HSP20 Antibody - #AF6003

Fig. 4. Identification of Hspb6 as a direct target of miR-3085-3p in ATDC5s. (a) Venn diagram display the overlapping of the mice target genes of miR-3085-3p, as predicted by miRDB, miRPathDB and microT-CDs starbase. (b) Venn diagrams display the overlapping miR-3085-3p target genes and transcriptome sequencing differential gene set (Degs) caught from the mRNA sequencing of bipedal standing model. (c) Heat map of 6 genes after intersection of MiR-3085-3p and Degs. (d) A sequence alignment of a putative miR-3085-3p binding site within the 3′UTR of Hspb6 mRNA shows high sequence conservation and complementarity. (e) HEK-293T cells were co-transfected with miR-3085-3p mimics or scramble and luciferase reporter constructs of the wild-type Hspb6-3′UTR (3′UTR-wt) or the mutated Hspb6-3′UTR (3′UTR mut). After transfection, luciferase activity was measured. Luciferase reporter assay revealed that miR-3085-3p exclusively decreased luciferase activity of the wild-type reporter plasmids. ATDC5s were transfected with miR-3085-3p mimics(a) or scramble and MiR-3085-3p inhibitor (b) or negative control (NC). A cyclic tensile strain (CTS) test was performed after transfection, and the load condition of CTS group molding was 20 % strength, 0.5Hz, 48h. (f,g) qRT-PCR analysis of Hspb6 transfected with miR-3085-3p mimics(f) or inhibitor(g) with or without CTS. (h,i) Western blotting analysis of Hspb6 protein levels in ATDC5s after miR-3085-3p overexpression (h) and inhibition(i) with or without CTS. (j) Representative images of Hspb6 assayed by immunofluorescence confocal microscopy in ATDC5s with miR-3085-3p knockdown or overexpression with CTS. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 50 μm. (k,i) Quantification of positive cells after miR-3085-3p overexpression(k) or inhibition (i) with CTS. ns: no significant difference, ∗P < 0.05, ∗∗∗P < 0.001,∗∗∗∗P < 0.0001. All data are shown as means ± SD of three independent experiments in (e) (f) (g) (k) (l), student's t-test and one-way analysis of variance (ANOVA) were used for comparison between two groups and multiple groups, respectively.

Fig. 6. MiR-3085-3p aggravates facet joint osteoarthritis in vivo. Mice in control group were injected with adeno-associated virus (AAV) miR-3085-3p mimics or AAV NC, while experiencing bipedal standing model (BSM) mice were injected with AAV-miR-3085-3p sponge or AAV NC. (a) 3D reconstruction view were created using Micro CT, Scale bars, 500 μm. (b,c) The articular cartilage was stained with Hematoxylin/Eosin (H&E) and Safranin-O/Fast green (SOFG), Scale bars, 50 μm. (d–f) Expression of Hspb6 (d), Atf4 (e), Grp78 (f) in articular cartilage were detected by immunohistochemistry, Scale bars, 50 μm. (i) OARSI scoring was based on the results of SOFG staining. (j–l) Statistical graph of expression of Hspb6(j), Atf4(k), Grp78(l) detected by immunohistochemistry. (m) Schematic of the scientific hypothesis. MiR-3085-3p is upregulated in mechanical-loading chondrocytes and FJOA cartilage tissues directly related to endoplasmic reticulum stress and cell apoptosis. MiR-3085-3p induces ER stress by directly targeting Hspb6. ∗∗P < 0.01, ∗∗∗∗P < 0.0001. All data are shown as means ± SD of six independent experiments in (g) (h) (i) (j) (k) (l), one-way analysis of variance (ANOVA) was used for comparison between multiple groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2. HSP20 overexpression inhibits BC cell proliferation. (A) Relative mRNA and protein expression levels of HSP20 in five BC cell lines compared with the MCF-10A normal mammary epithelial cell line. (B) Relative expression levels of HSP20 in MDA-MB-231 and MDA-MB-468 cells overexpressing HSP20. (C) Cell Counting Kit-8 proliferation assay. (D) Representative images and quantitative analysis of colony formation assays. Data are presented as the mean ± SD. **P<0.01; ***P<0.001 vs. MCF-10A or as indicated. BC, breast cancer; control, untransfected cells; vector, cells transfected with an empty vector; exHSP20, HSP20-overexpressing cells; HSP20, heat shock protein 20; OD, optical density.

Figure 2. HSP20 overexpression inhibits BC cell proliferation. (A) Relative mRNA and protein expression levels of HSP20 in five BC cell lines compared with the MCF-10A normal mammary epithelial cell line. (B) Relative expression levels of HSP20 in MDA-MB-231 and MDA-MB-468 cells overexpressing HSP20. (C) Cell Counting Kit-8 proliferation assay. (D) Representative images and quantitative analysis of colony formation assays. Data are presented as the mean ± SD. **P<0.01; ***P<0.001 vs. MCF-10A or as indicated. BC, breast cancer; control, untransfected cells; vector, cells transfected with an empty vector; exHSP20, HSP20-overexpressing cells; HSP20, heat shock protein 20; OD, optical density.