Journal: Advanced Science

Article Title: Targeting Intratumoral Copper Inhibits Tumor Progression via p62‐Mediated EZH2 Degradation and Potentiates Anti‐PD‐1 Immunotherapy in Oral Squamous Cell Carcinoma

doi: 10.1002/advs.202417795

Figure Lengend Snippet: Serum copper level and SLC31A1 indicated cancer progression and poor survival in OSCC and human cancers. A) Serum copper ion levels in OSCC patients (n = 92) were compared with those in a healthy control group (n = 16). B–D) The relationship between serum copper ion levels and tumor size (B), lymph node metastasis (C) and tumor clinical staging (D) in OSCC patients were analyzed. E) Expression levels of SLC31A1 protein in 9 pairs of OSCC tumor tissues and adjacent normal tissues. F–I) In an independent clinical sample of OSCC from the Shanghai Ninth People's Hospital (n = 105) (F), the correlation between SLC31A1 expression and tumor clinical staging (H), tumor size (I), and patient prognosis (G) were analyzed, Scale bars, 100 µm. J,K) In BRCA tissue microarrays (n = 132), the correlation between SLC31A1 expression and tumor size (K), as well as patient prognosis (J), were analyzed. L,M) In LUSC tissue microarrays (n = 85), the association between SLC31A1 expression and lymph node metastasis (M), as well as patient prognosis (L), were analyzed. N,O) In ESCA tissue microarrays (n = 112), the correlation between SLC31A1 expression and tumor size (O), as well as patient prognosis (N), were analyzed. Data in A‐D were calculated by two‐tailed unpaired Student's t test; Data in H, I, K, M, and O were calculated by the Wilcoxon rank‐sum test; Data in G, J, L, and N were analyzed by Kaplan–Meier plots, p values were determined by a two‐tailed log‐rank test.

Article Snippet: Antibodies against SLC31A1 (NB100‐402, Novus Biologicals, USA, dilution 1:1000, RRID: AB_10 003 309), β‐tubulin (#2128, CST, USA, dilution 1:1000, RRID: AB_823 664), β‐actin (66009‐1‐Ig, Proteintech, USA, dilution 1:50 000, RRID: AB_2 687 938), ERK1/2 (11257‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 139 822), Phospho‐ERK1/2 (Thr202/Tyr204) (28733‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 202), G9a (#3306, CST, USA, dilution 1:1000, RRID: AB_2 097 647), ESET (66293‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 676), EZH2 (#5246, CST, USA, dilution 1:1000, RRID: AB_10 694 683), SUV39H1 (#8729, CST, USA, dilution 1:1000, RRID: AB_10 829 612), TIP60 (10827‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 128 431), MOF (13842‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 146 894), HAT1 (67971‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 721), KDM2A (24311‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 879 488), KDM4A (29977‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 923 625), KDM6A (23984‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 935 460), SIRT1 (60303‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 417), HDAC1 (#5356, CST, USA, dilution 1:1000, RRID: AB_10 612 242), HDAC4 (#7628, CST, USA, dilution 1:1000, RRID: AB_10 860 255), HDAC11 (67949‐1‐lg, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 701), H3 (BS1174, Bioworld, China, dilution 1:1000, RRID: AB_1 663 967), H3K27me3 (#9733, CST, USA, dilution 1:1000, RRID: AB_2 616 029), mTOR Pathway Antibody Sampler Kit (#9964, CST, USA, RRID: AB_10 696 892), FLAG (20543‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 232 216), HA (51064‐2‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 042 321), p62 ( T59081 , Abmart, China, dilution 1:1500, RRID: AB_2 936 470), NDP52 (A7358, Abclonal, China, dilution 1:1000, RRID: AB_2 767 894), CCT2 (A4700, Abclonal, China, dilution 1:1000, RRID: AB_2 863 327), LC3B ( T55992 , Abmart, China, dilution 1:1000, RRID: AB_2 929 010), GAPDH (A19056, Abclonal, China, dilution 1:1000, RRID: AB_2 862 549), K48‐linkage Specific Polyubiquitin(#8081, CST, USA, dilution 1:1000, RRID: AB_10 859 893), K63‐linkage Specific Polyubiquitin(#5621, CST, USA, dilution 1:1000, RRID: AB_10 827 985), MYC (60003‐2‐Ig, Proteintech, USA, dilution 1:2000, RRID: AB_2 734 122), STUB1 (55430‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_10 949 225) and SMURF2 (18038‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_3 085 549) were used.

Techniques: Control, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Intratumoral Copper Inhibits Tumor Progression via p62‐Mediated EZH2 Degradation and Potentiates Anti‐PD‐1 Immunotherapy in Oral Squamous Cell Carcinoma

doi: 10.1002/advs.202417795

Figure Lengend Snippet: Decreasing intratumoral copper abrogated OSCC cell growth. A) Targeting intratumoral copper by silencing SLC31A1 or using copper ion chelators. B,C) Measurement of cellular copper ion concentrations in OSCC cells after silencing SLC31A1 (B) and using copper ion chelators (C). D,E) The effect of silencing SLC31A1 (D) and copper ion chelators (E) on cell proliferation ability in OSCC cell lines (HN6 and SCC7). F) The effect of copper ion chelators on sphere formation (sphere number) in OSCC cell lines (HN6 and CAL27), Scale bars, 200 µm. G) Schematic diagram of subcutaneous tumor (SCC7) and oral administration experiment in C3H mice. H) Macroscopic view of subcutaneous xenografts in C3H mice. I) Volume changes of subcutaneous xenografts in C3H mice. J) Changes in total body weight of C3H mice. K) Weight of subcutaneous xenografts in C3H mice. L) Copper concentration in tumor tissue. M) Multiplexed immunofluorescence of Epcam, Ki67 and Ezh2 in subcutaneous xenograft tissues from C3H mice, Scale bars, 200 µm (left), 50 µm (right). N) scRNA‐seq analysis of subcutaneous xenograft tumors in C3H mice. O) Dimensionality reduction clustering analysis of tumor epithelial cells in subcutaneous xenograft tumors in C3H mice. P) Proportions of various tumor epithelial cell clusters. Q) CytoTRACE analysis of tumor epithelial cell. R) Development potential of tumor epithelial cell clusters. S) Multiplexed immunofluorescence of Cxcr4 and Epcam in subcutaneous xenograft tissues from C3H mice, Scale bars, 200 µm (left), 50 µm (right). Data were calculated by two‐tailed unpaired Student's t test.

Article Snippet: Antibodies against SLC31A1 (NB100‐402, Novus Biologicals, USA, dilution 1:1000, RRID: AB_10 003 309), β‐tubulin (#2128, CST, USA, dilution 1:1000, RRID: AB_823 664), β‐actin (66009‐1‐Ig, Proteintech, USA, dilution 1:50 000, RRID: AB_2 687 938), ERK1/2 (11257‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 139 822), Phospho‐ERK1/2 (Thr202/Tyr204) (28733‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 202), G9a (#3306, CST, USA, dilution 1:1000, RRID: AB_2 097 647), ESET (66293‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 676), EZH2 (#5246, CST, USA, dilution 1:1000, RRID: AB_10 694 683), SUV39H1 (#8729, CST, USA, dilution 1:1000, RRID: AB_10 829 612), TIP60 (10827‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 128 431), MOF (13842‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 146 894), HAT1 (67971‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 721), KDM2A (24311‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 879 488), KDM4A (29977‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 923 625), KDM6A (23984‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 935 460), SIRT1 (60303‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 417), HDAC1 (#5356, CST, USA, dilution 1:1000, RRID: AB_10 612 242), HDAC4 (#7628, CST, USA, dilution 1:1000, RRID: AB_10 860 255), HDAC11 (67949‐1‐lg, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 701), H3 (BS1174, Bioworld, China, dilution 1:1000, RRID: AB_1 663 967), H3K27me3 (#9733, CST, USA, dilution 1:1000, RRID: AB_2 616 029), mTOR Pathway Antibody Sampler Kit (#9964, CST, USA, RRID: AB_10 696 892), FLAG (20543‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 232 216), HA (51064‐2‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 042 321), p62 ( T59081 , Abmart, China, dilution 1:1500, RRID: AB_2 936 470), NDP52 (A7358, Abclonal, China, dilution 1:1000, RRID: AB_2 767 894), CCT2 (A4700, Abclonal, China, dilution 1:1000, RRID: AB_2 863 327), LC3B ( T55992 , Abmart, China, dilution 1:1000, RRID: AB_2 929 010), GAPDH (A19056, Abclonal, China, dilution 1:1000, RRID: AB_2 862 549), K48‐linkage Specific Polyubiquitin(#8081, CST, USA, dilution 1:1000, RRID: AB_10 859 893), K63‐linkage Specific Polyubiquitin(#5621, CST, USA, dilution 1:1000, RRID: AB_10 827 985), MYC (60003‐2‐Ig, Proteintech, USA, dilution 1:2000, RRID: AB_2 734 122), STUB1 (55430‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_10 949 225) and SMURF2 (18038‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_3 085 549) were used.

Techniques: Concentration Assay, Immunofluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Intratumoral Copper Inhibits Tumor Progression via p62‐Mediated EZH2 Degradation and Potentiates Anti‐PD‐1 Immunotherapy in Oral Squamous Cell Carcinoma

doi: 10.1002/advs.202417795

Figure Lengend Snippet: Intratumor copper is required for EZH2 protein stability. A) Differential gene expression analysis of HN6 cells with silenced SLC31A1 and control HN6 cells using RNA‐seq data. B) Pathway enrichment analysis of the differentially expressed genes. C,D) Detection of protein expression levels of histone‐modifying enzymes in HN6 cells after targeting intracellular copper. E) Changes in EZH2 mRNA levels under SLC31A1 silencing condition. F) Changes in EZH2 mRNA levels under treatment with copper ion chelator TEPA. G,H) Detection of EZH2 protein expression levels in SLC31A1‐KO HN6 (G) and SLC31A1‐silenced CAL27 (H) cells treated with CHX (20 µg mL −1 ) (0, 6, 12, and 18 h). I,J) Detection of EZH2 protein expression levels in TEPA‐treated HN6 (I) and CAL27 (J) cells treated with CHX (20 µg mL −1 ) (0, 6, 12, and 18 h). K,L) Ubiquitination immunoprecipitation experiments detecting changes in EZH2 ubiquitination levels in SLC31A1‐silenced (K) and TEPA‐treated (L) HN6 and CAL27 cells. Data in E‐J were calculated by two‐tailed unpaired Student's t test.

Article Snippet: Antibodies against SLC31A1 (NB100‐402, Novus Biologicals, USA, dilution 1:1000, RRID: AB_10 003 309), β‐tubulin (#2128, CST, USA, dilution 1:1000, RRID: AB_823 664), β‐actin (66009‐1‐Ig, Proteintech, USA, dilution 1:50 000, RRID: AB_2 687 938), ERK1/2 (11257‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 139 822), Phospho‐ERK1/2 (Thr202/Tyr204) (28733‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 202), G9a (#3306, CST, USA, dilution 1:1000, RRID: AB_2 097 647), ESET (66293‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 676), EZH2 (#5246, CST, USA, dilution 1:1000, RRID: AB_10 694 683), SUV39H1 (#8729, CST, USA, dilution 1:1000, RRID: AB_10 829 612), TIP60 (10827‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 128 431), MOF (13842‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 146 894), HAT1 (67971‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 721), KDM2A (24311‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 879 488), KDM4A (29977‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 923 625), KDM6A (23984‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 935 460), SIRT1 (60303‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 417), HDAC1 (#5356, CST, USA, dilution 1:1000, RRID: AB_10 612 242), HDAC4 (#7628, CST, USA, dilution 1:1000, RRID: AB_10 860 255), HDAC11 (67949‐1‐lg, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 701), H3 (BS1174, Bioworld, China, dilution 1:1000, RRID: AB_1 663 967), H3K27me3 (#9733, CST, USA, dilution 1:1000, RRID: AB_2 616 029), mTOR Pathway Antibody Sampler Kit (#9964, CST, USA, RRID: AB_10 696 892), FLAG (20543‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 232 216), HA (51064‐2‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 042 321), p62 ( T59081 , Abmart, China, dilution 1:1500, RRID: AB_2 936 470), NDP52 (A7358, Abclonal, China, dilution 1:1000, RRID: AB_2 767 894), CCT2 (A4700, Abclonal, China, dilution 1:1000, RRID: AB_2 863 327), LC3B ( T55992 , Abmart, China, dilution 1:1000, RRID: AB_2 929 010), GAPDH (A19056, Abclonal, China, dilution 1:1000, RRID: AB_2 862 549), K48‐linkage Specific Polyubiquitin(#8081, CST, USA, dilution 1:1000, RRID: AB_10 859 893), K63‐linkage Specific Polyubiquitin(#5621, CST, USA, dilution 1:1000, RRID: AB_10 827 985), MYC (60003‐2‐Ig, Proteintech, USA, dilution 1:2000, RRID: AB_2 734 122), STUB1 (55430‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_10 949 225) and SMURF2 (18038‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_3 085 549) were used.

Techniques: Gene Expression, Control, RNA Sequencing, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Intratumoral Copper Inhibits Tumor Progression via p62‐Mediated EZH2 Degradation and Potentiates Anti‐PD‐1 Immunotherapy in Oral Squamous Cell Carcinoma

doi: 10.1002/advs.202417795

Figure Lengend Snippet: Intracellular copper deficiency promoted p62‐mediated selective autophagic degradation of EZH2. A,B) Detection of EZH2 protein expression differences with or without MG‐132 treatment in SLC31A1‐silenced HN6 (A) and CAL27 (B) cells. C,D) Detection of EZH2 protein expression differences with or without MG‐132 treatment in TEPA‐treated HN6 (C) and CAL27 (D) cells. E,F) Detection of EZH2 protein expression differences with or without CQ treatment in SLC31A1‐KO HN6 (E) and SLC31A1‐silenced CAL27 (F) cells. G,H) Detection of EZH2 protein expression differences with or without CQ treatment in TEPA‐treated HN6 (G) and CAL27 (H) cells. I,J) Detection of EZH2 protein expression differences with or without BafA1 treatment in SLC31A1‐KO HN6 (I) and SLC31A1‐silenced CAL27 (J) cells. K,L) Detection of EZH2 protein expression differences with or without BafA1 treatment in TEPA‐treated HN6 (K) and CAL27 (L) cells. M) Immunoprecipitation experiments were conducted to detect autophagy receptors interacting with EZH2 in HN6 and CAL27 cells. N) Overexpression of p62 and EZH2 alone or together in HEK293T cells, and immunoprecipitation experiments were performed to detect the interaction between EZH2 and p62. O) Immunoprecipitation experiments were carried out to detect the interaction between p62 and EZH2 in HN6 and CAL27 cells. P) Overexpression of p62 and EZH2 alone or together in HEK293T cells, and immunoprecipitation experiments were conducted to detect the interaction between p62 and EZH2. Q,R) Co‐localization experiments were performed to detect the co‐localization of EZH2 and p62 after targeting intracellular copper, Scale bars, 5 µm (top), 10 µm (bottom). S) Overexpression of EZH2 and p62 or mutant p62 in HEK293T cells, and immunoprecipitation experiments were conducted to detect the ubiquitination level of EZH2. T,U) Detection of EZH2 ubiquitination level in SLC31A1‐silenced (T) and TEPA‐treated (U) cells after rescuing trace amounts of copper ions by immunoprecipitation experiments in HN6 and CAL27 cells.

Article Snippet: Antibodies against SLC31A1 (NB100‐402, Novus Biologicals, USA, dilution 1:1000, RRID: AB_10 003 309), β‐tubulin (#2128, CST, USA, dilution 1:1000, RRID: AB_823 664), β‐actin (66009‐1‐Ig, Proteintech, USA, dilution 1:50 000, RRID: AB_2 687 938), ERK1/2 (11257‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 139 822), Phospho‐ERK1/2 (Thr202/Tyr204) (28733‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 202), G9a (#3306, CST, USA, dilution 1:1000, RRID: AB_2 097 647), ESET (66293‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 676), EZH2 (#5246, CST, USA, dilution 1:1000, RRID: AB_10 694 683), SUV39H1 (#8729, CST, USA, dilution 1:1000, RRID: AB_10 829 612), TIP60 (10827‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 128 431), MOF (13842‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 146 894), HAT1 (67971‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 721), KDM2A (24311‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 879 488), KDM4A (29977‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 923 625), KDM6A (23984‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 935 460), SIRT1 (60303‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 417), HDAC1 (#5356, CST, USA, dilution 1:1000, RRID: AB_10 612 242), HDAC4 (#7628, CST, USA, dilution 1:1000, RRID: AB_10 860 255), HDAC11 (67949‐1‐lg, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 701), H3 (BS1174, Bioworld, China, dilution 1:1000, RRID: AB_1 663 967), H3K27me3 (#9733, CST, USA, dilution 1:1000, RRID: AB_2 616 029), mTOR Pathway Antibody Sampler Kit (#9964, CST, USA, RRID: AB_10 696 892), FLAG (20543‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 232 216), HA (51064‐2‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 042 321), p62 ( T59081 , Abmart, China, dilution 1:1500, RRID: AB_2 936 470), NDP52 (A7358, Abclonal, China, dilution 1:1000, RRID: AB_2 767 894), CCT2 (A4700, Abclonal, China, dilution 1:1000, RRID: AB_2 863 327), LC3B ( T55992 , Abmart, China, dilution 1:1000, RRID: AB_2 929 010), GAPDH (A19056, Abclonal, China, dilution 1:1000, RRID: AB_2 862 549), K48‐linkage Specific Polyubiquitin(#8081, CST, USA, dilution 1:1000, RRID: AB_10 859 893), K63‐linkage Specific Polyubiquitin(#5621, CST, USA, dilution 1:1000, RRID: AB_10 827 985), MYC (60003‐2‐Ig, Proteintech, USA, dilution 1:2000, RRID: AB_2 734 122), STUB1 (55430‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_10 949 225) and SMURF2 (18038‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_3 085 549) were used.

Techniques: Expressing, Immunoprecipitation, Over Expression, Mutagenesis, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: Targeting Intratumoral Copper Inhibits Tumor Progression via p62‐Mediated EZH2 Degradation and Potentiates Anti‐PD‐1 Immunotherapy in Oral Squamous Cell Carcinoma

doi: 10.1002/advs.202417795

Figure Lengend Snippet: Intracellular copper depletion recruited and stabilized E3 ligase SMURF2 to ubiquitinate EZH2. A,B) Detection of EZH2 protein expression and localization in SLC31A1‐silenced (A) and TEPA‐treated (B) cells by nuclear‐cytoplasmic fractionation experiments in CAL27 and HN6 cells. C) Detection of EZH2 ubiquitination level by combining nuclear‐cytoplasmic fractionation and immunoprecipitation experiments in 293T cells co‐overexpressing EZH2 and p62 and treated with TEPA. D) Intersecting the datasets from E3 Ligases and UbiBrowser with mass spectrometry analysis results for p62 protein. E) Immunoprecipitation experiments were carried out to detect the interaction between SMURF2 and EZH2 in 293T cells. (F,G) Treated with or without TEPA and detected EZH2 protein expression levels in SMURF2‐KO (F) and SMURF2‐overexpression (G) HN6 cells treated with CHX (20 µg mL −1 ) (0, 6, 12, and 18 h). H) Treated with or without TEPA and detected the EZH2 ubiquitination level in SMURF2‐KO and SMURF2‐overexpression HN6 cells. I) Predict the binding site of SMURF2 with copper ions using AlphaFold 3. J) Detection of copper ion concentration in SLC31A1‐silenced and TEPA‐treated HN6 (ip‐SMURF2) cells. K) Detection of copper ion concentration in SLC31A1‐silenced and TEPA‐treated CAL27 (ip‐SMURF2) cells. L,M) Detection of EZH2 and SMURF2 protein expression levels in TEPA‐treated (L) and SLC31A1‐KO (M) HN6 cells treated with CHX (20 µg mL −1 ) (0, 6, 12, and 18 h) in the case of SMURF2 mutation. N,O) Detection of SMURF2 thermal stability treated with TEPA via Cellular Thermal Shift Assay (CETSA) in 293T cell. P,Q) Detection of SMURF2 and STUB1 thermal stability via CETSA in SLC31A1‐KO HN6 cell. Data in J‐K were calculated by two‐tailed unpaired Student's t test; Data in O and Q were calculated by nonlinear regression (curve fit).

Article Snippet: Antibodies against SLC31A1 (NB100‐402, Novus Biologicals, USA, dilution 1:1000, RRID: AB_10 003 309), β‐tubulin (#2128, CST, USA, dilution 1:1000, RRID: AB_823 664), β‐actin (66009‐1‐Ig, Proteintech, USA, dilution 1:50 000, RRID: AB_2 687 938), ERK1/2 (11257‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 139 822), Phospho‐ERK1/2 (Thr202/Tyr204) (28733‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 202), G9a (#3306, CST, USA, dilution 1:1000, RRID: AB_2 097 647), ESET (66293‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 676), EZH2 (#5246, CST, USA, dilution 1:1000, RRID: AB_10 694 683), SUV39H1 (#8729, CST, USA, dilution 1:1000, RRID: AB_10 829 612), TIP60 (10827‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 128 431), MOF (13842‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 146 894), HAT1 (67971‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 721), KDM2A (24311‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 879 488), KDM4A (29977‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 923 625), KDM6A (23984‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 935 460), SIRT1 (60303‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 417), HDAC1 (#5356, CST, USA, dilution 1:1000, RRID: AB_10 612 242), HDAC4 (#7628, CST, USA, dilution 1:1000, RRID: AB_10 860 255), HDAC11 (67949‐1‐lg, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 701), H3 (BS1174, Bioworld, China, dilution 1:1000, RRID: AB_1 663 967), H3K27me3 (#9733, CST, USA, dilution 1:1000, RRID: AB_2 616 029), mTOR Pathway Antibody Sampler Kit (#9964, CST, USA, RRID: AB_10 696 892), FLAG (20543‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 232 216), HA (51064‐2‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 042 321), p62 ( T59081 , Abmart, China, dilution 1:1500, RRID: AB_2 936 470), NDP52 (A7358, Abclonal, China, dilution 1:1000, RRID: AB_2 767 894), CCT2 (A4700, Abclonal, China, dilution 1:1000, RRID: AB_2 863 327), LC3B ( T55992 , Abmart, China, dilution 1:1000, RRID: AB_2 929 010), GAPDH (A19056, Abclonal, China, dilution 1:1000, RRID: AB_2 862 549), K48‐linkage Specific Polyubiquitin(#8081, CST, USA, dilution 1:1000, RRID: AB_10 859 893), K63‐linkage Specific Polyubiquitin(#5621, CST, USA, dilution 1:1000, RRID: AB_10 827 985), MYC (60003‐2‐Ig, Proteintech, USA, dilution 1:2000, RRID: AB_2 734 122), STUB1 (55430‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_10 949 225) and SMURF2 (18038‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_3 085 549) were used.

Techniques: Expressing, Fractionation, Ubiquitin Proteomics, Immunoprecipitation, Mass Spectrometry, Over Expression, Binding Assay, Concentration Assay, Mutagenesis, Thermal Shift Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Intratumoral Copper Inhibits Tumor Progression via p62‐Mediated EZH2 Degradation and Potentiates Anti‐PD‐1 Immunotherapy in Oral Squamous Cell Carcinoma

doi: 10.1002/advs.202417795

Figure Lengend Snippet: Targeting intracellular copper enhances the therapeutic efficacy of anti‐PD‐1 therapy in OSCC. A,B) The expression level of B2m, H2‐Aa, H2‐Ab1, H2‐Eb1 and Cd40 in tumor epithelial cell in C3H mice. B) Multiplexed immunofluorescence of Epcam and B2m in subcutaneous xenograft tissues from C3H mice. D) Schematic diagram of subcutaneous tumor (SCC7) formation in C3H mice, TEPA oral administration, and PD‐1 monoclonal antibody intraperitoneal injection experiment. E) Macroscopic view of subcutaneous transplanted tumors in C3H mice. F) Changes in volume of subcutaneous transplanted tumors in C3H mice. G) Weight of subcutaneous transplanted tumors in C3H mice. H) Body weight of C3H mice. I,J) Multiplexed immunofluorescence was performed on paraffin sections of transplant tumor tissues from C3H mice to detect Cd3, F4/80 and Cd49b. K,L) Through Multiplexed immunofluorescence, the relationship between SLC31A1 expression and immune response is evaluated in OSCC patients who are resistant or sensitive to immunotherapy. The area outlined by the white dotted line represents the tumor tissue. Data in F‐H, J, and L were calculated by two‐tailed unpaired Student's t test.

Article Snippet: Antibodies against SLC31A1 (NB100‐402, Novus Biologicals, USA, dilution 1:1000, RRID: AB_10 003 309), β‐tubulin (#2128, CST, USA, dilution 1:1000, RRID: AB_823 664), β‐actin (66009‐1‐Ig, Proteintech, USA, dilution 1:50 000, RRID: AB_2 687 938), ERK1/2 (11257‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 139 822), Phospho‐ERK1/2 (Thr202/Tyr204) (28733‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 202), G9a (#3306, CST, USA, dilution 1:1000, RRID: AB_2 097 647), ESET (66293‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 676), EZH2 (#5246, CST, USA, dilution 1:1000, RRID: AB_10 694 683), SUV39H1 (#8729, CST, USA, dilution 1:1000, RRID: AB_10 829 612), TIP60 (10827‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 128 431), MOF (13842‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 146 894), HAT1 (67971‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 721), KDM2A (24311‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 879 488), KDM4A (29977‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 923 625), KDM6A (23984‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_2 935 460), SIRT1 (60303‐1‐Ig, Proteintech, USA, dilution 1:1000, RRID: AB_2 881 417), HDAC1 (#5356, CST, USA, dilution 1:1000, RRID: AB_10 612 242), HDAC4 (#7628, CST, USA, dilution 1:1000, RRID: AB_10 860 255), HDAC11 (67949‐1‐lg, Proteintech, USA, dilution 1:1000, RRID: AB_2 918 701), H3 (BS1174, Bioworld, China, dilution 1:1000, RRID: AB_1 663 967), H3K27me3 (#9733, CST, USA, dilution 1:1000, RRID: AB_2 616 029), mTOR Pathway Antibody Sampler Kit (#9964, CST, USA, RRID: AB_10 696 892), FLAG (20543‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 232 216), HA (51064‐2‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_11 042 321), p62 ( T59081 , Abmart, China, dilution 1:1500, RRID: AB_2 936 470), NDP52 (A7358, Abclonal, China, dilution 1:1000, RRID: AB_2 767 894), CCT2 (A4700, Abclonal, China, dilution 1:1000, RRID: AB_2 863 327), LC3B ( T55992 , Abmart, China, dilution 1:1000, RRID: AB_2 929 010), GAPDH (A19056, Abclonal, China, dilution 1:1000, RRID: AB_2 862 549), K48‐linkage Specific Polyubiquitin(#8081, CST, USA, dilution 1:1000, RRID: AB_10 859 893), K63‐linkage Specific Polyubiquitin(#5621, CST, USA, dilution 1:1000, RRID: AB_10 827 985), MYC (60003‐2‐Ig, Proteintech, USA, dilution 1:2000, RRID: AB_2 734 122), STUB1 (55430‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_10 949 225) and SMURF2 (18038‐1‐AP, Proteintech, USA, dilution 1:1000, RRID: AB_3 085 549) were used.

Techniques: Drug discovery, Expressing, Immunofluorescence, Injection, Two Tailed Test

Journal: Materials Today Bio

Article Title: PKA activation rescues myocardial injury elicited by silica nanoparticles through improving oxidative stress, mitochondrial health, and copper homeostasis

doi: 10.1016/j.mtbio.2026.103021

Figure Lengend Snippet: PKA mitigated copper overload triggered by SiNPs. (A) In the SiNPs-instilled rats, enhanced copper contents in both serum (a) and myocardial tissues (b) were detected, but PKA agonist Forskolin reversed it. n = 9-10 per group. (B) The dose-dependent elevation of intracellular Cu 2+ content by SiNPs treatment in the in vitro cultured AC16 cardiomyocytes (a), which could be greatly reduced by PKA stimulator 8-Br-cAMP but increased by PKA inhibitor H89 (b). (C) The expressions of copper metabolism-related protein (FDX1, SLC31A1, and ATP7B) in AC16 cells were measured (C-a, protein bands; C-b, bands analysis). Also, the expression of ATP7B was measured in rat myocardial tissue (D). n = 3. ∗ p < 0.05 vs control, # p < 0.05 vs SiNPs or SiNPs + Forskolin.

Article Snippet: Primary antibodies for PKA, p -PKA, p -DRP1 s637 , SLC31A1, and ATP7B were purchased from ABclonal, China; FDX1 from Proteintech, USA; and DRP1 and GAPDH from CST, USA.

Techniques: In Vitro, Cell Culture, Expressing, Control

Journal: Oncotarget

Article Title: Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth.

doi: 10.18632/oncotarget.10201

Figure Lengend Snippet: Figure 2: Levels of CTR1 protein expression and intracellular copper concentrations vary between neuroblastoma cell lines. (A) Representative Western blot for CTR1 protein on whole-cell extracts from IMR-32, IMR-32-CisRes, BE(2)-C, and MRC-5 cells. GAPDH expression was used as a protein loading control. (B) Densitometry graph of Western blots showing higher expression of CTR1 in IMR-32 and BE(2)-C cells compared to IMR-32-CisRes and normal MRC-5 cells. Values are normalized to GAPDH protein expression and shown relative to CTR1 expression in IMR-32 cells (100%). (C) Intracellular copper levels are higher in IMR-32 and BE(2)-C cells compared to IMR-32-CisRes and MRC-5 cells. Columns, means of at least three independent experiments; Bars, SEM (*P < 0.05, **P < 0.01).

Article Snippet: Twelve hours post-transfection with two different CTR1 specific siRNAs (siRNA A and siRNA B Origene, Rockville, MD, USA) or scrambled non-silencing siRNA, cells were treated with Dextran-Catechin for 24 hours.

Techniques: Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth.

doi: 10.18632/oncotarget.10201

Figure Lengend Snippet: Figure 3: Knockdown of CTR1 in IMR-32 and BE(2)-C cells significantly reduced their sensitivity to Dextran-Catechin. (A) Cell death in IMR-32 and (B) BE(2)-C cells after knockdown of CTR1 and subsequent treatment with Dextran-Catechin for 24 hours. Columns, means of at least three independent experiments; Bars, SEM (***p < 0.001, ****p < 0.0001).

Article Snippet: Twelve hours post-transfection with two different CTR1 specific siRNAs (siRNA A and siRNA B Origene, Rockville, MD, USA) or scrambled non-silencing siRNA, cells were treated with Dextran-Catechin for 24 hours.

Techniques: Knockdown