibody, and PLS3 pulled down by Ni beads was probed with phosphothreonine antibody.

Article Snippet: The polyclonal antibody to PLS3 was raised in rabbits against fulllength recombinant PLS3 (Proteintech Group, Inc., Chicago, IL).

Techniques: Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Incubation, Western Blot

Journal: Cancer Research

Article Title: N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

doi: 10.1158/0008-5472.can-05-1688

Figure Lengend Snippet: Figure 4. PLS3 interacts with wild-type PKC-y but not with the inactive kinase-defective PKC-y mutant. A, HeLa cells expressing His-tagged PLS3 were transfected with PKC-y or kinase-defective PKC-y vectors. They were then incubated with AD198 for 0, 2, 4, and 6 hours. Whole cell lysates were incubated with Ni beads to pull down His-tagged PLS3 and probed with antibodies against PLS3 and PKC-y. B, HeLa-PLS3 cells transfected with pcDNA, PKC-y, or kinase-defective PKC-y vectors were harvested before and 2 hours after AD198 treatment. PKC-y was immunoprecipitated from whole cell lysates and analyzed with antibodies against PKC-y and PLS3. Western blotting of whole cell lysates revealed that the level of PLS3 expression was roughly similar before and after AD198 treatment.

Article Snippet: The polyclonal antibody to PLS3 was raised in rabbits against fulllength recombinant PLS3 (Proteintech Group, Inc., Chicago, IL).

Techniques: Mutagenesis, Expressing, Transfection, Incubation, Immunoprecipitation, Western Blot

Journal: Cancer Research

Article Title: N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

doi: 10.1158/0008-5472.can-05-1688

Figure Lengend Snippet: Figure 5. Identification of Thr21 as the phosphorylation site of PLS3 induced by PKC-y. A, in vitro phosphorylation of recombinant PLS3 by PKC-y. Equal amounts of recombinant PLS3 and a 6 kDa NH2-terminal fragment of PLS3 were mixed together and phosphorylated by purified PKC-y in the presence of [g-32P]ATP. The phosphorylated sample was analyzed with SDS gel and exposed by autoradiography. B, PLS3 phosphorylation by PKC-y is suppressed by T21A mutation in PLS3. Recombinant His-tagged PLS3 (wild type) and T21A mutant was isolated by Ni beads from two elutions and analyzed with SDS gel. Top, yield of each protein in the two-step elution by Coomassie staining. Bottom, in vitro phosphorylation of equal amounts of PLS3 protein (elution 1) by recombinant PKC-y. C, subcellular fraction of cells transfected with PLS3 or PLS3(T21A) expression vectors. D, T21A mutation abolished in vivo PLS3 phosphorylation after AD198 treatment. HeLa cells were transfected with the control vector or vectors expressing His-tagged PLS3 or PLS3(T21A). His-tagged PLS3 protein was pulled down by Ni beads from cells with or without AD198 treatment. Whole cell lysates and pulldown samples by Ni beads were analyzed with Western blotting using antibodies against PLS3 and phosphothreonine. E, interaction of PLS3(T21A) with PKC-y. HeLa cells were cotransfected with the PKC-y vector and pcDNA, PLS3, or PLS3(T21A) vector. Whole cell lysates were incubated with Ni beads to pull down PLS3. The pulldown samples were analyzed with Western blotting using antibodies against PLS3 and PKC-y. F, HeLa cells were cotransfected as in (E). Whole cell lysates were immunoprecipitated with PKC-y antibody. Whole cell lysates and immunoprecipitates were analyzed by Western blotting with the antibody against PLS3.

Article Snippet: The polyclonal antibody to PLS3 was raised in rabbits against fulllength recombinant PLS3 (Proteintech Group, Inc., Chicago, IL).

Techniques: Phospho-proteomics, In Vitro, Recombinant, Purification, SDS-Gel, Autoradiography, Mutagenesis, Isolation, Staining, Transfection, Expressing, In Vivo, Control, Plasmid Preparation, Western Blot, Incubation, Immunoprecipitation

Journal: Cancer Research

Article Title: N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

doi: 10.1158/0008-5472.can-05-1688

Figure Lengend Snippet: Figure 6. PLS3(T21A) does not enhance AD198-induced apoptosis like the wild-type PLS3. A, HeLa cells were transfected with the empty vector or vectors expressing PLS3 or PLS3(T21A). Cells were harvested for Western blotting with antibodies against PLS3 and actin. B, cells then treated with DMSO or 5 Amol/L AD198 for 16 hours followed by flow cytometry analysis with propidium iodide staining. The representatives of flow cytometry analysis were shown. The sub-G0 apoptotic population was determined and indicated in each panel. C, histogram to show the average of apoptotic population in control cells and cells transfected with wild-type PLS3 or PLS3(T21A). Results are the averages of three independent experiments. Statistical significance (P < 0.05) was achieved by the paired t test between wild-type PLS3 versus control or PLS3(T21A).

Article Snippet: The polyclonal antibody to PLS3 was raised in rabbits against fulllength recombinant PLS3 (Proteintech Group, Inc., Chicago, IL).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Flow Cytometry, Staining, Control

Journal: Cancer Research

Article Title: N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

doi: 10.1158/0008-5472.can-05-1688

Figure Lengend Snippet: Figure 7. AD198-induced PLS3 phosphorylation is an upstream event of caspase activation and independent of mitochondrial permeability transition. A, Z-VAD and cyclosporine A (CsA) could not block AD198-induced loss of mitochondrial potential. HeLa cells were treated with 50 Amol/L Z-VAD or 5 Amol/L cyclosporine A for 30 minutes, and then AD198 was added at 0, 1 or 5 Amol/L for 16 hours. Cells were incubated with MitoTracker Green at 37jC for 20 minutes followed by flow cytometry analysis. Columns, mean from three independent experiments; bars, SD. B, Z-VAD, but not cyclosporine A, inhibits AD198-induced apoptosis. HeLa cells were treated as in (A) and collected for the apoptosis study with propidium iodide staining. C, percentage of apoptosis from three independent experiments in (B). Columns, mean; bars, SD. D, Z-VAD or cyclosporine A could not suppress AD198-induced PLS3 phosphorylation. HeLa cells were transfected with the vector expressing His-tagged PLS3. Cells were treated with AD198 (5 Amol/L) along with Z-VAD or cyclosporine A for 2 hours and harvested for Ni bead pulldown. Whole cell lysates and pulldown samples were probed with antibodies against phosphothreonine and PLS3.

Article Snippet: The polyclonal antibody to PLS3 was raised in rabbits against fulllength recombinant PLS3 (Proteintech Group, Inc., Chicago, IL).

Techniques: Phospho-proteomics, Activation Assay, Permeability, Blocking Assay, Incubation, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Expressing