Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: Primers used for qPCR analyses

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: Expression of ANGPTL4 is up-regulated during chondrogenic differentiation of MSCs. A, MSCs were differentiated into chondrocytes by micromass culture in the presence of BMP-2 or TGF-β-3 for the indicated days. Transcriptomic analysis was performed using DNA microarrays. Data obtained with the two probe sets corresponding to ANGPTL4 variant 1 are shown (black and white bars) and correspond to the mean values of the three donors. B, MSCs were differentiated into the chondrogenic lineage by micromass culture in the presence of TGF-β-3, and expression of the two ANGPTL4 variants was analyzed by qPCR. C, MSCs were differentiated into the chondrogenic, adipogenic, or osteogenic lineage. Relative expression of ANGPTL4 was measured by qPCR. D, cumulative release of ANGPTL4 in culture supernatants of MSCs undergoing chondrogenic differentiation was determined by ELISA. *, p < 0.05 versus value at day 0.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Expressing, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: Culture conditions required for induction of ANGPTL4 expression. Monolayer cultures of MSCs were subjected to normoxic (21% O2) or hypoxic (2% O2) conditions in the absence or presence of TGF-β-3. A, relative ANGPTL4 mRNA levels were determined by qPCR. B, the amounts of ANGPTL4 in culture supernatants were measured by ELISA. C, cells were cultured in micromass in the absence or presence of TGF-β-3. Expression of ANGPTL4 was analyzed by qPCR. *, p < 0.05.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: Exogenous ANGPTL4 inhibits expression of cartilaginous matrix components by differentiating MSCs. Pellets of MSCs were cultured for 21 days in the presence or absence of TGF-β-3 or ANGPTL4 as indicated. A–D, relative mRNA levels of aggrecan (ACAN), COL2A1 (α-1 type II collagen, transcript variant 2), HAPLN1 (hyaluronan and proteoglycan link protein 1), and α-1 type X collagen (COL10A1) were measured by qPCR. Level of mRNA in TGF-β-3-treated cells was given the nominal value 100%. E, pellets were sectioned and analyzed by immunohistochemistry using aggrecan antibodies. Nonimmune IgG served as negative control to check for specific staining. Representative illustrations are shown. Scale bar, 300 μm. F, acid and pepsin-soluble collagens were quantified by colorimetry. G, the amount of DNA was determined by a fluorimetric assay. *, p < 0.05.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Expressing, Cell Culture, Variant Assay, Immunohistochemistry, Negative Control, Staining, Colorimetric Assay, Fluorimetry Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: Exogenous ANGPTL4 up-regulates expression of MMPs. Pellets of MSCs were cultured for 21 days in the presence or absence of TGF-β-3 or ANGPTL4 as indicated. A–C, relative mRNA levels of MMPs were determined by qPCR. Level of mRNA in TGF-β-3-treated cells was given the nominal value 100%. D, activity corresponding to activatable pro-MMP13 and endogenous active MMP13 (black bars) or endogenous active MMP13 only (white bars) was measured in culture supernatants using a fluorimetric assay. E and F, the amounts of MMP1 and MMP3 released in culture supernatants were quantified by ELISA. *, p < 0.05.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Expressing, Cell Culture, Activity Assay, Fluorimetry Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: Extinction of ANGPTL4 expression by RNA interference. MSCs were transfected with negative control siRNA (siCont) or ANGPTL4-targeted siRNA (siANGPTL4) and induced into chondrogenesis. A, expression of ANGPTL4 was measured by qPCR at various time points after induction of differentiation as indicated. Level of ANGPTL4 mRNA in control siRNA-transfected cells at day 2 (D2) of differentiation was given the nominal value 100%. B, culture supernatants at day 2 of differentiation were analyzed by Western blotting using an antibody to ANGPTL4. Full-length ANGPTL4 and the C-terminal fragment containing the fibrinogen-like domain (FLD) are indicated. C, concentration of ANGPTL4 in supernatants collected at the indicated days of differentiation was determined by ELISA. *, p < 0.05.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Expressing, Transfection, Negative Control, Control, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: ANGPTL4 siRNA knockdown increases expression of cartilaginous matrix components by differentiating MSCs. MSCs were transfected with negative control siRNA (siCont) or ANGPTL4-targeted siRNA (siANGPTL4) and induced into chondrogenesis in the absence or presence of recombinant ANGPTL4 as indicated. A, the volume of the micromasses was calculated by considering that these have the shape of ellipsoids. B–E, relative mRNA levels of aggrecan (ACAN), COL2A1 (α-1 type II collagen, transcript variant 2), HAPLN1 (hyaluronan and proteoglycan link protein 1), and α-1 type X collagen (COL10A1) were determined by qPCR and represented as percentage of maximum. F, expression of ANGPTL4 was measured by qPCR and represented as induction over value at day 0 (D0). G, pellets were analyzed by immunohistochemistry at days 14 and 21 with antibodies to aggrecan and type II collagen as indicated. Nonimmune IgG served as negative control to check for specific staining. Scale bar, 200 μm. H, acid and pepsin-soluble collagens were quantified by colorimetry. I, the amount of DNA was determined by a fluorimetric assay. *, p < 0.05.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Recombinant, Variant Assay, Immunohistochemistry, Staining, Colorimetric Assay, Fluorimetry Assay

Journal: The Journal of Biological Chemistry

Article Title: Involvement of Angiopoietin-like 4 in Matrix Remodeling during Chondrogenic Differentiation of Mesenchymal Stem Cells *

doi: 10.1074/jbc.M113.539825

Figure Lengend Snippet: ANGPTL4 siRNA knockdown inhibits expression of MMP13, MMP1, and MMP3 in MSCs undergoing chondrogenic differentiation. MSCs were transfected with negative control siRNA (siCont) or ANGPTL4-targeted siRNA (siANGPTL4) and induced into chondrogenesis in the absence or presence of recombinant ANGPTL4 as indicated. A–C, relative mRNA levels of MMP13, MMP1, and MMP3 were measured by qPCR and represented as percentage of maximum. D, MMP13 activity corresponding to activatable and endogenously active MMP13 was quantified in culture supernatants using a fluorimetric assay. E and F, the amounts of MMP1 and MMP3 released in culture supernatants were measured by ELISA. *, p < 0.05. D0, day 0.

Article Snippet: Recombinant ANGPTL4 partially rescued the effect of siANGPTL4 on micromass size, total soluble collagens, and on expression of aggrecan, COL2A1, HAPLN1, COL10A1, MMP1, and MMP13 ( , A–E and H , and A ).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Recombinant, Activity Assay, Fluorimetry Assay, Enzyme-linked Immunosorbent Assay