Fig 1: MARCH7 regulated VAV2-RAC1-CDC42 pathway. (A) The interaction between MARCH7 and VAV2 was identified by Co-IP assay. Cells were co-transfected with Flag-VAV2 and HA-MARCH7, and control group was established simultaneously, cells were then harvested 24 h later. Anti-HA antibodies were used to pull the interaction protein. Then, they were detected by anti-Flag antibodies. Results showed that Flag bands could not be detected in the cells transfected with Flag-VAV2 (lane 1) or HA-MARCH7 (lane 3) only. However, it can be detected in cells co-transfected with both Flag-VAV2 and HA-MARCH7 (lane 2), which indicated that there existed interaction between MARCH7 and VAV2 in vivo. (B) The expression of VAV2, RAC1 and CDC42 were determined by western blot analysis. The VAV2, RAC1 and CDC42 expression in cells infected with LV3-shMARCH7-1 or LV3-shMARCH7-2 was significantly lower than in control cells. LV3-shMARCH7-1, lentiviral vector expressing shRNA targeting MARCH7; LV, lentiviral vector.
Fig 2: JSH-23 reduces RANKL-induced ROS production during osteoclastogenesis by downregulating the TRAF6/Rac1/NOX1 signaling pathway and enhancing the expression of Nrf2/HO-1. (A) Representative images of RANKL-induced ROS generation in BMMs with or without JSH-23 treatment at different concentrations. (B) Quantification of the average DCF intensity per well. (C) BMMs were stimulated with RANKL in the absence or presence of JSH-23 (20 and 40 μM) for 48 h, and then the ROS production pathway was analyzed by western blotting. (D) BMMs were cultured in α-MEM containing M-CSF and RANKL in the presence of JSH-23 (40 μM) at the indicated times (1, 3, 5 days). The expression of Nrf2 was analyzed by western blotting. (E) Western blotting images of the effects of JSH-23 on antioxidant enzyme, HO-1, catalase, NQO1, and GSR expression. (F–H) Quantitative analysis of the western blotting results. (I) Representative immunofluorescence images showing Nrf2 translocation and (J) HO-1 expression. (K–L), BMMs were transfected with siRNA against Nrf2 or (M, N) HO-1 for 48 h. The silencing efficiency was evaluated by western blotting. (O) Transfected BMMs were treated with RANKL and JSH-23 (40 μM) for 5 days. TRAP staining was measured, and (P) the osteoclasts were counted. The values are shown as the means ± SDs, n = 3; *p < 0.05, **p < 0.01.
Fig 3: Mouse model assays further confirmed that BPB exposure induced miscarriage by directly suppressing MI and MF. A) Western blot analysis of the protein levels of murine Pkca, Rac1, Cxcl12, Tspan4, and Ndst1 in BPB‐exposed mouse placental tissues, with Gapdh as internal standard, and their relative quantification (each n = 6). B) WGA staining of BPB‐exposed mouse placenta tissues (scale bar, 50 µm). C) The scheme for the construction of a mouse miscarriage intervention model. D,E) Embryo resorption (D, indicated by red arrows, scale bar, 1 cm) and the average miscarriage rates (E) in 100 mg kg−1 d BPB‐exposed mice with Pkca supplement (each n = 6). F,G) Western blot (F) analysis of the protein levels of Pkca, Rac1, Cxcl12, Tspan4, and Ndst1 in placental tissues in 100 mg kg−1 d−1 BPB‐exposed mice with supplement with Pkca, with Gapdh as internal standard, and their relative quantification (G, each n = 6). H) WGA staining of the placenta tissues of BPB‐exposed mice with supplement with Pkca (scale bar, 50 µm). I,J) Embryo resorption (I, indicated by red arrows, scale bar, 1 cm) and the average miscarriage rates (J) in 100 mg kg−1 d−1 BPB‐exposed mice with Tspan4 supplement (each n = 6). K) WGA staining of the placenta tissues of BPB‐exposed mice with supplement with Tspan4 (scale bar, 50 µm). Data were shown as means ± SD (standard deviation). p <0.05 meant significant differences compared with the control.
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