Fig 1: Overexpression of piR-36741 alleviated ovariectomy-induced osteoporosis in mice. Ovariectomy was used to construct a mouse model of osteoporosis. 10 mg/kg mimic-NC or mimic-piR-36741 in 50 µL volumes were respectively injected into mice 14 days after the ovariectomy through the tail vein once a week until the 8th week. (A) Representative images of HE staining of mouse distal femur tissue sections (100×). (B–D) Bone mineral density, bone strength and elastic modulus were evaluated. (E, F) The expression of piR-36741 and the protein levels of METTL3 and BMP2 in femoral tissues were measured. (G) The protein levels of RUNX2, COL1A1, OCN and OPN were analyzed with Western blotting. N = 6 in sham group, and N = 8 in OVX, OVX+mimic-NC, and OVX+mimic-piR-36741 groups. *P < 0.05. Each test was independently repeated at least three times.
Fig 2: Silencing METTL3 impeded osteogenic differentiation of BMSCs. BMSCs were infected with Lv-sh-NC or Lv-sh-METTL3, and then cultured in osteogenic differentiation medium for 14 days. (A) The expression of piR-36741 was measured on day 14. (B) The global m6A level of BMSCs with or without METTL3 knockdown was analyzed with the EpiQuik™ m6A RNA methylation quantification kit. (C) The mRNA levels of METTL3, RUNX2, COL1A1, OCN and OPN were detected on day 14. (D) Images of ALP staining on day 14 (100×). (E) ALP activity was determined on day 14. (F) Images of ARS staining on day 14 (100×). (G) Quantitative analysis of ARS accumulation on day 14. N = 5 in each group. *P < 0.05, **P < 0.01. Each test was independently repeated at least three times.
Fig 3: Silencing piR-36741 hindered osteogenic differentiation of BMSCs. BMSCs were infected with Lv-sh-NC and Lv-sh-piR-36741, and then cultured in osteogenic differentiation medium for 14 days. (A–C) The expression of piR-36741, and the mRNA and protein levels of METTL3, RUNX2, COL1A1, OCN and OPN were measured on day 14. (D) Images of ALP staining on day 14 (100×). (E) ALP activity was determined on day 14. (F) Images of ARS staining (100×). (G) Quantitative analysis of ARS accumulation on day 14. N = 5 in each group. *P < 0.05, **P < 0.01. Each test was independently repeated at least three times.
Fig 4: Verification of the expression of genes in the TLR-based signature in HCC and normal tissue specimens. (A) Western blot for detecting MAP2K2, IRAK1, RAC1, TRAF, and MAP3K7 proteins in three paired HCC and normal tissues. (B–G) Quantification of the expression of (B) MAP2K2, (C) IRAK1, (D) RAC1, (E) TRAF, (F) MAP3K7, and (G) SPP1 proteins in three paired HCC and normal tissues. Comparisons between groups were evaluated with Student’s t tests. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 5: Downregulation of Spp1 expression in ischemia/reperfusion after PCI. (a) qRT-PCR analysis showing the expression of Spp1 in ischemia-reperfusion samples after PCI (square shape) compared to normal samples (circle shape). *p < 0.05. (b) Western blot indicating that Spp1 protein levels were significantly downregulated in ischemia-reperfusion cells after PCI compared with normal cells.
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