Journal: Oxidative medicine and cellular longevity

Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3.

doi: 10.1155/2021/2896674

Figure Lengend Snippet: Figure 1: ULK1 is associated with osteoclast differentiation and bone loss. (a) Heat map of differentially expressed mRNA of ULK1 in osteoclasts (OC) and bone marrow macrophage (BMM) from dataset GSE54779 in GEO. (b) Statistical analysis of ULK1 expression between BMM and OC in a heat map. (c) Unc-51-like autophagy activating kinase 1 (ULK1) expression during osteoclast differentiation of RAW264.7 cells. (d) ULK1 expression during osteoclast differentiation of mouse BMM. (e, f) ULK1 mRNA (e) or protein (f) expression in BMM from the sham and OVX groups. (g) Immunofluorescence analysis of ULK1 expression (green) in BMM between the sham and OVX groups. BMM was stained with integrin alpha-M (CD11b) (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μm). (h) Immunofluorescence analysis of ULK1 expression (green) in OC between sham and OVX mice. OC was stained with cathepsin K (CTSK) (red). Nucleus was stained with DAPI (blue). The yellow line points to OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μm). (i) Quantification of ULK1 expression in BMM in (g). (j) Quantification of ULK1 expression in OC in (h). (k) TRAP staining (left) and immunohistochemistry (right) of ULK1 in femur sections from the OVX and sham groups. The yellow arrowhead points to OC (scale bar, 250 μm). All data are means ± SEM; ∗P < 0:05, ∗∗P < 0:01, ∗∗∗P < 0:001.

Article Snippet: DAPI staining (Servicebio, Cat# G1012) and phalloidin staining (Servicebio, Cat# G1041) were performed according to the manufacturer’s protocols.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Oxidative medicine and cellular longevity

Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3.

doi: 10.1155/2021/2896674

Figure Lengend Snippet: Figure 2: ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μm). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μm). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μm). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μm). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μm). All data are means ± SEM; ∗P < 0:05, ∗∗P < 0:01, ∗∗∗P < 0:001.

Article Snippet: DAPI staining (Servicebio, Cat# G1012) and phalloidin staining (Servicebio, Cat# G1041) were performed according to the manufacturer’s protocols.

Techniques: In Vitro, Staining, Control, Knockdown, Gene Expression

Journal: Oxidative medicine and cellular longevity

Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3.

doi: 10.1155/2021/2896674

Figure Lengend Snippet: Figure 4: ULK1 regulates the activation of Syk/JNK through DOK3. (a) The correlation of ULK1 and docking protein 3 (DOK3) expression in osteoclast differentiation (GSE56815 dataset). (b) The expression of DOK3 in Si-NC and Si-ULK1 in BMM. (c) DOK3 expression in control and ULK1 overexpressing BMM. (d) Western blotting to detect the expression of DOK3 in Si-NC and Si-ULK1 BMM. (e) Western blotting to detect the expression of DOK3 in control and ULK1 overexpressing BMM. (f) Immunofluorescence analysis of DOK3 expression (green) in BMM between the sham and OVX groups. BMM was stained with CD11b (red). Nucleus was stained with DAPI (blue). The red arrowhead points to BMM. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μm). (g) Immunofluorescence analysis of DOK3 expression (green) in OC between the sham and OVX groups. OC was stained with CTSK (red). Nucleus was stained with DAPI (blue). The yellow line marks OC. The white line showed the boundary between bone and bone marrow cavity (scale bar, 50 μm). (h) Expression of DOK3 during osteoclast differentiation of RAW264.7 cells. (i) Western blotting analysis of JNK signalling in Si-NC and Si-DOK3 RAW264.7 cells treated with 50 ng/ml RANKL for 0–30 minutes. (j) p-Syk, total Syk levels of Si-NC and Si-DOK3 in RAW264.7 cells. (k, l) TRAP staining (k) and quantification (l) in Si-NC and Si-DOK3 OC (scale bar, 50 μm). (m) TRAP staining and quantification in control and ULK1 overexpressing and ULK1 overexpressing OC with Si-DOK3 (scale bar, 50 μm). All data are means ± SEM; ∗∗∗P < 0:001.

Article Snippet: DAPI staining (Servicebio, Cat# G1012) and phalloidin staining (Servicebio, Cat# G1041) were performed according to the manufacturer’s protocols.

Techniques: Activation Assay, Expressing, Control, Western Blot, Staining

Journal: Oxidative medicine and cellular longevity

Article Title: ULK1 Suppresses Osteoclast Differentiation and Bone Resorption via Inhibiting Syk-JNK through DOK3.

doi: 10.1155/2021/2896674

Figure Lengend Snippet: Figure 2: ULK1 suppresses osteoclast differentiation in vitro. (a, b) TRAP staining (a) and quantification (b) to show osteoclast differentiation in control (Si-NC) and ULK1 knockdown (Si-ULK1) cells (scale bar, 100 μm). (c) Actin staining with phalloidin (Cy3, red) in Si-NC and Si-ULK1 OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μm). (d) OC-specific gene expression in Si-NC and Si-ULK1 cells. (e) TRAP staining in vehicle-treated and SBI-6965-treated OC. (f, g) TRAP staining (f) and quantification (g) to show osteoclast differentiation in control and ULK1 overexpressing OC (scale bar, 100 μm). (h) Actin staining with phalloidin (Cy3, red) in control and ULK1 overexpressing OC. Nuclei were stained with DAPI (blue) (scale bar, 100 μm). (i) OC-specific gene expression in control and ULK1-overexpressing cells. (j) TRAP staining in vehicle-treated and LYN-1604-treated OC (scale bar, 100 μm). All data are means ± SEM; ∗P < 0:05, ∗∗P < 0:01, ∗∗∗P < 0:001.

Article Snippet: DAPI staining (Servicebio, Cat# G1012) and phalloidin staining (Servicebio, Cat# G1041) were performed according to the manufacturer’s protocols.

Techniques: In Vitro, Staining, Control, Knockdown, Gene Expression