anti sdc4 primary antibody  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Inhibition of MMP 2&9 protects the endothelial glycocalyx and improves diastolic function in diabetic cardiomyopathy

doi: 10.1101/2023.12.18.572183

Figure Lengend Snippet: Cells were pre-incubated with SB-3CT for 2 hours before being treated with TNF-α for 6 hours. (a) SDC4 cell surface expression. Fluorescence intensity was normalized to cell number (n=5 for all groups; control vs TNF-α, *p<0.05; TNF-α vs TNF-α + SB-3CT, **p<0.01; One-way ANOVA). (b) SDC4 mRNA expression (n=5 for all groups; Control vs TNF-α, ****p<0.0001; TNF-α vs TNF-α + SB-3CT, *P<0.05; One-way ANOVA). (c) SDC4 concentration in the conditioned media (n=4 for all groups; Control vs TNF-α, ***p<0.001; TNF-α vs TNF-α + SB-3CT, **p<0.01; One-way ANOVA). (d) GAG concentration in the conditioned media (n=4 for all groups; Control vs TNF-α, *p<0.05; TNF-α vs TNF-α + SB-3CT, **p<0.01; One-way ANOVA)

Article Snippet: The cells were incubated with an anti-SDC4 primary antibody at 10 mg/ml in blocking buffer (AF2918-SP, Bio-Techne) for 1h at room temperature.

Techniques: Incubation, Expressing, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Inhibition of MMP 2&9 protects the endothelial glycocalyx and improves diastolic function in diabetic cardiomyopathy

doi: 10.1101/2023.12.18.572183

Figure Lengend Snippet: (a) MMP9 mRNA expression (n=5 for both groups; *p<0.05; unpaired t test). (b) MMP9 activity in the conditioned media (n=5 for all groups; control vs TNF-α, *p<0.05; TNF-α vs TNF-α + SB-3CT, **p< 0.01; One-way ANOVA). (c) MMP9 mRNA expression from shRNA MMP9 cells (n=4 for both groups; **p<0.01; unpaired t test). (d) SDC4 mRNA expression (n=4 for all groups; scrambled control vs scrambled control + TNF-α, ****p<0.0001. Scrambled control + TNF-α vs shRNA MMP9 + TNF-α, **p<0.01; One-way ANOVA). No significant difference found between shRNA MMP9 and scrambled control (p=0.5). (e) SDC4 concentration in the conditioned media (n=4 for all groups; scrambled control vs scrambled control + TNF-α, ***p<0.001. Scrambled control + TNF-α vs shRNA MMP9 + TNF-α, *p<0.05)

Article Snippet: The cells were incubated with an anti-SDC4 primary antibody at 10 mg/ml in blocking buffer (AF2918-SP, Bio-Techne) for 1h at room temperature.

Techniques: Expressing, Activity Assay, shRNA, Concentration Assay

Journal: Oncogene

Article Title: Low expression of ZFP36L1 in osteosarcoma promotes lung metastasis by inhibiting the SDC4-TGF-β signaling feedback loop.

doi: 10.1038/s41388-023-02880-7

Figure Lengend Snippet: Fig. 5 Interaction between SDC4 and TGFBR3 protected against TGFBR3 cleavage by MMP. WB (A) and SBE-luciferase activity assays (B) were conducted to determine the role of SDC4 in regulating the TGF-β signaling pathway via the extracellular pathway in 143B cells. C ELISA tests were performed to measure the level of sTGFBR3 in the CM of oeSDC4 or shSDC4 143B cells. IP assays were carried out to reveal the interaction between SDC4 and TGFBR3 in exogenous (D) and endogenous (E) ways in 143B cells. F Immunofluorescence assay showed the colocalization of SDC4 and TGFBR3 on the cell surface in 143B cells or U2OS cells. Upper scale bar, 10 μm. Lower scale bar, 1 µm. G ELISA was conducted to measure TGFBR3 cleavage regulated by TAPI2 or DAPT in 143B cells. WB (H) and SBE-luciferase activity (I) assays were conducted to elucidate the role of TAPI2 in the modulation of the TGF-β signaling pathway attenuated by oeZFP36L in 143B cells. The data are shown as means ± SEMs; *P < 0.05, **P < 0.01.

Article Snippet: 143B cells were lysed with 500 μL of IP lysis buffer and incubated with BeyoMagTM Protein A+ G magnetic beads (Beyotime, China) and primary antibodies against SDC4 (CST, #12236), TGFBR3 (CST, #5544), HA-tag (CST, #2367) or FLAG-tag (CST, #14793) at 4 °C.

Techniques: Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Low expression of ZFP36L1 in osteosarcoma promotes lung metastasis by inhibiting the SDC4-TGF-β signaling feedback loop.

doi: 10.1038/s41388-023-02880-7

Figure Lengend Snippet: Fig. 6 ZFP36L1 accelerated SDC4 mRNA degradation by binding to the ARE element. mRNA stability assays (A) were performed to determine the stability of SDC4 mRNA that was regulated by ZFP36L1, and the half-life of SDC4 mRNA (B) was analyzed. RIP assays (C) and RNA pull-down assays (D) were carried out to confirm the binding relationship between ZFP36L1 and SDC4 mRNA. E Schematic representation of the predicted AREs in the 3’UTR of SDC4 mRNA (NM_002999.4) and construction of corresponding reporter plasmids. The 3’UTR in TNF mRNA (NM_000594.4) was used as a positive control. F Renilla luciferase activity assays were conducted to determine the role of AREs in the 3’UTR of SDC4 mRNA in the regulation of mRNA decay. RIP assays (G) and RNA pull-down assays (H) confirmed that ARE3 was the most important element for ZFP36L1 binding. I, J, RFP plasmids were constructed by fusing 3 AREs with RFP genes. mRNA stability assays (I) were performed to determine the function of AREs in regulating RFP mRNA decay, and the half-life of RFP mRNA (J) was calculated. The data are shown as means ± SEMs; *P < 0.05, **P < 0.01.

Article Snippet: 143B cells were lysed with 500 μL of IP lysis buffer and incubated with BeyoMagTM Protein A+ G magnetic beads (Beyotime, China) and primary antibodies against SDC4 (CST, #12236), TGFBR3 (CST, #5544), HA-tag (CST, #2367) or FLAG-tag (CST, #14793) at 4 °C.

Techniques: Binding Assay, Positive Control, Luciferase, Activity Assay, Construct

Journal: Oncogene

Article Title: Low expression of ZFP36L1 in osteosarcoma promotes lung metastasis by inhibiting the SDC4-TGF-β signaling feedback loop.

doi: 10.1038/s41388-023-02880-7

Figure Lengend Snippet: Fig. 7 Impact of the ZFP36L1-SDC4-TGF-β loop on osteosarcoma lung metastasis in vivo. A Representative IHC images of ZFP36L1, SDC4 and p-SMAD3 in individual samples from among 70 OS specimens. Scale bar, 100 μm. Percentage of samples with high or low expression of ZFP36L1 compared to SDC4 (B) and p-SMAD3 (C). Lung metastasis of osteosarcoma was measured by in vivo bioluminescence (D), lung tumor counts (E), ex vivo bioluminescence (F) and HE staining (G). Scale bar, 120 μm. The bioluminescence levels of in vivo bioluminescence images (H) and ex vivo bioluminescence images (I) were analyzed by LivingImage software. J The number of lung tumors in different groups of mice was counted. K Representative osteosarcoma metastatic lesion in the lungs from different groups of mice were analyzed by IHC staining for ZFP36L1, p-ZFP36L1, SDC4, and p-SMAD3. Scale bar, 60 μm. The data are shown as means ± SEMs; *P < 0.05, **P < 0.01.

Article Snippet: 143B cells were lysed with 500 μL of IP lysis buffer and incubated with BeyoMagTM Protein A+ G magnetic beads (Beyotime, China) and primary antibodies against SDC4 (CST, #12236), TGFBR3 (CST, #5544), HA-tag (CST, #2367) or FLAG-tag (CST, #14793) at 4 °C.

Techniques: In Vivo, Expressing, Ex Vivo, Staining, Software, Immunohistochemistry

Journal: Oncogene

Article Title: Low expression of ZFP36L1 in osteosarcoma promotes lung metastasis by inhibiting the SDC4-TGF-β signaling feedback loop.

doi: 10.1038/s41388-023-02880-7

Figure Lengend Snippet: Fig. 8 Blocking the ZFP36L1-SDC4-TGF-β loop inhibited osteosarcoma EMT and lung metastasis. In bone lesions, the high expression of ZFP36L1 expedited SDC4 mRNA degradation and reduced SDC4 interacted with TGFBR3, leading to increased free TGFBR3 cleavage by MMP. The increased soluble TGFBR3 (sTGFBR3) subsequently inhibited activation of TGF-β signaling pathway via blocking TGF-β1 binding to TGFBR1/2. Conversely, in metastatic OS cells and lung metastases, the low expression of ZFP36L1 reduced SDC4 mRNA degradation, leading to an abnormal accumulation of SDC4. The interaction between SDC4 and TGFBR3 protected against TGFBR3 cleavage by MMP, resulting in a decrease in extracellular sTGFBR3. The decrease in sTGFBR3 facilitated the activation of the TGF-β signaling pathway by enhancing the binding of TGF-β1 to TGFBR1/2, ultimately resulting in osteosarcoma EMT and lung metastasis. Intriguingly, activation of the TGF-β signaling pathway further upregulated SDC4 expression, which caused a vicious circle. Targeting the ZFP36L1-SDC4-TGF-β loop with MK2 inhibitor III or SB431542 effectively inhibited osteosarcoma EMT and lung metastasis.

Article Snippet: 143B cells were lysed with 500 μL of IP lysis buffer and incubated with BeyoMagTM Protein A+ G magnetic beads (Beyotime, China) and primary antibodies against SDC4 (CST, #12236), TGFBR3 (CST, #5544), HA-tag (CST, #2367) or FLAG-tag (CST, #14793) at 4 °C.

Techniques: Blocking Assay, Expressing, Activation Assay, Binding Assay