Journal: Muscle & nerve

Article Title: Amyotrophic Lateral Sclerosis and swim training affect copper metabolism in skeletal muscle in a mouse model of disease.

doi: 10.1002/mus.28237

Figure Lengend Snippet: FIGURE 2 Effects of amyotrophic lateral sclerosis (ALS) disease progression and swim training on ATP7a, CTR1, and DMT1 protein level in skeletal muscle. ATP7a (A), CTR1 (B), and DMT1 (C) levels were measured in mice skeletal muscle. The cropped blots were used in the figure. Full-length blots are shown in Figures S2–S4. There were significant differences between the groups: ap < .05, bp < .01, dp < .001 versus ALS BEFORE group, Ap < .05, Bp < .01 versus ALS ONSET untrained group of mice, **p < .01, ***p < .001 versus wild type (WT) group of mice (Tukey's post hoc test), #p < .05, ##p < .01 between the indicated groups (Student t-test). The data are presented as the means ± SD (n = 6 in each group).

Article Snippet: Next, the membranes were rinsed three times in 1 TBST for 5 min, incubated with primary antibodies dissolved in blocking buffer with gentle shaking, and left overnight at 4 C. The following rabbit polyclonal antibody was used: CTR1/ SLC31A1 (Cat. No. 13086, Cell Signaling, Danvers, MA, USA, 1:1000), DMT1/anti-SLC11A1 (Cat. No. FNab07905, Fine Test, Wuhan, China, 1:500), anti-ATP7a (Cat. No. ab 125,137, Abcam, Cambridge, UK, 1:1000), and rabbit monoclonal antibody; CCS/CCS antibody (Cat. No. ab137131, Abcam, Cambridge, UK, 1:1000).

Techniques: Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Theaflavin-3,3′-Digallate Enhances the Inhibitory Effect of Cisplatin by Regulating the Copper Transporter 1 and Glutathione in Human Ovarian Cancer Cells

doi: 10.3390/ijms19010117

Figure Lengend Snippet: Treatment with TF3 potentiated inhibitory effect of cisplatin against ovarian cancer A2780/CP70 and OVCAR3 cells via upregulating CTR1 protein expression. ( A ) The effect of TF3 at the designated concentrations on the protein levels of MRP2, ATP7A, ATP7B and CTR1 in ovarian cancer cells; ( B ) treatment with 7.5 μM TF3 upregulated CTR1 protein levels in ovarian cancer cells pretreated with 7.5 μM cisplatin; ( C ) transfection with CTR1 siRNA decreased CTR1 protein levels in ovarian cancer cells; ( D ) transfection with CTR1 siRNA enhanced the resistance of ovarian cancer cells to 7.5 μM cisplatin. Results are expressed as mean ± SD from three independent experiments. Significant differences among different treatments are marked with different letters ( p < 0.05), * ( p < 0.05) and ** ( p < 0.01).

Article Snippet: CTR1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Transfection

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