Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 1 STEAP4 expression in HCC cohorts. A Box plots showing STEAP4 mRNA expression in liver hepatocellular carcinoma (LIHC) tumor cohort versus TCGA normal and GTEx data, analyzed by t-test in the GEPIA database (*, p < 0.01). B Scatter plots of STEAP4 mRNA expression in tumor and non-tumor samples from HCC patients, as presented in the Oncomine database across multiple datasets, including Chen Liver, Roessler Liver, Roessler Liver 2, Wurmbach Liver, and Mas Liver (*, p < 0.05; ****, p < 0.0001). C Representative IHC images (10X and 40X) illustrating STEAP4 protein expression in HCC and adjacent non-tumor tissue. Scale bar = 50 µm for 40X images. Overall statistical analysis of STEAP4 protein expression in HCC patient samples based on IHC staining (****, p < 0.0001)

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 2 Prognostic evaluation of STEAP4 in HCC patients. A, B Analysis of overall survival (OS) and disease-free survival (DFS) based on high and low STEAP4 expression in LIHC patients using the GEPIA database. C Kaplan–Meier analysis of OS for high and low STEAP4 expression in TCGA-LIHC samples. D Multivariate Cox proportional hazards analysis of OS, visualized through a forest plot

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Expressing

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 3 DEGs of key cell cycle-related genes in high and low STEAP4 expression groups in LIHC cohort. A Volcano plots display differentially expressed genes (DEGs). B KEGG pathway enrichment analysis of DEGs, conducted using DAVID, is visualized in bubble plots, with the y-axis showing significantly enriched KEGG pathways and the x-axis showing various gene ratios. DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes. C The top 5 negatively correlated cell cycle-related genes with STEAP4, analyzed using TIMER

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Expressing

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 4 Representative IHC images showing the expression of STEAP4, PLK1, CDC25C, CDC20, CCNB2, CCNB1, and CDK1 proteins in HCC and adjacent non-tumor tissues. Compared to peritumoral tissues, HCC tissues exhibit lower STEAP4 expression and higher levels of PLK1, CDC20, CDC25C, and CDK1 (n = 7). T: tumor; P: peritumoral tissue. Scale bar: 100 µm

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Expressing

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 5 Efficacy of STEAP4 overexpression in Hep G2 Cells via copper reductase activity. A STEAP4 overexpression in Hep G2 cells shows no significant cytotoxicity. B, C Copper incubation induces a dose-dependent cytotoxic effect on Hep G2 cells, with 50 μM copper identified as a non-cytotoxic concentration. D Co-treatment with STEAP4 overexpression and 50 μM copper decreases cell viability in Hep G2 cells. E, F Co-treatment with STEAP4 overexpression and 50 μM copper reduces colony formation in Hep G2 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ##p < 0.0001

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Over Expression, Activity Assay, Incubation, Concentration Assay

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 7 Effects of STEAP4 overexpression and 50 μM copper on cell cycle distribution in Hep G2 cells. A–C Cell cycle distribution analysis shows that co-treatment with STEAP4 overexpression and 50 μM copper increases the percentage of Hep G2 cells in sub-G1 and G2/M phases. The synergistic effect is eliminated by STEAP4 knockdown via siRNA, indicating G2/M cell cycle arrest associated with STEAP4 copper reductase activity. * p < 0.05

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Over Expression, Knockdown, Activity Assay

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 8 STEAP4 and 50 μM Copper co-treatment downregulates indicated cell cycle genes in Hep G2 cells. A STEAP4 with copper reductase activity reduces PLK1, CDK1, CDC20, CDC25C, and CCNB2 mRNA expression in Hep G2 cells. This synergistic effect is abolished by STEAP4 knockdown via siRNA. * p < 0.05, ** p < 0.01. B STEAP4 with copper reductase activity reduces PLK1, CDK1, CDC20, CDC25C, and CCNB2 protein expression in Hep G2 cells as indicated by representative western blot results

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Activity Assay, Expressing, Knockdown, Western Blot

Journal: Cell division

Article Title: STEAP4 with copper reductase activity suppresses tumorigenesis by regulating the cell cycle in hepatocellular carcinoma cells.

doi: 10.1186/s13008-024-00140-y

Figure Lengend Snippet: Fig. 11 Schematic diagram of proposed mechanism of STEAP4-mediated copper reductase activity in HCC tumorigenesis suppression via cell cycle regulation. This schematic illustrates STEAP4’s functional impact on HCC cells in vitro. STEAP4-mediated copper reduction leads to downregulation of key cell cycle genes, inhibiting G2/M progression in HCC cells. Further investigation into the underlying mechanisms of STEAP4-mediated regulation of cell cycle genes is warranted. (Figure created with Figdraw)

Article Snippet: The tissue section was then incubated overnight at 4 °C with an anti-STEAP4 antibody (A17767, 1:100, Abclonal, Germany), anti-CDC20 antibody (A15656, 1:100, Abclonal, Germany), anti-CDK1 antibody (A22347, 1:100, Abclonal, Germany), anti-Cyclin B2 antibody (DF9047, 1:100, Affinity Biosciences, USA), anti-PLK1 antibody (AF2685, 1:100, Affinity Biosciences, USA), and anti-CDC25C antibody (AF6258, 1:100, Affinity Biosciences, USA).

Techniques: Activity Assay, Functional Assay, In Vitro

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: FTY720 has an affinity for TCP1 and can inhibit AML cell proliferation. ( A ) The chemical structure of FTY720. ( B ) TCP1 subunit protein and FTY720 molecular docking virtual screening. ( C ) Surface plasmon resonance detected binding between FTY720 and TCP1. ( D ) The effect of FTY720 on the viability of AML cells. ( E ) TCP1 protein levels in the three cell lines were examined by Western blotting and ( F ) normalized to the corresponding density of GAPDH. ( G ) The sensitivity of HL-60 cells to FTY720 increased as the TCP1 protein level increased. ( H ) HL-60-G4 cells formed many more colony units than HL-60 cells, and their colony numbers decreased as FTY720 increased. ( I ) The quantification and plot were created using ImageJ software (Version 1.8.0) for the image shown in H. Data are presented as the means ± SDs from three independent experiments. *** p < 0.001.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207 ); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: SPR Assay, Binding Assay, Western Blot, Software

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia.

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: Figure 1. TCP1 is positively related to the tumorigenesis and development of AML. (A) In the succes- sive generations, the tumor formation rate increased in the HL-60 cell line. (B) Mass spectrometry

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Mass Spectrometry

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia.

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: Figure 2. The effect of TCP1 on AML cell development and proliferation in vitro and in vivo. (A) The efficiency of shTCP1 was measured in HL-60-G4 cells by Western blotting. (B) A clonogenic cell survival assay was performed to evaluate the growth of HL-60-G4-shTCP1 cells in vitro. (C,D) Rep- resentative cell cycle distribution of HL-60-G4 cells after knocking down TCP1 with lentivirus transfection. (E) Detection of xenograft tumor formation in nude mice using Tet-on technology. (F) Representative image of Dox- and Dox+ groups of tumors at the end of the experiment. (G) West- ern blotting was used to analyze the protein level of TCP1 in tumor tissues in each group. Data are presented as the means ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: In Vitro, In Vivo, Western Blot, Clonogenic Cell Survival Assay, Transfection

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia.

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: Figure 3. TCP1 regulated the stability of PLK1 and AML1-ETO. (A) Western blotting analysis of TCP1, PLK1, and AML1-ETO expression in HL-60-G4-shTCP1 and Kasumi-1-shTCP1 cells. (B) Cells were incubated with 10 µM CHX alone or combined with 10 µM MG132, and WB assays were used to determine the levels for TCP1 and PLK1. (C) The image shown in Figure 3B was quantified using ImageJ software (Version 1.8.0). (D) Co-IP assays were performed to observe the pull-down levels of PLK1 and AML1-ETO in the shTCP1 group and scramble group. Data are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Western Blot, Expressing, Incubation, Software, Co-Immunoprecipitation Assay

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia.

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: Figure 5. FTY720 has an affinity for TCP1 and can inhibit AML cell proliferation. (A) The chemical structure of FTY720. (B) TCP1 subunit protein and FTY720 molecular docking virtual screening. (C) Surface plasmon resonance detected binding between FTY720 and TCP1. (D) The effect of FTY720 on the viability of AML cells. (E) TCP1 protein levels in the three cell lines were examined by Western blotting and (F) normalized to the corresponding density of GAPDH. (G) The sensitivity of HL-60 cells to FTY720 increased as the TCP1 protein level increased. (H) HL-60-G4 cells formed many more colony units than HL-60 cells, and their colony numbers decreased as FTY720 increased. (I) The quantification and plot were created using ImageJ software (Version 1.8.0) for the image shown in Figure 5H. Data are presented as the means ± SDs from three independent experiments. *** p < 0.001.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: SPR Assay, Binding Assay, Western Blot, Software

Journal: Pharmaceutics

Article Title: The Molecular Chaperone TCP1 Affects Carcinogenicity and Is a Potential Therapeutic Target for Acute Myeloid Leukemia.

doi: 10.3390/pharmaceutics17050557

Figure Lengend Snippet: Figure 7. FTY720 induces cell cycle arrest and decreases protein levels by inhibiting TCP1 chaperone function in AML cells. (A) Representative cell cycle distribution images. (B) Column chart of the cell cycle ratio of HL-60-G4, HL-60, Kasumi-1, and KG1α cells. (C) Changes in intracellular protein levels in HL-60-G4 and KG1α cells incubated with FTY720 for 24 h. (D) The image shown in Figure 8C was quantified using ImageJ software (Version 1.8.0). (E) The interaction between the PLK1 protein and TCP1 protein in HL-60-G4 and KG1α cells exposed to FTY720 for 24 h was assayed by Co-IP. (F) The interaction between the CDC20 protein and TCP1 protein in HL-60-G4 and KG1α cells exposed to FTY720 for 24 h was assayed by Co-IP. (G) Changes in intracellular protein levels in Kasumi-1 cells incubated with FTY720 for 24 h were detected by Western blotting. (H) TCP1 interacts with AML1-ETO in Kasumi-1 cells, but FTY720 disrupts the interaction. Means ± SDs of three independent experiments are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: The following antibodies were used: anti-TCP1 polyclonal antibody (Proteintech, 10320-1-AP); anti-cleaved-caspase-3 (Wanleibio, WL02117); anti-CDC20 Rabbit pAb (Abclonal, A15656); anti-PKMYT1 Rabbit pAb (Abclonal, A20525); anti-GAPDH monoclonal antibody (ImmunoWay, YM3029); anti-AML1-ETO (CST, 4334); anti-cleaved-caspase-9 (CST, 52873); anti-cleaved-PARP (CST, 5625); anti-PLK1 (Abcam, ab189139); anti-pan-myc (Abcam, Ab195207); anti-mouse IgG, HRP-conjugated (CST, 7076); and anti-rabbit IgG, HRP-conjugated (CST, 7074).

Techniques: Incubation, Software, Co-Immunoprecipitation Assay, Western Blot