Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: Deprivation of SPARC in conditioned media from SPARC-expressing Ishikawa cells still activates normal fibroblasts. (A) Schematic presentation of the production of conditioned media from SPARC-expressing Ishikawa cells immunodepleted of SPARC using anti-FLAG antibody. (B) Successful immunodepletion of SPARC in conditioned media from control and SPARC-expressing Ishikawa cells was confirmed using immunoblotting. (C) Protein expression of ACTA2 and CDH2 in NF cultured in the conditioned media with or without immunodepletion of SPARC. Intensity of the bands was quantified using Image J. Values of the protein-of-interest were corrected using the intensity of GAPDH bands. NF were also analyzed for cell proliferation on day 6 (D) and in vitro contraction (E) in the conditioned media. (F–H) NF were treated with the indicated concentration of recombinant SPARC. Protein expression of ACTA2 and CDH2 (F), cell number on day 6 (G) and in vitro contraction ability (H) were assessed. NF, normal fibroblasts; Ctrl, control; rh SPARC, recombinant human SPARC; CM, conditioned media; n.s., not significant; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in Supplementary Fig. 7 , and (B, C, F). The experiments were independently repeated three times using identical fibroblasts and representative data were shown

Article Snippet: The slides were immunolabeled with primary antibodies against SPARC (1:500, M125, TAKARA, Tokyo, Japan) and FN1 (1:300, ab2413, Abcam, Cambridge, UK) overnight at 4 °C after incubation with Antibody Diluent Solution (DAKO).

Techniques: Expressing, Western Blot, Cell Culture, In Vitro, Concentration Assay, Recombinant

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: FN1 secreted from SPARC-expressing Ishikawa cells activates normal fibroblasts. (A) Amount of FN1 secreted from SPARC-expressing Ishikawa cells was measured by ELISA. (B) Protein levels of ACTA2 and CDH2 in NF cultured on FN1-coated dishes in the presence of recombinant SPARC were analyzed using immunoblotting. (C) NF were also analyzed for cell proliferation on day 6 in the same culture condition as (B). (D) NF cultured in the same condition as (B) were moved to collagen gel for in vitro contraction assays. (E) Successful knockdown of FN1 by siRNA (siFN1) was confirmed using ELISA. (F) Protein levels of ACTA2 and CDH2 in NF cultured in conditioned media from SPARC-expressing Ishikawa cells with siFN1 or immunodepleted of SPARC were analyzed using immunoblotting. NF were also analyzed for cell proliferation on day 6 (G) and in vitro contraction (H) in the conditioned media. (B, F) Intensity of the bands was quantified using Image J. Values of the protein-of-interest were corrected using the intensity of ACTB bands. Ctrl, control; rh SPARC, recombinant human SPARC; siCtrl, control siRNA; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in Supplementary Fig. 10 and (B, F). The experiments were independently repeated three times using identical fibroblasts and representative data were shown

Article Snippet: The slides were immunolabeled with primary antibodies against SPARC (1:500, M125, TAKARA, Tokyo, Japan) and FN1 (1:300, ab2413, Abcam, Cambridge, UK) overnight at 4 °C after incubation with Antibody Diluent Solution (DAKO).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Western Blot, In Vitro

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: Characteristics of isolated fibroblasts from normal endometrium and endometrial cancer specimens. (A) Fibroblasts isolated from normal endometrium sample #1 (top, NF1) and those from endometrial cancer sample #1 (bottom, CAF1) were used for immunocytochemistry. Anti-pan-cytokeratin (pan-CK) and anti-VIM antibodies were used to visualize protein expression. (B) Isolated NF ( n = 6) and CAF lines ( n = 7) were used for immunoblotting to detect the expression of ACTA2, FAP, VIM, CDH2 and SPARC. GAPDH was used as an internal control. Full-length blot images are presented in . (C) Semi-quantification of the immunoblotting data in (B). Intensity of the bands was quantified using Image J ( https://imagej.nih.gov/ij/ ). Values of the protein-of-interest were corrected using the intensity of GAPDH bands. (D) ELISA analysis of SPARC secreted from the NF and CAF lines. (E) In vitro contraction analysis of the NF and CAF lines. The images show representative results of the experiments. The graphs on the right show quantification data of the results. The scale bars indicate 100 μm. 488, Alexa Fluor 488; pan-CK, pan-cytokeratin; NF, normal fibroblasts; CAF, cancer-associated fibroblasts; n.s., not significant; *, P < 0.05

Article Snippet: The slides were immunolabeled with primary antibodies against SPARC (1:500, M125, TAKARA, Tokyo, Japan) and FN1 (1:300, ab2413, Abcam, Cambridge, UK) overnight at 4 °C after incubation with Antibody Diluent Solution (DAKO).

Techniques: Isolation, Immunocytochemistry, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: Forced expression of SPARC in Ishikawa cells induces EMT and cell migration. Successful lentiviral transduction of SPARC in Ishikawa cells was confirmed using immunoblotting (A), RT-qPCR (B) and ELISA (C). ACTB was used as an internal control (A). (D) The expression of FN1, VIM, CDH2 and CDH1 protein and mRNA in SPARC-expressing Ishikawa cells was analyzed using immunoblotting (D) and RT-qPCR (E). (F) In vitro cell migration was analyzed using transwell chamber assays. Images on the left show representative results. Graphs on the right show quantification data of the results. The scale bars indicate 100 μm. Ctrl, control; n.s., not significant; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in and (A, D). The experiments were independently repeated three times and representative data were shown

Article Snippet: The slides were immunolabeled with primary antibodies against SPARC (1:500, M125, TAKARA, Tokyo, Japan) and FN1 (1:300, ab2413, Abcam, Cambridge, UK) overnight at 4 °C after incubation with Antibody Diluent Solution (DAKO).

Techniques: Expressing, Migration, Transduction, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: SPARC expression sites were adjacent to areas with FN1 expression in endometrial cancer tissues. Three cases of endometrial cancer, (A, B) endometrioid carcinoma grade 3 at stage IA (case #1), (C, D) endometrioid carcinoma grade 1 at stage IA (case #2), and (E–H) endometrioid carcinoma grade 1 at stage IA (case #3) were examined for SPARC (A, C, E, G) and FN1 (B, D, F, H) expression by immunohistochemistry. In the third case, a SPARC-positive area (E) and SPARC-negative area (G) were examined for FN1 expression (F, H). The scale bars indicate 100 μm

Article Snippet: The slides were immunolabeled with primary antibodies against SPARC (1:500, M125, TAKARA, Tokyo, Japan) and FN1 (1:300, ab2413, Abcam, Cambridge, UK) overnight at 4 °C after incubation with Antibody Diluent Solution (DAKO).

Techniques: Expressing, Immunohistochemistry

Journal: BMC Cancer

Article Title: Fibronectin mediates activation of stromal fibroblasts by SPARC in endometrial cancer cells

doi: 10.1186/s12885-021-07875-9

Figure Lengend Snippet: Induction of EMT and cell migration by SPARC is mediated by the AKT pathway. (A) Immunoblotting analysis of AKT and phosphorylated-AKT in SPARC-expressing Ishikawa cells. (B) Immunoblotting analysis of SPARC-expressing Ishikawa cells treated with indicated concentrations of the highly selective AKT inhibitor, MK2206. Antibodies against AKT, phosphorylated-AKT (p-AKT, Ser473), CDH2, VIM, FN1 and SPARC were used for immunoblotting. (A, B) Intensity of the bands was quantified using Image J. Values of the protein-of-interest were corrected using the intensity of GAPDH and ACTB bands, respectively. (C) In vitro cell migration of SPARC-expressing Ishikawa cells treated with indicated concentrations of MK2206. Numbers of migrated cells are shown in the top graph. Inhibition ratio of migrated cells compared with numbers of migrated cells at 0 nM is shown in the bottom graph. Ctrl, control; n.s., not significant; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in Supplementary Fig. 4 and (A, B). The experiments were independently repeated three times and representative data were shown

Article Snippet: The slides were immunolabeled with primary antibodies against SPARC (1:500, M125, TAKARA, Tokyo, Japan) and FN1 (1:300, ab2413, Abcam, Cambridge, UK) overnight at 4 °C after incubation with Antibody Diluent Solution (DAKO).

Techniques: Migration, Western Blot, Expressing, In Vitro, Inhibition

Journal: Scientific Reports

Article Title: Bone augmentation after ectopic implantation of a cell-free collagen-hydroxyapatite scaffold in the mouse

doi: 10.1038/srep36399

Figure Lengend Snippet: ( A – F ) Alizarin Red S staining showing the time-course of increasing calcium mineralization within the implanted scaffolds. It is evident that the calcium deposits are mainly localized in the bony layer. ( G – E ′) Panel of fluorescence microscope images showing the time-course of expression of crucial osteogenic and angiogenic markers by the host cells populating the Coll-MHA layer of the scaffolds. ( F′–J′ ) Examples of fluorescence microscope images showing the expression of osteogenic and angiongenic markers in the Coll layer: ALPL and osteocalcin have shown a low signal at all time-points (examples in F′,I′ ), whereas an increased expression of osteopontin ( B′ ) and a little increase of osteonectin ( H′ ) and CD31 ( J′ ) could be observed at week 16. Scale bars: 500 μm in A for ( A–F ); 100 μm in ( G ) for all fluorescence images.

Article Snippet: For staining, sections were blocked with 0.4% Triton-X100 and 4% bovine serum albumin and then incubated overnight at 4 °C with one of the following rabbit polyclonal antibodies: anti-osteopontin (1:250, Novus Biological), anti-osteocalcin (1:50, LSBio), anti-alkaline phosphatase (ALPL; 1:50, LSBio), anti-osteonectin (1:50, LSBio) and anti-CD31 (1:50, Novus Biological).

Techniques: Staining, Fluorescence, Microscopy, Expressing

Journal: Scientific Reports

Article Title: Bone augmentation after ectopic implantation of a cell-free collagen-hydroxyapatite scaffold in the mouse

doi: 10.1038/srep36399

Figure Lengend Snippet: Primer sequences.

Article Snippet: For staining, sections were blocked with 0.4% Triton-X100 and 4% bovine serum albumin and then incubated overnight at 4 °C with one of the following rabbit polyclonal antibodies: anti-osteopontin (1:250, Novus Biological), anti-osteocalcin (1:50, LSBio), anti-alkaline phosphatase (ALPL; 1:50, LSBio), anti-osteonectin (1:50, LSBio) and anti-CD31 (1:50, Novus Biological).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

doi: 10.1186/bcr1874

Figure Lengend Snippet: Alteration in the secreted proteome of MRJ(L) expressor of MDA-MB-435

Article Snippet: We used the following antibodies (dilutions given in parenthesis): anti-DNAJB6 antibody (1:5,000) from Abnova Corporation (Taipei City, Taiwan); anti-osteopontin (1:2,500) from Sigma (St. Louis, MO, USA); anti-KiSS1 antibody (1:500) from Abcam (Cambridge, MA, USA); anti-osteonectin (1:1,000) from Haematologic Technologies Inc. (Essex Junction, VT, USA); anti-nucleophosmin (1:500) from Abcam; anti-zinc α 2 -glycoprotein (1:500); anti-VGF nerve growth factor (1:500) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and β-actin (1:30,000) from BioRad.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

doi: 10.1186/bcr1874

Figure Lengend Snippet: Confirmation of changes in level of secreted proteins. (a) The changes in the secreted proteome of mammalian relative of DnaJ (MRJ) long isoform (MRJ [L]) expressor observed by mass spectrometry were confirmed by Western blot analysis. Serum-free medium from equal number of MDA-MB-435 parent (P), vector (V), MRJ(L) expressors (M) or MDA-MB-231 vector (V), and MRJ(L) expressors (M) was probed for presence of osteopontin (SPP1), osteonectin (SPARC), VGF nerve growth factor inducible (VGF), and zinc binding α 2 -glycoprotein 1 (AZGP1). Equal amount of total protein extract (20 μg) of the same cells was probed for the level of KiSS1 (melanoma metastasis suppressor); simultaneously, the expression of MRJ(L) was confirmed. β-Actin was used to verify equal loading of the lysate. Apparent molecular weights of the proteins detected are given in parenthesis: VGF (90 kDa), SPP1 (62 kDa), AZGP1 (47 kDa), SPARC (40 kDa), nucleophosmin (NPM1; 37 kDa), MRJ(L) (38 kDa), and KiSS1 (15 kDa). (b) Real-time quantitative RT-PCR analysis was performed using RNA from the MDA-MB-435-vector and MDA-MB-435-MRJ(L) expressor. The PCR primers and probes for KiSS1, NPM1, AZGP1, SPARC, SPP1, and VGF and endorse control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used. The reaction was performed for up to 40 cycles in triplicate. The gene expression Δ CT values of mRNAs from each sample were calculated by normalizing with internal control GAPDH. The fold change is represented as 2 -ΔΔCT . Real-time quantitative RT-PCR analysis was performed on the experimental mRNAs in triplicate and the experiment was repeated once from an independent passage.

Article Snippet: We used the following antibodies (dilutions given in parenthesis): anti-DNAJB6 antibody (1:5,000) from Abnova Corporation (Taipei City, Taiwan); anti-osteopontin (1:2,500) from Sigma (St. Louis, MO, USA); anti-KiSS1 antibody (1:500) from Abcam (Cambridge, MA, USA); anti-osteonectin (1:1,000) from Haematologic Technologies Inc. (Essex Junction, VT, USA); anti-nucleophosmin (1:500) from Abcam; anti-zinc α 2 -glycoprotein (1:500); anti-VGF nerve growth factor (1:500) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and β-actin (1:30,000) from BioRad.

Techniques: Mass Spectrometry, Western Blot, Plasmid Preparation, Binding Assay, Expressing, Quantitative RT-PCR