Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: CPAP overexpression contributes to tumor growth and metastasis in vivo. a For orthotopic liver injection, GFP/Hep3B, or GFP-CPAP/Hep3B cells were injected into the left liver lobe of BALB/c nude mice. After 24 days of injection, the mice were sacrificed, and the liver tissues were collected for further analysis. Images of orthotopic liver xenograft tumors obtained from BALB/c nude mice injected with GFP/Hep3B ( n = 7) or GFP-CPAP/Hep3B cells ( n = 6) are shown (left); the tumor volume was calculated using the formula length × width 2 × 0.5. Quantitative results are shown as the mean ± SEM. Student’s t test, * p = 0.0475. b The liver tissues injected with GFP-CPAP/Hep3B but not GFP/Hep3B cells showed intrahepatic metastatic tumor growth. c After 8 weeks of injection, the mouse liver and lung tissues from orthotopically injected mice were collected for H&E staining. The incidence of lung metastasis is shown. d Seventy-two HCC specimens (T) and their adjacent normal tissues (NT) were collected to analyze the expression level of CPAP mRNA by RT-qPCR, which was presented as a ratio (T/NT). HCC tissues were divided into two size groups, small (≤2 cm, n = 33) and large (≥5 cm, n = 39). Mann–Whitney U test, p = 0.0473. RT-qPCR results were normalized to actin . e GFP/Hep3B ( n = 5) or GFP-CPAP/Hep3B ( n = 5) cells were intra-splenically injected into BALB/c nude mice. After 8 weeks of injection, the mice were sacrificed, and the spleen, liver, and lung tissues were collected for analysis. Representative images of the spleen and liver tumors, H&E staining of the liver and lung tissues, and the incidence of liver and lung metastasis are shown. f A representative image of H&E staining shows vascular invasion of the GFP-CPAP/Hep3B-derived liver tumor in the splenically injected mice. Arrows indicate vascular invasion of the GFP-CPAP/Hep3B cells. Scale bars, 100 μm

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Over Expression, In Vivo, Injection, Staining, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: CPAP promotes cancer cell and HUVEC migration. a , b HCC cells stably expressing GFP, GFP-CPAP, or knocked-down CPAP by pSUPER-SP1 or pSUPER-SP5 were used to perform Transwell migration assays ( a ) or invasion assay ( b ). pSUPER-NS2 is a negative control of shRNA. c Conditioned media from IL-6-treated Hep3B (Hep3BST) and HepG2 (HepG2ST) cells stably expressing GFP or GFP-CPAP were collected to perform HUVEC migration assays. d Hep3B/GFP-CPAP or Huh7/GFP-CPAP stable cells were starved and treated without (–) or with IL-6 or TNF-α for 24 h, and then cell migration (left) and invasion (right) assays were performed. The number of migrated or invasive cells was counted, and the quantitative results are shown. Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Migration, Stable Transfection, Expressing, Invasion Assay, Negative Control, shRNA

Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: CPAP enhances STAT3 activity in HCC. a, b Immunoblotting analysis showed the expression of CPAP, p-STAT3, and STAT3 in paired HCC tumor (T) and adjacent nontumor tissues (N) ( a ). A total of twenty-one paired HCC specimens were analyzed. GAPDH was used as a loading control. A positive correlation between CPAP and p-STAT3 expression is shown ( b ). Pearson correlation coefficient, R = 0.4848, p = 0.0259. c (Upper) STAT3 transcriptional activity was determined in GFP- or GFP-CPAP-expressing Hep3B cells by a STAT3-driven luciferase reporter assay. STAT3 transcriptional activity is shown in relative luciferase activity as a fold change. (Lower) The level of phosphor-STAT3/Y705 (p-STAT3/Y705) in GFP/Hep3B or GFP-CPAP/Hep3B stable cells under normal culture conditions was determined by Western blot analysis. IL-6-treated GFP-CPAP/Hep3B cells were used as a positive control to detect p-STAT3/Y705. α-tubulin was used as a loading control. d STAT3 transcriptional activity in CPAP-knocked-down cells was determined by STAT3-driven luciferase reporter assay as described above. e GFP- or GFP-CPAP-expressing Hep3B or Huh7 cells were serum starved and then treated with (■) or without (□) IL-6 (25 ng/ml) for 6 h to determine the transcriptional activity of STAT3 by reporter assay as described above. Western blot analysis showed the expression of the indicated proteins. f Total lysates of GFP/Hep3B or GFP-CPAP/Hep3B stable cells treated with 25 ng/ml IL-6 for 10 min were collected for Western blot as described above. g CPAP-knocked-down Hep3B cells were treated with (■) or without (□) IL-6 to determine STAT3 activity by reporter assay (left) or Western blot analysis (right) as described above. h After serum starvation, GFP/Hep3B or GFP-CPAP/Hep3B cells were pretreated with (+) or without (−) 200 μM AG490 for 30 min and then treated with 25 ng/ml IL-6 for 6 h. STAT3 transcriptional activity was then determined by a STAT3-driven luciferase reporter assay. Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Activity Assay, Western Blot, Expressing, Control, Luciferase, Reporter Assay, Positive Control

Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: CPAP promotes the activity of the IL-6/STAT3 pathway. a, b The promoter activity ( a ) and gene expression level ( b ) of IL-8 , ICAM-1 , IL-6 , and HIF1-α were determined in Hep3B cells expressing GFP or GFP-CPAP by reporter assay and RT-qPCR, respectively. Cells were serum starved, treated with (■) or without (□) IL-6 for 24 h, and then harvested for analysis. c GFP- or GFP-CPAP-expressing Hep3B cells were treated with IL-6 for 24 h; the medium was then replaced by fresh culture medium, and the cells were cultured for an additional 24 h. The conditioned media were collected to determine the expression of IL-8 by enzyme-linked immunosorbent assay (ELISA). d Sera from HCC patients ( n = 31) were used to perform IL-8 ELISA. HCC tissues (T) and paired nontumorous tissues (NT) were used to analyze the CPAP mRNA expression level by RT-qPCR. The serum expression level of IL-8 is positively correlated with CPAP mRNA overexpression (T/NT) in HCC patients; Pearson correlation coefficient, R = 0.5162, p = 0.003. Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001. e GFP-CPAP-expressing Huh7 cells were treated with (+) or without (−) IL-6 for 4 min, and then total cell lysates were collected to perform a coimmunoprecipitation assay using anti-STAT3 antibodies. The immunoprecipitated complexes were subjected to Western blot analysis using the indicated antibodies. f (i) In vivo PLA using anti-STAT3 and anti-CPAP antibodies in IL-6-treated (+) or untreated (−) GFP- or GFP-CPAP-expressing Huh7 cells. (ii) Huh7 cells were cotransfected with Myc-STAT3/C1 and GFP-PN1 or GFP-A5C and then treated with (+) or without (−) IL-6 for 4 min. The interaction between PN1 or A5C and Myc-STAT3/C1 was detected by in situ PLA using anti-GFP and anti-Myc antibodies. The red signals indicate positive interactions. DAPI is a DNA-specific dye. The quantitative results of PLA red signals are shown

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Activity Assay, Gene Expression, Expressing, Reporter Assay, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Over Expression, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, In Vivo, In Situ

Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: CPAP enhances angiogenesis via the IL-6/STAT3 pathway. a , b GFP/Hep3B, GFP-CPAP/Hep3B stable cells ( a ) or CPAP-knocked-down Hep3B cells ( b ) were treated with IL-6 for 24 h and then collected to perform Transwell migration and invasion assays. The number of migrated or invasive cells was counted, and the quantitative results are shown. c HUVEC Transwell migration assay (i) and tube formation assay (ii) were performed using conditioned medium (CM) collected from IL-6-treated Hep3B stable cells. The quantitative results of migration assay and tube formation assay are shown. d GFP/Hep3B or GFP-CPAP/Hep3B cells were used to perform Matrigel plug assay in NOD-SCID mice. The Matrigel plugs were excised (i) and analyzed by H&E staining and IHC assay using anti-CD31 antibodies (see Supplementary Fig. ). The IHC quantitative results are shown (ii). e, f GFP/Hep3B or GFP-CPAP/Hep3B cells transfected with si IL-6 R were treated with IL-6 for 24 h and used in the cell migration assay ( e ) and HUVEC migration assay ( f ). Control siRNA (siCtrl) was used as a negative control. g GFP/Hep3B or GFP-CPAP/Hep3B stable cells were treated with 10 μM Sorafenib as indicated and the cell viability was determined by CCK-8. Two-way ANOVA analysis, *** p < 0.01 (i). Cells treated with 10 μM Sorafenib for 48 h were collected to check the apoptotic status by immunoblotting analysis using anti-caspase 3 antibodies (ii) and evaluating the caspase3/7 activity (iii). Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Migration, Transwell Migration Assay, Tube Formation Assay, Matrigel Assay, Staining, Transfection, Cell Migration Assay, Control, Negative Control, CCK-8 Assay, Western Blot, Activity Assay

Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: PN1 reduces cell proliferative, migrative, and angiogenic abilities by downregulating the IL-6/STAT3 pathway. a, b Hep3B cells transfected with GFP, GFP-CPAP, or GFP-PN1 were treated with IL-6 for the STAT3-driven reporter assay ( a -i), Western blot analysis ( a -ii), and RT-qPCR ( b ) as described above. c Huh7 cells were co-transfected with HA-CPAP and GFP or GFP-PN1; after serum starvation, cells were treated with (+) IL-6 and then used to perform the co-immunoprecipitation (co-IP) assay. d Huh7 cells transfected with GFP or GFP-PN1 were serum starvation (column 1), treated with IL-6 (columns 2–4), and then used to perform the in situ PLA using anti-STAT3 and anti-CPAP (columns 1–3) or anti-GFP (column 4) antibodies. The red signals show the interaction between STAT3 and CPAP or GFP-PN1. e Hep3B cells stably expressing GFP, GFP-CPAP, or GFP-PN1 were subjected to BrdU incorporation (i), colony formation (ii). (iii) GFP/Hep3B or GFP-PN1/Hep3B cells were subcutaneously injected into NOD-SCID mice. Tumor size was measured, and tumor volume was calculated. The quantitative results of the tumor weights are shown. Two-way ANOVA analysis, ** p < 0.01. f After IL-6 treatment, GFP/Hep3B, GFP-CPAP/Hep3B, or GFP-PN1/Hep3B cells were collected for HUVEC migration (i) and Matrigel plug (ii) assays as described above. The Matrigel plugs were collected to perform H&E staining and an IHC analysis using the anti-CD31 antibody. Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Transfection, Reporter Assay, Western Blot, Quantitative RT-PCR, Co-Immunoprecipitation Assay, In Situ, Stable Transfection, Expressing, BrdU Incorporation Assay, Injection, Migration, Staining

Journal: Cell Death and Differentiation

Article Title: CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity

doi: 10.1038/s41418-019-0413-7

Figure Lengend Snippet: Several target genes of the IL-6/STAT3 pathway mediate CPAP-enhanced metastasis. a Expression of IL-8 mRNA was determined in Hep3B cells cotransfected with siCtrl or si IL-6R and GFP or GFP-CPAP. b GFP/Hep3B or GFP-CPAP/Hep3B stable cells (Hep3BST) were transfected with si IL-8 or siCtrl, treated with IL-6, and then used in the cell migration (upper) and HUVEC migration (lower) assays. Migrated cells were counted, and the quantitative results are shown. c The GFP- or GFP-CPAP-derived plug tumors (from Fig. , n = 5 for either group) were pulled together for RNA purification and determined the expression level of IL-8 mRNA by RT-qPCR. d Expression of IL-8 mRNA was determined in GFP-CPAP-derived liver tumors with ( n = 3) or without ( n = 2) lung metastasis (from Fig. ) by RT-qPCR. e CD44 mRNA level was determined by RT-qPCR in GFP/Hep3B and GFP-CPAP/Hep3B cells. f, g Expression of CD44 mRNA in HCC tumor tissues and paired nontumor (Non-T) tissues was determined by RT-qPCR ( f ). Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001. A total of 95 HCC paired specimens were collected for analysis. The expression of CD44 mRNA is positively correlated with CPAP mRNA in HCC tumor tissues ( g ). Pearson correlation coefficient, R = 0.3904, p < 0.0001. h The expression of CD44 mRNA (left, Pearson correlation coefficient, R = 0.403, p < 0.03.) and IL-8 mRNA (right, Pearson correlation coefficient, R = 0.4729, p < 0.0227.) is positively correlated with CPAP mRNA in HCC tumor tissues with vascular invasion. A total of 28 HCC specimens with vascular invasion were collected for analysis

Article Snippet: Cells were cotransfected with STAT3-driven, IL-6 promoter, IL-8 promoter, or HIF-1α promoter luciferase reporter constructs and GFP, GFP-CPAP, or GFP-PN1 by PolyJet TM (SL100688, SignaGen).

Techniques: Expressing, Transfection, Migration, Derivative Assay, Purification, Quantitative RT-PCR