Fig. 6: Autophagy inducers Rapamycin and Torin 1 rescue the inhibitory effects of Mul-A on osteoclasts.

A BMMs were treated with 30 ng/mL M-CSF, 50 ng/mL RANKL, and Rapamycin (Rapa) (0.1 nM) in the presence or absence of Mul-A for 6 days. The cells were then fixed in 4% paraformaldehyde for 15 min and stained with Trap (n = 5). Scale bar = 200 μm. B, C Quantitative analysis of the number of Trap-positive multinuclear cells per well (n = 5). D BMMs were treated with 30 ng/mL M-CSF, 50 ng/mL RANKL, and Torin 1 (5 nM) in the presence or absence of Mul-A for 6 days. Then the cells were fixed in 4% paraformaldehyde for 15 min and stained with Trap (n = 5). Scale bar = 200 μm. E, F Quantitative analysis of the number of Trap-positive multinuclear cells per well (n = 5). G BMMs were incubated with 30 ng/mL M-CSF and 50 ng/mL RANKL for 6 days. After the formation of mature osteoclasts, the cells were treated with or without Rapa (0.1 nM), Torin 1 (5 nM), and Mul-A for 4-5 days continuously. Scanning electron microscopy was utilized to observe the number and area of absorption pits (n = 5). Scale bar = 50 μm. H Quantitative analysis was conducted to determine the area of bone resorption pits (n = 5). I BMMs were treated with 30 ng/mL M-CSF, 50 ng/mL RANKL, Rapamycin (0.1 nM), and Torin 1 (5 nM) in the presence or absence of Mul-A for 6 days to extract cell lysates. Western blot analysis was performed to assess the expression of Nfatc1, c-Fos, Ctsk, and Lc3 (n = 3). J–M Quantitative analysis was conducted to determine the relative grayscale intensity of the protein bands (n = 3). The control group was added with an equivalent DMSO. Data were presented as the median and interquartile range (IQR). *P < 0.05, **P < 0.01, ***P < 0.001.