Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Immunofluorescence of SPARC and immunohistochemistry of TNC and S100A4 in mouse uteri on D4 (n=5), D4.5 (n=5), D5 (n=5), and D5.5 (n=5) of pregnancy. LE, luminal epithelium; St, stroma; * Embryo. Scale bar, 50 μm. ( B ) Western blot analysis of α-SMA, SPARC, TNC protein level under in vitro decidualization (EP) for 24 hr. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 1—source data 1. Raw data of all western blots from . Figure 1—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Immunofluorescence, Immunohistochemistry, Western Blot, In Vitro

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Western blot analysis on the effects of TNC on decidualization markers (BMP2, WNT4, E2F8 and CYCLIN D3) after stromal cells were treatment with TNC for 72 hr. ( B ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with TNC for 72 hr. ( C ) Western blot analysisof the effects of S100A4 on decidualization markers after stromal cells were treated with S100A4 for 72 hr. ( D ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with S100A4 for 72 hr. ( E ) Western blot analysis on the effects after stromal cells were treated with SPARC for 72 hr. ( F ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated SPARC for 72 hr. ( G ) Western blot analysis on ACTIVIN A protein levels in mouse uteri on D4, D4.5, PD4, and PD4.5, respectively. ( H ) Western blot analysis on the effects of ACTIVIN A on decidualization markers after stromal cells were treated with ACTIVIN A for 72 hr. ( I ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with ACTIVIN A for 72 hr. ( J ) Western blot analysis on the effects of ACTIVIN A on decidualization markers after stromal cells were treated with ACTIVIN A for 48 hr under in vitro decidualization. EP, 17β-estradiol+progesterone. All data were is presented as means ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. CYC D3: CYCLIN D3; ACT-A: ACTIVIN A. Figure 2—source data 1. Raw data of all western blots from . Figure 2—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Western Blot, In Vitro

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) AA concentration in uterine luminal fluid flushed on D3 (n=20 mice), D4 (n=20 mice), and D4.5 (n=20 mice) of pregnancy. ( B ) P-CPLA2α immunofluorescence in mouse uteri on D4 (n=6) and D4.5 (n=6). * Embryo. Scale bar = 50 μm. ( C ) P-CPLA2α immunofluorescence in mouse uteri at implantation sites and inter-implantation sites on D5 (n=6 mice). * Embryo. NI, inter-implantation site; IS, implantation site. Scale bar = 50 μm. ( D ) p-CPLA 2α immunofluorescence of in mouse uteri 12 and 24 h after delayed implantation was activated by estrogen treatment, respectively (n=4 mice). * Embryo. Scale bar = 0 μm. ( E ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels in mouse uteri on D4, D4.5 PD4 and PD4.5 (n=4 mice), respectively. ( F ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels in mouse uteri 12 and 24 hr after delayed implantation was activated by estrogen treatment (n=4 mice). ( G ) Immunostaining of TNC and S100A4 in mouse uteri 12, 24, 36, and 48 hr after delayed implantation was activated by estrogen treatment (n=4 mice). * Embryo. ( H ) Western blot analysis α-SMA, TNC, and SPARC protein levels in mouse uteri on D4 and PD4 (n=4 mice). *, p<0.05; **, p<0.01; ***, p<0.001. Figure 5—source data 1. Raw data of all western blots from . Figure 5—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Concentration Assay, Immunofluorescence, Western Blot, Immunostaining

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet:

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Software

Journal: PLoS ONE

Article Title: Analytical Performance of ELISA Assays in Urine: One More Bottleneck towards Biomarker Validation and Clinical Implementation

doi: 10.1371/journal.pone.0149471

Figure Lengend Snippet: Summary of analytical performance.

Article Snippet: SPARC (R&D Systems, DSP00) and PR3 (Cusabio Biotech Co. LTD, E13058h) results are presented as examples of successful or poor analytical validation performance respectively (Figs – , Tables and ), and detailed experimental data for each kit can be found in the supplementary information section.

Techniques:

Journal: PLoS ONE

Article Title: Analytical Performance of ELISA Assays in Urine: One More Bottleneck towards Biomarker Validation and Clinical Implementation

doi: 10.1371/journal.pone.0149471

Figure Lengend Snippet: Standard curve validation of A) SPARC (R 2 = 0.999) and B) PR3 (R 2 = 0.996).

Article Snippet: SPARC (R&D Systems, DSP00) and PR3 (Cusabio Biotech Co. LTD, E13058h) results are presented as examples of successful or poor analytical validation performance respectively (Figs – , Tables and ), and detailed experimental data for each kit can be found in the supplementary information section.

Techniques: Biomarker Discovery

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. Genes and assays included in the low-density array for the study of the expression of angiogenic factors by quantitative RT-PCR.

Article Snippet: 61 , SPARC , Hs00277762_m1 , 122 , 182120.

Techniques: Expressing, Amplification

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. 2- DDCt values corresponding to the Nu335 series.

Article Snippet: 61 , SPARC , Hs00277762_m1 , 122 , 182120.

Techniques:

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. 2- DDCt values corresponding to the Nu407 series.

Article Snippet: 61 , SPARC , Hs00277762_m1 , 122 , 182120.

Techniques:

Journal: Cell reports

Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms

doi: 10.1016/j.celrep.2024.114527

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SPARC , Cell Signaling Technology , Cat# 5420S; RRID: AB_10692794.

Techniques: Recombinant, Membrane, Cell Culture, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Protein Extraction, Extraction, Bicinchoninic Acid Protein Assay, In Situ, Blocking Assay, Migration, shRNA, Control, Software, Pyromark Assay, Western Blot, Simple Western

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) Experimental set-up of the MDA-MB-231 cell-HMF co-culture system. MDA-MB-231 TNBC cells and HMFs were co-cultured in serum-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37°C for 24h. The 24h-conditioned medium was then concentrated to 0.2 mg/ml, and incubated in cleavage buffer with or without pepstatin A (Pepst.) (12.5 µM) at pH 5.5 and 37° for 60 min. A representative image of an MDA-MB-231/HMF co-culture is shown in the right panel (x 200). (B) Box and whisker plot of the normalized ratios of the N-terminal peptides identified by TAILS in the MDA-MB-231/HMF co-culture secretome. 3091 peptides quantified at t = 60 min of incubation with cleavage buffer were used to generate the graph. The without/with pepstatin A ratios corresponding to SPARC peptides are highlighted in red; whiskers correspond to the 2.5 th and 97.5 th percentiles. (C) Validation of SPARC cleavage in the MDA-MB-231/HMF co-culture secretome. Secretome samples (2 µg) from the MDA-MB-231/HMF co-culture incubated in cleavage buffer with or without pepstatin A (Pepst.) (12.5 µM) at pH 5.5 and at 37° for 60 min were separated on 13.5% SDS-PAGE followed by immunoblotting with anti-SPARC antibody (clone AON-5031).

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Co-Culture Assay, Cell Culture, Incubation, Whisker Assay, Biomarker Discovery, SDS Page, Western Blot

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) Time-course of cath-D-induced SPARC cleavage. Recombinant human FL SPARC was incubated with recombinant human auto-activated pseudo-cath-D (51-kDa) in cleavage buffer at pH 5.9 with or without pepstatin A (Pepst.) at 37°C for the indicated times. SPARC cleavage was analyzed by 13.5% SDS-PAGE and immunoblotting with an anti-SPARC antibody (clone AON-5031). (B) pH dependence of cath-D-induced SPARC cleavage. Recombinant human FL SPARC was incubated with recombinant human auto-activated pseudo-cath-D (51-kDa) in cleavage buffer with or without pepstatin A (Pepst.) at the indicated pH at 37°C overnight. SPARC cleavage was analyzed as in ( A ). (C) Detection of the cath-D-induced SPARC fragments by silver staining. Recombinant SPARC was incubated with recombinant auto-activated pseudo-cath-D (51-kDa) or fully-mature cath-D (34+14-kDa) at pH 5.9 for the indicated times. SPARC cleavage was analyzed by 17% SDS-PAGE and silver staining. (D) Cath-D cleavage sites in SPARC extracellular Ca 2+ binding domain. The entire C-terminal extracellular Ca 2+ binding domain of human SPARC (amino acids 154-303) is shown. SPARC cleaved peptides generated in the extracellular Ca 2+ binding domain by auto-activated pseudo-cath-D (51-kDa) and fully-mature (34+14-kDa) cath-D at pH 5.9 were resolved by iTRAQ-ATOMS. Arrows, cleavage sites. (E) Schematic representation of the SPARC fragments generated by cath-D according to (C) and (D).

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Silver Staining, Binding Assay, Generated, Multiplex sample analysis

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) SPARC and cath-D in a TNBC TMA. Representative images of SPARC and cath-D expression analyzed in a TNBC TMA (n=147 samples) using anti-SPARC (clone AON-5031) and anti-cath-D (clone C-5) monoclonal antibodies. Scale bars, 50 µm (top panels) and 20 µm (bottom panels; higher magnifications of the boxed regions). (B) SPARC and cath-D expression and secretion in TNBC cell lines and breast fibroblasts. Whole cell extracts (10 µg proteins) (left panel) and 24-hour conditioned media in the absence of FCS (40 µl) (right panel) were separated by 13.5% SDS-PAGE and analyzed by immunoblotting with anti-cath-D antibodies for cellular (clone 49, #610801) and secreted cath-D (H-75) detection, respectively, and anti-SPARC (clone AON-5031) antibody. β-actin, loading control. (C) Co-localization of SPARC and cath-D in TNBC PDX. PDX 1995 tumor sections were co-incubated with an anti SPARC polyclonal antibody (15274-1-AP) (red; panel a) and an anti-cath-D monoclonal antibody (C-5) (green; panel b). Nuclei were stained with 0.5 µg/ml Hoechst 33342 (blue). Panel c (left): SPARC, cath-D and Hoechst 33342 merge. Panel c (middle and right): higher magnification of the boxed areas (right panels: Z projections of 3 x 0.25 µm slices). Arrows indicate SPARC and cath-D co-localization. Scale bar, 10 µm.

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Bioprocessing, SDS Page, Western Blot, Control, Incubation, Staining

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) Time-course of SPARC degradation in MDA-MB-231/HMF conditioned medium. MDA-MB-231 TNBC cells and HMFs were co-cultured in serum-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37°C for 24h. The 24h conditioned medium from co-cultured MDA-MB-231/HMF was incubated at 37°C in cleavage buffer with or without pepstatin A (Pepst.) at pH 5.5 for the indicated times. SPARC cleavage in conditioned medium was analyzed by 13.5% SDS-PAGE and immunoblotting with an anti-SPARC antibody (15274-1-AP). O/N, overnight. (B) Influence of the milieu acidity on SPARC degradation in MDA-MB-231/HMF conditioned medium. MDA-MB-231 TNBC cells and HMFs were co-cultured as in ( A ). The 24h conditioned medium was incubated at 37°C in cleavage buffer with or without pepstatin A at the indicated pH overnight. SPARC cleavage was analyzed as described in ( A ). (C and D) Time-course of SPARC cleavage in in TNBC cell conditioned medium. HS578T TNBC cells (C) and SUM159 TNBC cells (D) were cultured in serum-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37°C for 24h.The 24h conditioned medium was incubated at 37°C in cleavage buffer with or without pepstatin A at pH 5.5 for the indicated times. SPARC cleavage was analyzed as described in ( A ). (E) SPARC cleavage by cath-D secreted by MDA-MB-231 cells. MDA-MB-231 cells were transfected with Luc or cath-D siRNAs. At 48h post-transfection, siRNA-transfected MDA-MB-231 cells were co-cultured with HMFs as described in ( A ). Then, the 24h conditioned media from co-cultured siRNA-transfected MDA-MB-231/HMF were incubated at 37°C in cleavage buffer with or without pepstatin A at pH 5.5 for 120 min. Cath-D secretion by siRNA-transfected MDA-MB-231 cells was analyzed with an anti-cath-D antibody (H-75). SPARC cleavage was analyzed as described in ( A ). (F) SPARC cleavage by cath-D secreted by inducible Ctsd knock-out MMTV-PyMT mammary tumor cells. Inducible Ctsd knock-out MMTV-PyMT breast cancer cells were incubated or not with 4-hydroxytamoxifen (OH-Tam; 3 µM) for 4 days to induce Ctsd knock-out. Then, cells were cultured in FCS-free DMEM without sodium bicarbonate and phenol red and buffered with 50 mM HEPES [pH 7.5] at 37°C for 24h. The 24h-conditioned medium was incubated at 37°C in cleavage buffer with or without pepstatin A at pH 5.5 for 120 min or O/N. Cath-D secretion was analyzed with an anti-cath-D antibody (AF1029). SPARC cleavage was analyzed as described in ( A ).

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Incubation, SDS Page, Western Blot, Transfection, Knock-Out

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) SPARC expression in mammary tumors from MMTV-PyMT Ctsd knock-out mice . Left panel, whole cytosols (40 µg) of mammary tumors from MMTV-PyMT Ctsd+/+ (N°1-3) and MMTV-PyMT Ctsd-/- ( Ctsd knock-down in mammary glands) (N°4-6) mice were analyzed by 13.5% SDS-PAGE and immunoblotting with anti-mouse cath-D (clone 49, #610801) and anti-SPARC (AON-5031) antibodies. β-actin, loading control. Right panel, total RNA was extracted from mammary tumors from MMTV-PyMT Ctsd+/+ (N°1-3) and MMTV-PyMT Ctsd-/- (N°4-6) mice, and Sparc expression was analyzed by RT-qPCR. P =0.1 (Student’s t -test). (B and C) SPARC expression in TNBC PDXs and TNBC biopsies . Top panels, cath-D expression was determined in whole cytosols from two TNBC PDXs (B) and two TNBC biopsies (C) by sandwich ELISA with the immobilized anti-human cath-D D7E3 antibody and the anti-human cath-D M1G8 antibody coupled to HRP. Bottom panels, whole cytosols (40 µg) from these PDXs (B) and TNBC biopsies (C) were analyzed by 13.5% SDS-PAGE and immunoblotting with anti-cath-D (H-75) and anti-SPARC (15274-1-AP) antibodies. β-actin (B) and tubulin (C) , loading controls.

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Knock-Out, Knockdown, SDS Page, Western Blot, Control, Quantitative RT-PCR, Sandwich ELISA

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) Cell adhesion. MDA-MB-231 cells were let to adhere for 30 min on a fibronectin matrix in the absence or presence of recombinant FL SPARC (SPARC), or recombinant cath-D-induced cleaved SPARC fragments (cleaved SPARC) at the final concentration of 240 nM. Left panels, representative images of adherent cells. Right panel, adherent cells were stained with crystal violet, and adhesion was quantified at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n=3); ***, p<0.001, ANOVA and Bonferroni’s post hoc test. Similar results were obtained in four independent experiments. (B) Cell migration. MDA-MB-231 cells were let to migrate for 16h on a fibronectin matrix in the absence or presence of recombinant FL SPARC, or cleaved SPARC at a final concentration of 240 nM. Left panels, representative images of migrating cells. Right panel, migrating cells were quantified by MTT staining and absorbance was read at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n=3); *, p<0.05; **, p<0.01; ***, p<0.001, ANOVA and Bonferroni’s post hoc test. Similar results were obtained in three independent experiments. (C) Endothelial transmigration. MDA-MB-231 cells were let to transmigrate for 16h through a HUVEC monolayer in the absence or presence of recombinant FL SPARC, or cleaved SPARC at a final concentration of 240 nM. Left panels, representative images of transmigrating cells. Right panel, transmigrating cells were stained with MTT and quantified at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n=3); *, p<0.05, **, p<0.01, ***, p<0.001, ANOVA and Bonferroni’s post hoc test. Similar results were obtained in two independent experiments. (D) Cell invasion. MDA-MB-231 cells were let to invade for 16h on a Matrigel matrix in the absence or presence of recombinant FL SPARC, or cleaved SPARC at a final concentration of 240 nM. Left panels, representative images of invading cells. Right panel, invading cells were stained with MTT and quantified at 570 nm. CTRL, PBS in cleavage buffer. Data are the mean ± SD (n=3); ***, p<0.001, ANOVA and Bonferroni’s post hoc test. Similar results were obtained in three independent experiments.

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Recombinant, Concentration Assay, Staining, Migration, Transmigration Assay

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) Production of Myc/His-tagged FL SPARC, and Myc/His-tagged 34-, 27-, 16-, 9-, and 6-kDa SPARC fragments. Left panel, equimolar concentrations (240 nM each) of purified Myc/His-tagged FL SPARC and SPARC fragments were analyzed by SDS-PAGE (17%) and immunoblotting with an anti-Myc antibody (clone 9B11). Right panel, schematic representation of the purified Myc/His-tagged SPARC fragments. AC, acidic domain; FL, follistatin-like domain; EC, Ca 2+ -extracellular binding domain. (B) Cell adhesion. MDA-MB-231 cells were let to adhere for 30 min on a fibronectin matrix in the presence of purified Myc/His-tagged FL SPARC, or individual Myc/His-tagged SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa) at an equimolar final concentration (240 nM each). Upper panels, representative images of adherent cells stained with crystal violet after incubation with the indicated SPARC variants. Lower panel, cell adhesion was quantified as described in A and expressed as percentage relative to the value in control (SPARC-immunodepleted control for each SPARC fragment). Data are the mean ± SD of three independent experiments; ***, p<0.001, ANOVA and Bonferroni’s post hoc test.

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Purification, SDS Page, Western Blot, Binding Assay, Concentration Assay, Staining, Incubation, Control

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: (A) Cell adhesion. MDA-MB-231 cells were let to adhere for 30 min on a fibronectin matrix in the presence of recombinant FL SPARC, recombinant cleaved SPARC fragments (cleaved SPARC), or purified 9-kDa C-terminal SPARC fragment at a final concentration of 240 nM. Left panels, representative images of adherent cells stained with crystal violet. Right panel, adhesion was quantified as described in A. Data are the mean ± SD (n=3); ns, not significant; ***, p<0.001, ANOVA and Bonferroni’s post hoc test. CTRL, PBS in cleavage buffer and SPARC-immunodepleted supernatant from the 9-kDa SPARC fragment purification. Similar results were obtained in three independent experiments. (B) Cell migration. MDA-MB-231 cells were let to migrate for 16h on a fibronectin matrix in the absence or presence of FL SPARC, cleaved SPARC fragments, or the 9-kDa C-terminal SPARC fragment at a final concentration of 240 nM. Left panels, representative images of migrating cells stained with crystal violet. Right panel, migration was quantified as described in B. Data are the mean ± SD (n=3); ***, p<0.001, ANOVA and Bonferroni’s post hoc test. CTRL, PBS in cleavage buffer and SPARC immunodepleted supernatant from the 9-kDa SPARC fragment purification. Similar results were obtained in two independent experiments. (C) Endothelial transmigration. MDA-MB-231 cells were let to transmigrate for 16h through a HUVEC monolayer in the absence or presence of FL SPARC, cleaved SPARC fragments, or the 9-kDa C-terminal SPARC fragment at a final concentration of 240 nM. Left panels, representative images of transmigrating cells. Right panel, transmigrating cells were stained with MTT and quantified by absorbance at 570 nm. Data are the mean ± SD (n=3); *, p<0.05, **, p<0.01, ***, p<0.001, ANOVA and Bonferroni’s post hoc test. CTRL, PBS in cleavage buffer and SPARC-immunodepleted supernatant from the 9-kDa SPARC fragment purification. Similar results were obtained in two independent experiments. (D) Cell invasion. MDA-MB-231 cells were let to invade for 16h on a Matrigel matrix in the absence or presence of FL SPARC, cleaved SPARC fragments, or the 9-kDa C-terminal SPARC fragment at a final concentration of 240 nM. Left panels, representative images of invading cells stained with crystal violet. Right panel, invading cells were quantified by absorbance at 570 nm. Data are the mean ± SD (n=3); *, p<0.05, **, p<0.01, ***, p<0.001, ANOVA and Bonferroni’s post hoc test. CTRL, PBS in cleavage buffer and SPARC immunodepleted supernatant from the 9-kDa SPARC fragment purification. Similar results were obtained in two independent experiments.

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Recombinant, Purification, Concentration Assay, Staining, Migration, Transmigration Assay

Journal: bioRxiv

Article Title: A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

doi: 10.1101/2020.10.22.350082

Figure Lengend Snippet: TNBC-secreted cath-D triggers limited proteolysis of SPARC at the acidic pH of the tumor microenvironment. Among the SPARC fragments cleaved by cath-D, the 9-kDa C-terminal SPARC fragment inhibits TNBC cell adhesion and spreading. This might lead to an intermediate adhesive state, and stimulate TNBC cell migration, endothelial transmigration and invasion.

Article Snippet: The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Adhesive, Migration, Transmigration Assay