Fig 2: APPL1 binds to MYOF and inhibits MYOF degradation by the ubiquitination-protease pathway. a hMSCs lysates were immunoprecipitated with MYOF, APPL1 or IgG antibodies. The endogenous interactions between the APPL1 and MYOF proteins in hMSCs were detected using Western blot analysis. b Immunofluorescence staining revealed the colocalization of APPL1 (red) and MYOF (green), and the nucleus was stained with DAIP (blue). c Diagram showing the structural domains and sequence of APPL1. d The binding sites of APPL1 that interacted with MYOF were detected using Western blot analysis. e Changes in protein levels after siRNA intervention were detected using Western blot analysis. Quantification of the data is shown in the right panel. f APPL1 and MYOF mRNA levels were not different between the APPL1 knockdown group and the MYOF knockdown group in the qRT–PCR analysis. g Western blot analysis of MYOF protein levels in the siControl group or APPL1 knockdown group treated with lysosome inhibitors (BafA1, 10 μM; CQ, 10 mM) and the proteasome inhibitor (MG132, 10 μM). Quantification of the data is shown in the right panel. h Western blot analysis showed whether proteasome inhibitors MG132 could reverse MYOF protein degradation during APPL1 knockdown. Quantification of the data is shown in the right panel. i Immunoprecipitation showed the levels of MYOF ubiquitination, and the control group and APPL1 knockdown group were treated with or without MG132. Scale bar = 50 μm. All data are presented as the means ± SD, n = 6 per group. Statistical differences were determined using Student’s t test or ANOVA. ns not statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 3: MYOF deficiency in hMSCs inhibits autophagy flux by altering lysosomal function. a Western blot analysis of autophagy flux and autophagy activation after MYOF knockdown under adipogenic induction conditions with or without BafA1. b hMSCs were infected with a GFP-mCherry-LC3B lentivirus for 24 h, and fluorescent staining was detected using confocal microscopy after MYOF knockdown. c Transmission electron microscopy (TEM) was used to reveal autophagosomes in hMSCs under adipogenic induction conditions. d Protein levels of LC3B and adipogenic differentiation markers were evaluated using Western blotting after transfection with siControl and siMYOF and treatment with or without 3-MA. e ORO staining and quantification were performed to reveal adipogenesis after transfection with siControl and siMYOF and treatment with or without 3-MA. b, e Scale bar = 50 μm. c Scale bar = 1 μm. All data are presented as the means ± SD, n = 6 per group. Statistical differences were determined using Student’s t test or ANOVA. ns not statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 4: A schematic of the role of the APPL1/MYOF/lysosome/autophagy axis in hMSCs adipogenic differentiation. APPL1 deficiency enhanced the ubiquitination-mediated degradation of MYOF, and downregulation of MYOF expression enhanced the risk of lysosome damage during the adipogenic differentiation of hMSCs. Subsequently, lysosome damage inhibited autophagy flux by suppressing autophagosome degradation, and autophagosomes accumulated inside the cell. Finally, hMSCs differentiation towards the adipocyte lineage was promoted, and the adipogenic-osteogenic differentiation balance was disordered in osteoporosis