Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: The inhibition of PKM2 activity by IgSF11 is required for osteoclast differentiation. a Effect of shikonin on osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The indicated dose of shikonin was added and incubated for three days. b Effect of TEPP46 on osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The indicated dose of TEPP46 was added and incubated for three days. TRAP staining and the frequency of TRAP + multinucleated cells (3 nuclei or more per cell) are shown. Scale bars represent 100 μm. c Lactate production during osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The lactate concentration in the culture supernatants was measured daily. d Lactate production in response to the modulation of PKM2 activity. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. Control DMSO or TEPP46 was added to IgSF11 +/+ cultures and incubated for three days. Control DMSO or shikonin was added to IgSF11 −/− cultures and incubated for three days. The lactate concentrations in the culture supernatants were measured. e Effect of glycolysis intermediates on osteoclast differentiation. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days. The indicated dose of glycolysis intermediates was added to IgSF11 −/− cultures and incubated for three days. IgSF11 +/+ cultures were used as a control. The frequency of TRAP + multinucleated cells (3 nuclei or more per cell) is shown. The data are presented as the means ± S.D. Each dot represents a technical replicate. The results are representative of at least three independent experiments

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Inhibition, Activity Assay, Cell Culture, Incubation, Staining, Concentration Assay

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: Proper PKM2 activity is required for bone homeostasis. a , d Representative microcomputed tomography (μCT) images of femurs from mice treated with shikonin ( n = 5 mice per group) for 4 weeks ( a ) or TEPP46 ( n = 5 mice per group) for 6 weeks ( d ). Bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), bone mineral density (BMD), and cortical thickness (Ct.Th) are shown. Scale bars represent 0.5 mm. b , e Representative TRAP staining images of the tibias of mice treated with shikonin for 4 weeks ( b ) or TEPP46 for 6 weeks ( e ). The scale bar represents 200 μm. GP Growth plate, Metap. Metaphysis. c , f Representative dynamic histomorphometry of the tibias of mice treated with shikonin for 4 weeks ( c ) or TEPP46 for 6 weeks ( f ). The mineral apposition rate (MAR) and bone formation rate (BFR) are shown. The scale bar represents 50 μm. Each dot represents the result of a single mouse ( n = 5 mice per group). Each dot in OC size represents the result of a single osteoclast. The data are presented as the means ± S.D

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activity Assay, Tomography, Staining

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: The activation of PKM2 attenuates LPS-induced osteoclastic bone loss. a Representative μCT images of mouse calvaria 5 days after LPS injection with or without TEPP46 ( n = 5–6 mice per group). The scale bar represents 1 mm. Percentages of the bone destruction areas (Des. area) in calvaria are shown. b Representative TRAP staining images of calvarial coronal sections in ( a ). TRAP + osteoclast size (OC size) and number (OC.N/BS) are shown. Scale bar represents 200 μm. c Serum levels of TNF-α, IL-6, and IL-17AF in the mice in ( a ) are shown. d Representative μCT images of mouse calvaria at 4 days after RANKL injection with or without TEPP46 are shown ( n = 4–6 mice per group). The scale bar represents 1 mm. e Representative TRAP staining images of calvarial coronal sections in ( d ). The scale bar represents 200 μm. Each dot represents the result of a single mouse. Each dot in OC size represents the result of a single osteoclast. The data are presented as the means ± S.D

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activation Assay, Injection, Staining

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: The activation of PKM2 mitigates colitis-induced osteoclastic bone loss. a Representative μCT images of the femurs of mice treated with DSS or the control with or without TEPP46. Bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), bone mineral density (BMD), and cortical thickness (Ct.Th) are shown ( n = 4–6 mice per group). Scale bars represent 0.5 mm. b Representative TRAP staining images of tibias in ( a ). TRAP + osteoclast size (OC size) and number (OC.N/BS) are shown. The scale bar represents 200 μm. GP Growth plate, Metap. Metaphysis. c Representative dynamic histomorphometry of tibias in Panel ( a ). The mineral apposition rate (MAR) and bone formation rate (BFR) are shown. The scale bar represents 20 μm. d Serum levels of TNF-α, IL-6, and IL-17AF from the mice in ( a ) were measured by ELISA. Each dot represents the result of a single mouse. Each dot in OC size represents the result of a single osteoclast. The data are presented as the means ± S.D

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Bone Research

Article Title: IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

doi: 10.1038/s41413-023-00251-2

Figure Lengend Snippet: Schematic depicting the role of IgSF11-mediated regulation of PKM2 activity during osteoclast differentiation. a PKM2 activity is high in BMMs. RANKL stimulation enhances the expression of IgSF11. b IgSF11 mediates the phosphorylation of PKM2 through Src family kinases (c-Src, Fyn, and HcK) in a manner dependent on the C-terminal region. This phosphorylation leads to the inhibition of PKM2 activity. In the absence of IgSF11, the inhibition of PKM2 activity barely occurs, keeping PKM2 activity high. c IgSF11-mediated inhibition of PKM2 activity increases osteoclast differentiation. In the absence of IgSF11, osteoclast differentiation is impaired due to a lack of PKM2 inhibition, which can be rescued by the PKM2-specific inhibitor shikonin. Moreover, the PKM2-specific activator TEPP46 inhibits osteoclast differentiation in the presence of IgSF11

Article Snippet: TEPP46 (Cat.# ML-265) and Shikonin (Cat.# 14751-10) were purchased from Cayman Chemical.

Techniques: Activity Assay, Expressing, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Shikonin ameliorated mice colitis by inhibiting dimerization and tetramerization of PKM2 in macrophages

doi: 10.3389/fphar.2022.926945

Figure Lengend Snippet: Shikonin enhanced the anti-UC effect of TEPP-46 in colitis mice. The body weight (A) and DAI (B) of mice during the course of colitis. Representative images (C) and statistics (D) of colon length in each group. Representative images (E) of H&E-stained colon tissue samples (scale bar, 500 μm) and histological scores (F) of colonic tissues. Expression of PKM2 in colon of colitis mice were detected by immunohistochemistry assay (G) with the statistics (H) . Data are shown as the mean ± SD, n = 5–8. ## p < 0.01, compared to the control group. * p < 0.05, ** p < 0.01, compared to the DSS group.

Article Snippet: TEPP-46 (ML-265) were purchased from MedChemExpress (NJ, United States).

Techniques: Staining, Expressing, Immunohistochemistry, Control