Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 gene expression is decreased during adipocyte differentiation. ( a ) The expression of WISP1 mRNA was measured in subcutaneous (SAT) and epididymal adipose tissues (VAT) harvested from 12 weeks old C57BL6 mice (n = 4–5 per group). ( b ) Preadipocytes isolated from VAT and SAT were induced to differentiate. Total RNA was isolated and the expression of WISP1 and ADIPOQ mRNA were measured by real-time PCR. The values indicate the changes for the indicated samples compared to VAT on Day 0. ( c ) 3T3-F442A cells were induced to differentiate and the gene expression of WISP1 , and ADIPOQ was measured by real-time PCR. The values indicate the changes for the indicated samples compared to day 0. ( d ) Secreted and intracellular protein levels of WISP1 were analyzed during adipocyte differentiation by ELISA. ( e ) Expression of secreted and intracellular protein levels of WISP1 and adiponectin were measured by Western Blotting. Adiponectin serves as a positive control for adipocyte differentiation while actin was used as a loading control. All values are representative of data from 3 independent experiments each performed in duplicate. Results are presented as means ± SEM *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Gene Expression, Expressing, Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Control

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: Stimulation of Wnt-signaling increases the expression of WISP1 and inhibits adipocyte differentiation. 3T3-F442A preadipocytes were exposed to MDI media supplemented with 25 mM Lithium Chloride (LiCl) or, as negative control, 25 mM Sodium Chloride (NaCl). ( a ) Gene expression of WISP1, PPARG , and ADIPOQ were measured by real-time PCR. The values indicate the changes in the indicated sample compared to day 0. ( b ) The secretion of WISP1 following activation of Wnt signaling was determined by ELISA. The concentration of WISP1 is indicated as the ratio of LiCl-treated compared to the corresponding NaCl-treated sample. ( c ) 3T3-F442A preadipocytes at two days post confluence were treated with or without TNF-α at the indicated concentrations. After 24 hours, total RNA was isolated and the expression of WISP1 and ADIPOQ was analyzed by real-time PCR. The values indicate the changes for the indicated sample compared to the vehicle. All values are representative of data from 3 independent experiments each performed in duplicate. Results are presented as means ± SEM *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Expressing, Negative Control, Gene Expression, Real-time Polymerase Chain Reaction, Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Isolation

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 inhibits adipogenesis. ( a ) 3T3-F442A preadipocytes were transfected or not with 2.5 µg of empty (vector) or WISP1-encoding vector (WISP1) in the presence of MDI media for 72 hours. Alternatively, cells were incubated for 72 hours in MDI media (control) in the presence of recombinant WISP1 (Rec WISP1) or conditioned media from L cells expressing (Wnt3a-CM) or not (CM) Wnt3a. The differentiation process was monitored by microscopic imaging after Oil Red O-staining. Subsequently, Oil Red O-stained areas were quantified by ImageJ analysis (right). ( b ) 3T3-F442A preadipocytes were transfected with 2.5 µg of empty vector or human WISP1-encoding vector. Three, 5 and 8 days after transfection, endogenous murine WISP1, PPARG, ADIPOQ, LPL, FABP4, CD36, DLK1 and CEBPD mRNA levels were quantified by real-time PCR. The values indicate the changes for the indicated sample compared to day 0. All the values are averages of data from 3 independent experiments performed in duplicate. Results are presented as means ± SEM *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Transfection, Plasmid Preparation, Incubation, Control, Recombinant, Expressing, Imaging, Staining, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 silencing induces adipogenic differentiation. 3T3-F442A preadipocytes were transfected for 48 hours with siRNA control or siWISP1 (30 pmoles). Then, the influence of WISP1 knockdown on ( a ) endogenous WISP1 mRNA levels and ( b ) secreted WISP1 protein levels in the culture media was determined by real-time PCR and ELISA. ( c ) The effect of WISP1 knockdown on lipid accumulation was monitored by microscopic imaging after Oil Red O-staining. mRNA levels of PPARG, ADIPOQ, LPL, FABP4, CD36, DLK1 and CEBPD were measured by real-time PCR. The values indicate the changes for the indicated sample compared to the siRNA-control. ( d ) 3T3-F442A preadipocytes were incubated in MDI media or alternatively transfected with si-WISP1 (30 pmoles) in the presence or the absence of conditioned media from L cells expressing Wnt3a (Wnt3a-CM) for 96 hours. The differentiation process was monitored by microscopic imaging after Oil Red O-staining. Red-stained areas were quantified by ImageJ analysis. All values are averages of data from 3 independent experiments each performed in duplicate. Results are presented as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Transfection, Control, Knockdown, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Imaging, Staining, Incubation, Expressing

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 decreases PPARγ transcriptional activity. ( a ) Increasing amounts of expression vector for WISP1 (0.2, 0.4, 0.8 µg) were transfected into HEK or MEF cells along with 0.1 µg of PPRE-TK-Luc and 0.02 µg of an RSV-β-galactosidase construct as an internal control, alone or in combinations with 0.1 µg of PPARγ expressing vector. ( b ) Increasing amounts of WISP1 (0.2, 0.4, 0.8 µg) were transfected into HEK and MEF cells along with 0.1 µg of UAS-TK-Luc and 0.02 µg of an RSV-β-galactosidase construct as an internal control, alone or in combination with 0.1 µg of an expression vector for the Gal4-PPARγ-LBD fusion protein. Five hours after transfection, cells were treated (black bars) or not (open bars) with 1 µM rosiglitazone for 24 hours and assayed for luciferase and β-galactosidase activities. The results represent the average of at least three independent experiments each done in triplicate. Results are presented as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Transfection, Construct, Control, Luciferase

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 interacts with and down-regulates PPARγ ( a ) MEF cells were transfected with increasing amounts of Flag-WISP1 expression vector (0.65, 1.30 and 2.60 µg) in combination with 1 µg of PPARγ expression vector. After 4 hours, cells were treated with 1 µM of rosiglitazone and harvested 24 hours later. Lysates were subjected to immunoblotting with anti-PPARγ, anti-Flag, anti-active β-catenin or anti-β-catenin antibodies. Immunoblot signals were quantified by densitometry, and normalized with β-actin. Data were expressed as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005. Data are representative of 3 independent experiments ( b ) MEF cells were transfected with 2 µg of Flag-WISP1 expression vector in combination with 2 µg of PPARγ expression vector. After 24 hours, cells were treated with 10 µM MG132 for 6 hours and harvested. Lysates were subjected to immunoblotting with anti-PPARγ and anti-Flag antibodies. Immunoblot signals were quantified by densitometry, and normalized with β-actin. Data were expressed as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005. Data are representative of 3 independent experiments ( c ) MEF cells were transfected with 2 µg of Flag-WISP1expression vector in combination with 2 µg of PPARγ expression vector for 24 hours. Lysates were subjected to anti-Flag or anti-PPARγ immunoprecipitation (IP) and followed by immunoblotting (IB) with anti-PPARγ or anti-Flag antibodies respectively. Protein expressions were determined by direct immunoblotting (Input). Two independent experiments were performed. ( d ) MEF cells lysates were subjected to anti-WISP1 or anti-PPARγ immunoprecipitation (IP) and followed by immunoblotting (IB) with anti-PPARγ or anti-WISP1 antibodies, respectively. Immunoprecipitations with IgG were used as controls. Protein expressions were determined by direct immunoblotting (Input). Two independent experiments were performed.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 expression is up-regulated in adipose tissues from obese mice.( a ) WISP1 and A DIPOQ mRNA levels were quantified by RT-qPCR in visceral (epididymal) adipose tissue (VAT) from mice fed with a standard diet (SD) and a high fat diet (HFD) for 12 weeks (n = 7 animals per group). ( b ) WISP1 and ADIPOQ mRNA levels were quantified in VAT from ob/ob mice (n = 7 animals per group). Results are presented as means ± SEM *p < 0.05; ***p < 0.005 versus SD fed mice in panel (a) and versus WT mice in panel (b).

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway

doi: 10.3390/ijms242216037

Figure Lengend Snippet: The expression of WISP1 in KBD and control chondrocytes. ( a ) The scale bars are 500 μm and 100 μm individually. * p < 0.05. ( b ) The mRNA expression of WISP1 in KBD and control chondrocytes. * p < 0.05. n = 5. ( c ) The protein expression of WISP1 in KBD and control chondrocytes. * p < 0.05. n = 5.

Article Snippet: The protein suspension was equally added into the well of the SDS-polyacrylamid gelectrophoresis (SDS-PAGE) for separation, transferred to polyvinylidenedifluoride (PVDF) membranes, and blocked either in 5% nonfat milk (for total proteins) or in 3% FBS (for phosphorylated proteins) solution before being incubated with primary antibodies at 4 °C overnight, such as WISP1 (1:2000, GTX110512, Gene Tex, San Antonio, TX, USA), COL2A1 (1:1000, ab188570, abcam, Cambridge, UK), BECN1 (1:2000, ab207612, abcam), ATG4C (1:1000, ab183516, abcam), ATG4A (1:1000, ab108322, abcam), LC3B (1:2000, ab192890, abcam), and GAPDH (1:10,000, 10494-1-AP, proteintech, Wuhan, China), and the secondary antibody (1:5000, Xi’an Zhuangzhi Biotechnology Co., Ltd., Xi’an, China) for 1 h at RT.

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway

doi: 10.3390/ijms242216037

Figure Lengend Snippet: The functional role of WISP1 in KBD chondrocyte damage. ( a ) The TEM results of KBD chondrocytes. Red arrows represent ( a (i)) The autolysosome in KBD chondrocyte, ( a (ii)) The mitochondria in KBD chondrocyte, and ( a (iii)) The endoplasmic reticulum in KBD chondrocyte. The yellow arrows represent a great number of liposomes. The scale bars are 2 μm, 500 nm, and 200 nm, respectively, from left to right. ( b ) The mRNA expression of selected genes in KBD and control chondrocytes. n = 5. ( c ) The protein expression of markers in KBD and controls. n = 5. ( d ) The Western blot results of markers in KBD and controls. ( e ) The bright field and dark field of the efficiency of three targets of the WISP1 lentivirus in KBD chondrocytes. The magnification is 100×. ( f ) The mRNA expression of WISP1 in KBD chondrocytes and KBD with the WISP1 lentivirus of the negative control (NC) and three targets (WISP1-A, WISP1-B, and WISP1-C). ( g ) The mRNA expression of WISP1 and other genes in controls, KBD chondrocytes, and KBD of the WISP1-A lentivirus. n = 3. ( h ) The Western blot result from markers in WISP1 knock-down KBD chondrocytes and KBD chondrocytes. ( i ) The protein expression of WISP1 and other markers in WISP1 knock-down KBD chondrocytes and KBD chondrocytes. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The protein suspension was equally added into the well of the SDS-polyacrylamid gelectrophoresis (SDS-PAGE) for separation, transferred to polyvinylidenedifluoride (PVDF) membranes, and blocked either in 5% nonfat milk (for total proteins) or in 3% FBS (for phosphorylated proteins) solution before being incubated with primary antibodies at 4 °C overnight, such as WISP1 (1:2000, GTX110512, Gene Tex, San Antonio, TX, USA), COL2A1 (1:1000, ab188570, abcam, Cambridge, UK), BECN1 (1:2000, ab207612, abcam), ATG4C (1:1000, ab183516, abcam), ATG4A (1:1000, ab108322, abcam), LC3B (1:2000, ab192890, abcam), and GAPDH (1:10,000, 10494-1-AP, proteintech, Wuhan, China), and the secondary antibody (1:5000, Xi’an Zhuangzhi Biotechnology Co., Ltd., Xi’an, China) for 1 h at RT.

Techniques: Functional Assay, Liposomes, Expressing, Control, Western Blot, Negative Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway

doi: 10.3390/ijms242216037

Figure Lengend Snippet: The combined effect of rWISP1 with or without T-2 toxin in C28/I2 human chondrocytes. The mRNA expression of WISP1 ( a ), COL2A1 ( b ), ATG4A ( c ), ATG4B ( d ), ATG4C ( e ), BECN1 ( f ), and MAP1LC3B ( g ) in C28/I2 was influenced by the four different concentrations of rWISP1 protein with or without three different concentrations of T-2 toxin. The abscissa stands for the four different concentrations of rWISP1 protein (5, 20, 100, and 500 ng/mL). The black, green, red, and blue lines stand for 0, 2, 5, and 8 ng/mL of T-2 toxin separately. NS stands for no sense. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The protein suspension was equally added into the well of the SDS-polyacrylamid gelectrophoresis (SDS-PAGE) for separation, transferred to polyvinylidenedifluoride (PVDF) membranes, and blocked either in 5% nonfat milk (for total proteins) or in 3% FBS (for phosphorylated proteins) solution before being incubated with primary antibodies at 4 °C overnight, such as WISP1 (1:2000, GTX110512, Gene Tex, San Antonio, TX, USA), COL2A1 (1:1000, ab188570, abcam, Cambridge, UK), BECN1 (1:2000, ab207612, abcam), ATG4C (1:1000, ab183516, abcam), ATG4A (1:1000, ab108322, abcam), LC3B (1:2000, ab192890, abcam), and GAPDH (1:10,000, 10494-1-AP, proteintech, Wuhan, China), and the secondary antibody (1:5000, Xi’an Zhuangzhi Biotechnology Co., Ltd., Xi’an, China) for 1 h at RT.

Techniques: Expressing