Fig 2: Downregulated MYOF expression causes lysosomal dysfunction during the adipogenic differentiation of hMSCs. a Relative mRNA expression of adipogenic markers was detected using qRT–PCR after APPL1 overexpression or MYOF knockdown. b hMSCs adipogenesis was revealed with ORO staining and quantification after APPL1 overexpression or MYOF knockdown. c Protein levels of adipogenic markers were detected using Western blot analysis after APPL1 overexpression and MYOF knockdown. Quantification of the data is shown in the right panel. d After MYOF knockdown, the protein levels of lysosome markers (LAMP1) were analysed using Western blotting. Quantification of the data is shown in the lower panel. e The lysosomes of hMSCs with or without MYOF knockdown that were cultured in the control or adipogenic induction medium were stained with LysoTracker and revealed by immunofluorescence staining. Quantification of LysoTracker is shown in the lower panel. f Time course of LysoTracker staining in siControl and MYOF knockdown hMSCs following treatment with LLOMe for 0, 0.5, 1 and 2 h. Quantification of LysoTracker is shown in the right panel. g Immunofluorescence staining for GAL3 (green) and LAMP2 (red) along with adipogenic induction, and the nucleus was stained with DAIP (blue). Quantification of the data is shown in the right panel. 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: Decreasing APPL1 expression promoted the adipogenic differentiation of hMSCs in vivo. a Schematic diagram of the hMSCs adipogenic differentiation experiment in vivo. b H&E staining and Immunohistochemistry showing the fat vacuoles in the APPL1 knockdown group and overexpression group. c Quantification of fat vacuole numbers in hMSCs adipogenic differentiation experiments in vivo. d Quantification of the fat area percentage in hMSCs adipogenic differentiation experiment in vivo. Scale bar = 100 μm. All data are presented as the means ± SD, n = 6 per group. Statistical differences were determined using Student’s t test. ns not statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 4: Decreasing APPL1 expression promoted the adipogenic differentiation of hMSCs in vitro. a The Protein levels of adipogenic markers PPAR-γ, C/EBP-α, and FABP4 along with the day of hMSC adipogenic differentiation. b ORO staining and quantification along with the day of hMSC adipogenic differentiation. c Pearson correlation analysis revealed the correlation between APPL1 expression and ORO staining quantification, PPAR-γ, C/EBP-α, and FABP4 levels during hMSC adipogenic differentiation. d Relative RNA expression of the adipogenic markers PPAR-γ, C/EBP-α, and FABP4 were detected using qRT–PCR. e Protein levels of the adipogenic markers PPAR-γ, C/EBP-α, and FABP4 were detected by using Western blot analysis. Quantification of the data is shown in the right panel. f SiRNAs and APPL1 overexpression lentiviruses were transfected into hMSCs, and cells were then cultured in adipogenic medium. ORO staining and quantification on day 10. Scale bar = 50 μm. All data are presented as the means ± SD, n = 3 per group in (a, b), n = 15 in (c), n = 6 per group in (d–f). 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 5: 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