Fig. 3: Mul-A suppresses the occurrence of autophagy flux during osteoclastogenesis. | Cell Death Discovery

Fig. 3: Mul-A suppresses the occurrence of autophagy flux during osteoclastogenesis.

From: Restrained Mitf-associated autophagy by Mulberroside A ameliorates osteoclastogenesis and counteracts OVX-Induced osteoporosis in mice

Fig. 3

A BMMs were stimulated with 50 ng/mL RANKL and 30 ng/mL M-CSF for 0, 1, 2, 4, and 6 days. Cell lysates were extracted for western blot analysis to assess the expression of Lc3 and P62 proteins (n = 3). B, C Quantitative analysis of the relative grayscale intensity of Lc3 and P62 bands (n = 3). D, E BMMs were treated with 50 ng/mL of RANKL, 30 ng/mL of M-CSF, and different concentrations of Mul-A (0, 40, 80 μM) for 4 days. Cell lysates were then extracted for western blot analysis of autophagy-related proteins (n = 3). F–I Quantitative analysis of the relative grayscale intensity of autophagy-related protein bands (n = 3). J BMMs were treated with 50 ng/mL of RANKL and 30 ng/mL M-CSF for 4 days in the presence or absence of Mul-A, and the cell microstructure was fixed using 2.5% glutaraldehyde. Autophagosomes and lysosomes were observed using transmission electron microscopy (n = 5). Scale bars = 2 μm, Scale bars = 0.5 μm. K BMMs transfected with mRFP-GFP-Lc3 adenovirus were treated with 50 ng/mL of RANKL and 30 ng/mL of M-CSF for 4 days in the presence or absence of Mul-A (80 μM). The cells were fixed in 4% paraformaldehyde for 15 min, and fluorescent spots were observed using confocal microscopy (n = 5). Scale bar = 50 μm. 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.

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