Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: Expression of THBS1 in paired tumor and adjacent normal tissues, and correlation analysis between FGFR2 and THBS1 expression. (A–D) Representative micrographs showing higher THBS1 staining and lower THBS1 staining in human gastric cancer tissue (A and B) and adjacent normal tissues (C and D) (magnification, ×200). (E) Quantification of THBS1 expression in tumor tissues compared with adjacent normal tissues. Data are presented as the mean ± SD ( ** P<0.01). (F) Correlation analysis of FGFR2 and THBS1 expression.

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Expressing, Staining

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: Correlation of THBS1 expression with clinicopathological factors.

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: FGF7 elevates the expression of THBS1 in vitro . (A–D) Cells were incubated with FGF7 (10 ng/ml) for the indicated times. The expression of FGFR2, p-FGFR, THBS1 was detected by western blotting (A). The relative expression of THBS1 mRNA was detected by qRT-PCR (C). FGFR2 and THBS1 expression (B) and relative expression of THBS1 mRNA (D) were detected after treatment with FGF7 (10 ng/ml for 48 h) in shRNA-2 and shRNA-NC transfected cells and SGC7901 cells. (E) THBS1 secreted by cells treated with or without FGF7 for 12, 24 and 48 h was determined by ELISA. (F) THBS1 in the condition media of shRNA-2 and shRNA-NC transfected cells treated with or without FGF7 was detected by ELISA. Data are presented as the mean ± SD of three independent experiments ( * P<0.05, ** P<0.01).

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Expressing, In Vitro, Incubation, Western Blot, Quantitative RT-PCR, shRNA, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: THBS1 is required for the effect of FGF7 on invasion and migration in gastric cancer cells. (A) Cancer cell lines were treated with or without FGF7, THBS1 in the condition media were detected by ELISA. Western blotting (B) and qRT-PCR (C) were used to evaluate the efficiency of THBS1 knockdown in SGC7901 cells transfected with siRNA-1, siRNA-2, siRNA-3 and siRNA-NC. (D and E) Cells transfected with siRNA-2 and siRNA-NC were treated with FGF7 (10 ng/ml) and subjected to invasion and migration assays. Data are presented as the mean ± SD of three independent experiments ( ** P<0.01).

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Knockdown, Transfection

Journal: International Journal of Oncology

Article Title: FGF7/FGFR2 signal promotes invasion and migration in human gastric cancer through upregulation of thrombospondin-1

doi: 10.3892/ijo.2017.3927

Figure Lengend Snippet: FGF7 regulates THBS1 through PI3K/Akt/mTOR pathway in vitro . (A and B) Cells were pretreated with FGF7 (10 ng/ml for 48 h), and THBS1 was assessed by performing western blotting after treated with indicated concentration of LY294002, U0126, SB203580, SP600125 (A) and RAD001 (B) at different time-points. (C) Relative expression of THBS1 mRNA in cells treated with RAD001 (pretreated with FGF7) was assessed by qRT-PCR. Data are presented as the mean ± SD of three independent experiments ( ** P<0.01).

Article Snippet: At 12, 24 and 48 h, conditioned media were collected and ELISA for human THBS1 was conducted according to the manufacturer's protocol (Origene).

Techniques: In Vitro, Western Blot, Concentration Assay, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 2. Thrombospondin-1 (THBS1) is necessary and sufficient for the morphological ‘transformation’ of wildtype (WT) organoids. (A) Quantification of the percentage of WT cystic organoids in T-cM upon neutralisation with blocking antibodies against ceruloplasmin (CP), connective tissue growth factor (CTGF), hepatoma-derived growth factor (HDGF), galectin-3 (LGALS3), galectin-3 binding protein (LGALS3BP), thrombospondin-1 (THBS1), and transthyretin (TTR) (5 µg/ml). (B) Quantification of the percentage of WT cystic organoids in T-cM upon neutralisation with three different blocking

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: Transformation Assay, Blocking Assay, Derivative Assay, Binding Assay

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 3. Thrombospondin-1 (THBS1) is essential for the growth of tumoroids. (A–D) Representative bright-field images of wildtype (WT) organoids (A, B) or tumoroids (C, D) incubated with IgG1 isotype control antibodies (A, C) or anti-THBS1 A6.1-neutralising antibody (B, D) (10 µg/ml). (E, F) Representative images of tumoroids infected with a lentivirus CRISPR-GFP without sgRNA (control in E) or with an sgRNA targeting Thbs1 (Thbs1- KO in F) 48 hr after replacement of single-cell seeding medium (ENRC) by tumoroid medium (EN). (G, H) Quantification of the number of tumoroids

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: Incubation, Control, Infection, CRISPR

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 5. Thbs1 is expressed by Lgr5+ cancer stem cells in vivo and induces YAP activation in neighbouring epithelial cells. (A, C) Representative section of Apc mutant intestinal tumours analysed by single-molecule fluorescence in situ hybridisation (smFISH) for Thbs1 (pThbs1, red dots) and Lgr5 (pLgr5, green dots in A) or the YAP target Sca1 (pSca1, green dots in C). Examples of segmented and processed region of interest (ROI) that were automatically counted as co-localisation (Thbs1+/Lgr5+ in A or Thbs1+/Sca1+ cells in C outlined in yellow and indicated by yellow arrows) or single-probe expression (outlined in red or green and indicated by arrows of the corresponding colour) are shown. E-cadherin demarcates epithelial cells in white and DAPI labels nuclei in blue in (A) and (C). (B, D) Quantification of the frequency of tumour regions expressing exclusively one probe or co-expressing two probes (yellow): Thbs1 only in red or Lgr5 only in green (B); Thbs1 only in red or Sca1 only in green (D). The observed frequencies of co-localisation (yellow in B) or mutual exclusion (yellow in D) are statistically significant compared to the calculated probability of random co- expression (blue columns) (n = 22 sections from two tumours in B and n = 51 sections from five tumours in D). (E) Correlation of the number of RNA molecules (dots/mm²) detected by single-molecule RNA fluorescence in situ hybridisation (smRNA FISH) for the YAP target CTGF and Thbs1 in mouse intestinal tumours. Red dots indicate large tumours (≥ 8 mm), orange dots small tumours (<8 mm). Dashed lines indicate 95% confidence intervals. (F, G) Representative sections of tumours derived from VillinCreERT2;Apcflox/+ (Apc+/- in F) or VillinCreERT2;Apcflox/flox (Apc-/- in G) immunostained for YAP1 (in red)

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: In Vivo, Activation Assay, Mutagenesis, Fluorescence, In Situ, Hybridization, Expressing, Derivative Assay

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/elife.76541

Figure Lengend Snippet: Figure 6. The THBS1-YAP pathway operates in human low-grade adenomas. (A) Correlation matrix between the expression levels of THBS1 and the YAP targets CTGF, CYR61, and LGR5 in human colon tumours from the TCGA colon cancer bulk datasets. R indicates Spearman’s coefficient. (B– E) Representative sections of low-grade human adenomas (B, D) or advanced human carcinomas (C, E) processed by single-molecule RNA fluorescence in situ hybridisation (smRNA FISH) for Thbs1 (pThbs1, red dots) and Lgr5 (pLgr5, green dots in B, C) or immunostained with anti-YAP1 antibodies (D, E). White arrows highlight tumour cells presenting high nuclear YAP in (D, E). n = 5 human low-grade adenomas in (B, D) and n = 5 advanced human adenocarcinomas in (C, E). (F) Graphical summary of paracrine interactions between wildtype (WT) organoids and tumoroids along the THBS1-YAP axis. Mutant tumoroids ‘corrupt’ genetically WT organoids by secreting THBS-1 (orange arrows). This results in YAP1 nuclear translocation (black nuclei in organoids or tumoroids) and ectopic proliferation as well as cystic morphology in a subset of organoids.

Article Snippet: LentiThbs1Tg is a lentiORF- expressing mouse Thbs1 (NM_011580) –myc- DKK (Origene# MR211744L3V). pMD2.G was a gift from Didier Trono (Addgene plasmid# 12259; http:// n2t.net/addgene: 12259; RRID:Addgene_12259). psPAX2 was a gift from Didier Trono (Addgene plasmid# 12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260). sgRNA cloning Both lentivirus backbones used for the knockout experiments harboured the GeCKO cloning adaptors (Shalem et al., 2014; Sanjana et al., 2014).

Techniques: Expressing, Fluorescence, In Situ, Hybridization, Mutagenesis, Translocation Assay