Journal: Journal of Cancer

Article Title: FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10

doi: 10.7150/jca.125659

Figure Lengend Snippet: FAM188B expression is upregulated and associated with poor prognosis in hepatocellular carcinoma (HCC). (A) Expression of FAM188B mRNA in tumor and non-tumor tissues and its association with cancer stage and tumor grade in UALCAN database. (B) The expression of FAM188B protein was evaluated in normal hepatocytes (LX-2) and in hepatocellular carcinoma (HCC) cells (MHCC97H, Huh7, Hep3B and SNU387) by western blot. (C) Validation of FAM188B expression after overexpression in the indicated cell lines using western blotting (n = 3). (D) Proliferation of MHCC97H and Huh7 cells, as detected by performing CCK-8 assays (n = 10). (E) Migratory and invasive capacities of MHCC97H and Huh7 cells within 24 hours, as evaluated using Transwell assays (scale bar, 50 μm) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. The data are expressed as the mean ± SD of three independent experiments.

Article Snippet: Human liver tumor-derived cell lines SNU387 and Hep3B, human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA) in 2021.

Techniques: Expressing, Western Blot, Biomarker Discovery, Over Expression, CCK-8 Assay

Journal: Journal of Cancer

Article Title: FAM188B promotes progression of hepatocellular carcinoma by regulating YAP/TAZ via interaction with USP10

doi: 10.7150/jca.125659

Figure Lengend Snippet: FAM188B interacts with USP10. (A) Schematic illustration of IP and GO enrichment of identified proteins (using all proteins in the species database as the background). Fisher's exact test was used to analyze the significance and P value < 0.05 were considered significant. (B) Expression levels of USP10 mRNA in tumor (n = 374) and non-tumor (n = 50) tissues and its relationship with FAM188B, based on a public database. (C) The protein expression level of USP10 was evaluated in normal hepatocytes (LX-2) and hepatocellular carcinoma (HCC) cells (MHCC97H, Huh7, Hep3B and SNU387) by western blot. (D) Localization of USP10 in HCC cells (scale bar, 20 μm). (E) Co-IP assays to confirm the protein-protein interaction between FAM188B and USP10 in vitro . **p < 0.01, ***p < 0.001. The data are expressed as the mean ± SD of three independent experiments. IP, immunoprecipitation. Co-IP, co-immunoprecipitation.

Article Snippet: Human liver tumor-derived cell lines SNU387 and Hep3B, human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA) in 2021.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Immunoprecipitation

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylation determines the glucose metabolism reprogramming and tumor-promoting activity of sine oculis homeobox 1

doi: 10.1038/s41392-024-02034-5

Figure Lengend Snippet: SIX1 is phosphorylated at S225 by ERK. a Identification of S225 and S278 of SIX1 as potential phosphorylation sites by NanoLC-MS/MS. pho, phosphorylation. b ZR75-1 breast cancer cells were transfected with MYC-tagged SIX1, SIX1 (S225A), SIX1 (S278A) or SIX1 (Q177R). Cell lysates were immunoprecipitated with anti-MYC, followed by immunoblot with the indicated antibodies. pSer, anti-pan-phosphoserine antibody. pS225, anti-SIX1 S225 phosphorylation antibody. IP immunoprecipitation, IB immunoblot, MW molecular weight. c Co-immunoprecipitation (Co-IP) analysis of interaction between SIX1 and the indicated proteins in HepG2 and ZR75-1 cells. IgG, normal serum. d GST pull-down analysis of interaction between purified GST-SIX1 fusion protein and purified His-tagged ERK1/2. e Immunoblot analysis of HepG2 and ZR75-1 cells transfected with FLAG-tagged ERK1/2. β-actin was used as a loading control. f HepG2 or ZR75-1 cells were transfected with MYC-tagged ERK2 or ERK2 (K54R) and FLAG-tagged SIX1 or SIX1 (S225A) as indicated. Cell lysates were immunoprecipitated with anti-FLAG, followed by immunoblot with the indicated antibodies. g Immunoblot analysis of HepG2 and ZR75-1 cells transfected with MYC-tagged ERK2 or ERK2 (K54R). h Purified GST, GST-SIX1 or GST-SIX1 (S225A) was incubated with recombinant human ERK2 (rhERK2) protein in the presence of γ- 32 P-ATP. In vitro kinase assays were performed and analyzed by autoradiography. i Immunoblot analysis of HepG2 and ZR75-1 cells transfected with control siRNA, ERK2 siRNA1/2 or ERK2 siRNA1/2 plus siRNA-resistant ERK1/2 (ERK2-R1/2). j Immunoblot analysis of HepG2 and ZR75-1 cells treated with 20 μm PD98059

Article Snippet: Human breast ZR75-1 (CRL-1500) and liver HepG2 (HB-8065) cancer cell lines and embryonic kidney HEK293T cell line (CRL-11268) were purchased from American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, Tandem Mass Spectroscopy, Transfection, Immunoprecipitation, Western Blot, Molecular Weight, Co-Immunoprecipitation Assay, Purification, Control, Incubation, Recombinant, In Vitro, Autoradiography

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylation determines the glucose metabolism reprogramming and tumor-promoting activity of sine oculis homeobox 1

doi: 10.1038/s41392-024-02034-5

Figure Lengend Snippet: SIX1 is dephosphorylated at S225 by EYA4. a Immunoblot analysis of HepG2 and ZR75-1 cells transfected with GFP or GFP-tagged EYA1-4. b Immunoblot analysis of HepG2 and ZR75-1 cells transfected with control siRNA, EYA4 siRNA1/2 or EYA4 siRNA1/2 plus siRNA-resistant EYA4 (EYA4-R1/2). c Immunoblot analysis of HepG2 and ZR75-1 cells transfected with GFP, GFP-tagged wild-type (WT) EYA4 or GFP-tagged EYA4 mutants (DYY, Y4 and D352N). d Immunoblot analysis of purified His-tagged EYA4 or its mutant (DYY) incubated with purified FLAG-SIX1 from HepG2 cells. Coomassie blue staining shows loading levels of the purified proteins

Article Snippet: Human breast ZR75-1 (CRL-1500) and liver HepG2 (HB-8065) cancer cell lines and embryonic kidney HEK293T cell line (CRL-11268) were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Transfection, Control, Purification, Mutagenesis, Incubation, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylation determines the glucose metabolism reprogramming and tumor-promoting activity of sine oculis homeobox 1

doi: 10.1038/s41392-024-02034-5

Figure Lengend Snippet: SIX1 phosphorylation regulates its non-canonical ubiquitination and degradation via the ubiquitin-proteasome pathway. a Immunoblot analysis of HepG2 and ZR75-1 cells transfected with control siRNA (siCtrl), ERK2 siRNA1 (siERK2) or siERK2 plus ERK2-R1 (ERK2-R) and treated with or without 10 μM MG-132 for 4 h. b Immunoblot analysis of HepG2 and ZR75-1 cells transfected with GFP or GFP-EYA4 and treated with or without 10 μM MG-132 for 4 h. c Immunoblot analysis of SIX1 expression at the indicated times in HepG2 cells stably transfected with control vector (Ctrl), ERK2 shRNA (ERK2 KD) or GFP-EYA4 (EYA4 OE) and exposure to the protein synthesis inhibitor cycloheximide (20 μg/ml). Graphs show quantification of immunoblot data. Results shown are mean ± SD of 3 independent experiments. d Ubiquitination analysis of HepG2 and ZR75-1 cells transfected with siCtrl, siERK2 or siEYA4 and treated with 10 μM MG-132 for 4 h. Ub, ubiquitin. e Ubiquitination analysis of HepG2 and ZR75-1 cells transfected with HA-Ub and FLAG-tagged SIX1 or its mutants with or without MYC-tagged FZR1 and treated with 10 μM MG-132 for 4 h. f Co-IP analysis of HepG2 and ZR75-1 cells transfected with MYC-tagged FZR1 and FLAG-tagged SIX1 or its mutants. g Immunoblot analysis of SIX expression at the indicated times in HepG2 cells stably transfected with FLAG-tagged SIX1, SIX1 (S225A), SIX1 (S225K) and SIX1 (S247A) and exposure to 20 μg/ml cycloheximide. Graphs show quantification of immunoblot data. Results shown are mean ± SD of 3 independent experiments. **P < 0.01. h Immunoblot analysis of HepG2 and ZR75-1 cells transfected with siERK2, siFZR1 or siERK2 plus siFZR1 as indicated. i Immunoblot analysis of HepG2 and ZR75-1 cells transfected with GFP-EYA4, siFZR1 or GFP-EYA4 plus siFZR1 as indicated

Article Snippet: Human breast ZR75-1 (CRL-1500) and liver HepG2 (HB-8065) cancer cell lines and embryonic kidney HEK293T cell line (CRL-11268) were purchased from American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Western Blot, Transfection, Control, Expressing, Stable Transfection, Plasmid Preparation, shRNA, Co-Immunoprecipitation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylation determines the glucose metabolism reprogramming and tumor-promoting activity of sine oculis homeobox 1

doi: 10.1038/s41392-024-02034-5

Figure Lengend Snippet: SIX1 is phosphorylated in response to growth factors and important for growth factor-mediated glycolysis. a Immunoblot analysis of HepG2 and ZR75-1 cells treated with 50 ng/ml EGF, 100 ng/ml IGF1, 100 ng/ml TGFα, 5 ng/ml TGFβ, 10 ng/ml VEGF, 100 ng/ml IL-6, 20 ng/ml TNFα and 500 U/ml IFNγ for 12 h with BSA as a control. BSA, bovine serum albumin. b Immunoblot analysis of HepG2 and ZR75-1 cells treated with 5 ng/ml TGFβ for the indicated times. c Immunoblot analysis of HepG2 and ZR75-1 cells pretreated with 20 μm PD98059 and then treated with 5 ng/ml TGFβ for 12 h. d Immunoblot analysis of SIX1 WT or KO HepG2 or ZR75-1 cells treated with 5 ng/ml TGFβ for 12 h. e Analysis of glucose uptake and production of pyruvate, lactate and ATP in SIX1 WT or KO HepG2 or ZR75-1 cells treated with or without 5 ng/ml TGFβ for 12 h. PBS, phosphate buffered saline. f Immunoblot analysis of SIX1 KO HepG2 cells transfected with empty vector, FLAG-SIX1 or FLAG-SIX1 (S225A) and treated with or without 5 ng/ml TGFβ for 12 h. g Analysis of glucose uptake and production of pyruvate, lactate and ATP in cells from f . h The proliferation curve and lactate and ATP production of ZR75-1 and HepG2 cells transfected with EV or FLAG-tagged SIX1 (S225K) or SIX1 (S225A) and treated with 2.5 mM 2-DG or 0.1 mM Oligomycin in glucose (25 mM) or galactose (10 mM)-containing medium as indicated. Representative immunoblot with anti-FLAG indicates the expression of SIX1 (S225K) or SIX1 (S225A). Data shown are means ± SD of quintuplicate measurements that have been repeated three times with similar results ( e , g ). Data shown are mean ± SD of three independent experiments ( h ). Statistical significance was assessed by two-tailed Student’s t test. **P < 0.01 versus SIX1 WT cells treated with PBS ( e ) or SIX1 KO HepG2 cells transfected with empty vector and treated with PBS ( g ). *P < 0.05, **P < 0.01 ( h )

Article Snippet: Human breast ZR75-1 (CRL-1500) and liver HepG2 (HB-8065) cancer cell lines and embryonic kidney HEK293T cell line (CRL-11268) were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Control, Saline, Transfection, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylation determines the glucose metabolism reprogramming and tumor-promoting activity of sine oculis homeobox 1

doi: 10.1038/s41392-024-02034-5

Figure Lengend Snippet: Non-canonical phosphorylation-mimetic SIX1 mutant enhances glycolysis as well as tumor growth and metastasis. a Immunoblot analysis of SIX1 KO HepG2 cells transfected with FLAG-SIX1 or its mutants. b Analysis of lactate production and ATP level in cells from a . Data shown are means ± SD of quintuplicate measurements that have been repeated three times with similar results. Statistical significance was assessed by one-way ANOVA test. *P < 0.05, **P < 0.01. c The proliferation curve of SIX1 KO HepG2 cells stably transfected with FLAG-SIX1 or its mutants. Immunoblot shows the expression of HK2, LDHA and FLAG-SIX1 or its mutants. Data shown are means ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA test. **P < 0.01. d Cell invasion assay of SIX1 KO HepG2 cells stably transfected with FLAG-SIX1 or its mutants. The relative cell invasions are shown. Scale bar, 100 μm. Data shown are means ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA test. *P < 0.05, **P < 0.01. e The growth curve of xenograft tumors derived from SIX1 KO HepG2 cells stably transfected with FLAG-SIX1 or its mutants. Representative immunoblot with the indicated antibodies was shown in the representative excised tumors. Scale bar, 10 mm. Statistical significance was assessed by one-way ANOVA test. **P < 0.01 at day 28. f Representative lung tissues and hematoxylin and eosin (H&E)-stained sections of the lung tissues at 35 days from nude mice injected by tail vein with SIX1 KO MHCC97-H cells stably transfected with FLAG-SIX1 or its mutants (n = 7). Arrows indicate tumor foci. The number of tumor nodules spread throughout the pulmonary region is shown. Representative immunoblot with the indicated antibodies was shown in the representative excised tumors. Scale bar, 2.5 mm for lung tissues; Scale bar, 50 μm for H&E-stained sections. g SIX1 KO MHCC97-H cells stably expressing PKM2 shRNA, FLAG-tagged SIX1 (S225K) or PKM2 shRNA plus FLAG-tagged SIX1 (S225K) were injected into nude mice. 2-DG was used as indicated. Stripped tumors are shown (left). The growth curve was plotted (middle). The expression of the indicated proteins in representative excised tumors from the third column in each group was analyzed by immunoblot (right). Scale bar, 10 mm. *P < 0.05, **P < 0.01 at day 28. h Representative lung tissues and H&E-stained sections of the lung tissues at 35 days from nude mice injected by tail vein with SIX1 KO MHCC97-H cells harboring different constructs as in g . Arrows denote tumor foci. The number of tumor nodules spread throughout the pulmonary region is indicated. Scale bar, 2.5 mm for lung tissues; Scale bar, 50 μm for H&E-stained sections. Statistical significance was assessed by one-way ANOVA test. *P < 0.05, * *P < 0.05

Article Snippet: Human breast ZR75-1 (CRL-1500) and liver HepG2 (HB-8065) cancer cell lines and embryonic kidney HEK293T cell line (CRL-11268) were purchased from American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Transfection, Stable Transfection, Expressing, Invasion Assay, Derivative Assay, Staining, Injection, shRNA, Construct