THBS1 Antibody - #DF6848

Fig. 3. Fibrosis phenotype and THBS1/TGFβ1 expression were significantly elevated in the LFH group. (A) The thickness of LF was measured by MRI before operation. The thickness of LF at the L4/5 facet joint level was indicated by red arrow. (B) Representative tissues of LF acquired from patients with spinal surgery. The top three were from non-LFH patients and the bottom three were from LFH patients. Scale bar = 5 mm. (C) Representative images of H&E, MASSON, and EVG staining of the non-LFH group (n = 6) and the LFH group (n = 6). The yellow dotted area represents collagen fibers. Scale bar = 20 μm. (D) Immunohistochemical staining of THBS1, TGFβ1, COL1A2, and α-SMA in the LF samples from LFH group (n = 6) and non-LFH group (n = 6), and the percentage of positive cells was quantified. Scale bar = 20 μm. (E) TEM of LF ultrastructure of non-LFH and LFH groups. The yellow area represents elastin fibers. Scale bar = 8 μm. (F) The qRT-PCR analysis results showing that LFH group (n = 6) had a higher mRNA level of THBS1, TGFβ1, ACTA2 (α-SMA), and COL1A2 compared with non-LFH group (n = 6). The 2−ΔΔCt method was used to normalized with GAPDH. (G) Western blotting assay of THBS1, TGFβ1, COL1A2, and α-SMA protein levels of LF samples of the two groups. GAPDH served as the loading control. EVG: verhoeff's van gieson, TEM: transmission electron microscope, EF: elastic fiber, CF: collagen fiber. **p < 0.01, ***p < 0.001.

Fig. 7. The mechanical stress-THBS1 axis promotes hypertrophy and fibrosis of LF in vivo. (A) H&E staining of tissues from mice (n = 6 mice per group), and the quantitative analysis of LF area. Scale bar = 50 μm. (B) EVG staining of tissues from mice, and the quantitative analysis of volume fraction of elastin fibers in LF. Scale bar = 20 μm. (C, D) Immunohistochemical staining of α-SMA and THBS1 in mice tissues, and the percentage of positive cells was quantified. Scale bar = 20 μm. Error bars: mean ± S.D. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. THBS1 expression levels in SGC tissues and cells. (A) Immunohistochemical staining of THBS1 in SGC and para-carcinoma tissues. Expression of THBS1 was negative in SGC tissues, but positive in para-carcinoma tissues. Scale bar, 50 µm. (B) Positive expression rate of THBS1 in eyelid SGC and para-carcinoma formalin-fixed paraffin-embedded samples. (C) Relative mRNA expression levels of THBS1 in SGC cells were lower than those in normal SG cells. **P<0.01 and ***P<0.001. SGC, sebaceous gland cancer; THBS1, thrombospondin 1.

Figure 4. miR-3907 negatively regulates THBS1 expression. (A) Predicted binding sites for miR-3907 within the wild-type and mutant 3'UTR of THBS1. (B) Quantitative analysis of the relative luciferase activity of cells with reporter vectors containing the wild type or mutant THBS1 3'UTR, following miR-3907 co-transfection. (C) Reverse transcription-quantitative PCR and (D) western blotting with semi-quantification analysis revealed decreased THBS1 expression following miR-3907 mimics transfection. *P<0.05 and ***P<0.001. miR, microRNA; THBS1, thrombospondin 1; UTR, untranslated region; NC, negative control.

Figure 5. Inhibitory effects of THBS1 on SGC cell proliferation and migration. (A) THBS1-knockdown was induced by transfecting SGC cells with siRNA-THBS1. THBS1 expression level was determined by reverse transcription-quantitative PCR. Results of (B) CCK-8 and (C) wound-healing assays after transfection with siRNA-THBS1; siRNA-THBS1 increased the proliferation and migration of SGC cells. (D) THBS1 overexpression was induced by transfecting lentiviral vectors; THBS1 expression level was determined by reverse transcription-quantitative PCR. (E) Fluorescence assay results revealed that the THBS1 lentivirus vector was successfully transfected. (F) CCK-8 and (G) wound-healing assays results after transfection with THBS1 lentivirus revealed that THBS1 decreased the proliferation and migration of SGC cells. Scale bar, 500 µm. *P<0.05, **P<0.01 and ***P<0.001. SGC, sebaceous gland cancer; siRNA, small interfering RNA; CCK-8, Cell Counting Kit-8; THBS1, thrombospondin 1; NC, negative control.