Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) After 24-h culture of the RCC tumor cells, secreted STIP1 in the culture medium supernatant was detected, while the intracellular protein GAPDH was not detected in the culture medium supernatant indicating no leakage of intracellular components into the culture media. ( B – C ). STIP1 was detected in the purified cell surface protein, while no trace of HSP90 was identified, while HSP90 was detected in the total cell lysates (C). ( D ) Quantification of STIP1 protein in the culture medium supernatant as secreted STIP1 (left panel), and in the purified cell surface protein as outer cell surface STIP1 (right panel). Experiments were triplicated, and mean ± SD was presented. *p < 0.05, vs OS-RC-2; # p < 0.05, vs ACHN. In all panels, western blot images have been cropped to show the protein of interest, and all blots were performed under the same experimental conditions.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Purification, Western Blot

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) STIP1 protein was examined in 19 protein specimens from primary RCC tumors (P, n = 7) and bone metastatic samples (M, n = 12). Proteins were electrophoresed in two 10% SDS-PAGE gels, and were subsequently transferred to two PVDF membranes (10 and 9 specimens for gels 1and 2, respectively). ( B ) The intensity of STIP1 in each lane was normalized with the intensity of GAPDH. *p < 0.05. Experiments were duplicated, and western blot images shown have been cropped to show the protein of interest, and all blots were performed under the same experimental conditions. ( C ) Representative immunohistochemistry staining of STIP1 in primary RCC and bone metastasis tumors. Both intracellular and extracellular STIP1 immunoreactivity was examined as shown in the inset. Images were taken under 20× objective. Scale bar: 50 μm. ( D ) Correlation between the H scores of STIP1 in primary RCC and bone metastasis tumors of the 10 pairs of matched samples. R 2 = 0.6923.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: SDS Page, Western Blot, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) STIP1 mRNA expressed highly in the advanced stage RCC tumors. ( B ) STIP1 mRNA expressed highly in the high grades RCC tumors. ( C ) STIP1 mRNA expressed highly in the metastatic RCC tumors (M1+). Overexpression gene rank and P value were generated by the Oncomine algorithms. Fold change was log 2 based.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Over Expression, Generated

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Proliferation of OS-RC-BM5 cells under indicated treatment. ( B ) Cell cycle analysis of OS-RC-BM5 cells under indicated treatment. ( C ) shRNA knockdown of STIP1 in OS-RC-BM5 cells. ( D ) Representative images of Ki67 immunoreactivity in the bone metastasis tumors. Images were taken under 20× objective. Scale bar: 50 μm. ( E ) Correlation between the H scores of STIP1 and percentage of Ki67-positive cells in the same bone metastasis tumors ( n =). R 2 = 0.5868.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Cell Cycle Assay, shRNA, Knockdown

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Representative images of the Transwell membranes with tumor cells migrated to the counter side of the chamber. Note, cell migration was not affected when hrSTIP1 was added into the lower chamber (+ lower chamber). ( B ) Quantification of the migration analysis with three repeats. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1+anti-STIP1.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Migration

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) ALK2 expression was examined in the cell surface protein of indicated cell lines. ( B ) The Kaplan-Meier curve for overall survival of the TCGA RCC cohort ( n = 88) on the basis of ALK2 mRNA level. Patient was determined as ALK2-high or –low group when the ALK2 expression value was above or below the mean value in the dataset. The survival distributions were estimated by the Kaplan-Meier method, and the significance of differences between survival rates was ascertained using the log-rank test. ( C ) Expressions of p-SMAD1/5, SMAD1/5, ALK2 in the total cell lysate from indicated cell lines upon hrSTIP1 and/or LDN193189, or corresponding vehicle treatment. ( D ) Quantification of the western blot analysis (C) with three repeats. Expression of p-SMAD1/5 was normalized to the level of total SMAD1/5, and ALK2 expression was normalized to the level of GAPDH. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1. ( E ) Knockdown of endogenous SMAD1 and SMAD5 by siRNA decreased the stimulation of pSMAD1/5 protein levels by treatment with 500 nM hrSTIP1. F-G. Suppression of endogenous SMAD1/5 by siRNAs decreased the 96-h proliferation ( F ) and 24-h migration ( G ) of OS-RC-2-BM5 cells under treatment with 500 nM hrSTIP1. Results shown are the mean ± SE from three independent experiments. *p < 0.05, vs control siRNAs + hrSTIP1.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Expressing, Western Blot, Knockdown, Migration, Control

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) TRAP staining of osteoclasts induced in RAW264.7 cell line and primary bone marrow cells upon treatment with hrSTIP1 (500 nM). ( B ) Expressions of CTSK in the total cell lysate from indicated treatment in both RAW264.7 cell line and primary bone marrow cells. ( C ) Quantification of the western blot analysis (B) with three repeats. Expression of CTSK was normalized to the level of β-actin. *p < 0.05, vs vehicle; # p < 0.05, vs hrSTIP1. Western blots images shown are cropped to show the protein of interest, and all blots were performed under the same experimental conditions.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Staining, Western Blot, Expressing

Journal: Oncotarget

Article Title: Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma

doi: 10.18632/oncotarget.15222

Figure Lengend Snippet: ( A ) Expression of PrPc and ALK2 in the total cell lysate from hrSTIP1 treated-RAW264.7 cells. ( B ) TRAP staining of osteoclasts induced in RAW264.7 cell line upon treatment with hrSTIP1 (500 nM) or hrSTIP1+anti-PrPc (10 μg/ml) for 48 hours. ( C ) Expression of CTSK in the total cell lysate from indicated treatment in RAW264.7 cells. ( D ) Expression of p-ERK1/2 and total ERK1/2 in the total cell lysate from indicated treatment in RAW264.7 cells. ( E ) Quantification of the western blot analysis (A, C, D) with three repeats. Expression of PrPc and CTSK were normalized to the level of β-actin, and expression of pERK1/2 was normalized to total ERK1/2. *p < 0.05, vs control; * *p < 0.01, vs RANKL+hrATIP1. ( F ) Suppression the activation of endogenous ERK1/2 signaling by pre-treatment with 15 μM PD98059 for 2-h inhibited the hrSTIP1-induced CTSK protein expression in RAW264.7 cells. ( G ) Quantification of the western blot analysis of CTSK expression with three repeats. *p < 0.05, vs PD98059 -/hrSTIP1+.

Article Snippet: 500 nM recombinant human STIP1 (hrSTIP1) (Boster Biotechnology, Wuhan, China), 800 nM anti-STIP1 antibody (Abcam, Cambridge, USA), 10 μg/mL anti-PrPc antibody (Abcam, Cambridge, USA), and 20 μM PD98059 (Boster Biotechnology, Wuhan, China) were used to treat the tumor cells for certain assays.

Techniques: Expressing, Staining, Western Blot, Control, Activation Assay