Fig. 6: LYPLAL1-AS1 regulates adipogenic differentiation of hAMSCs potentially by modulating DSP protein stability. | Cell Death Discovery

Fig. 6: LYPLAL1-AS1 regulates adipogenic differentiation of hAMSCs potentially by modulating DSP protein stability.

From: Long noncoding RNA LYPLAL1-AS1 regulates adipogenic differentiation of human mesenchymal stem cells by targeting desmoplakin and inhibiting the Wnt/β-catenin pathway

Fig. 6

a RIP assay to assess the interaction between DSP and LYPLAL1-AS1 in hAMSCs. Antibodies against DSP (DSP) were used to immunoprecipitate lncRNAs, followed by qRT-PCR with LYPLALA-AS1-specific primers. IgG was used as the negative control. Relative abundances were compared to 1% of the input. b FISH assay of LYPLAL1-AS1 transcripts followed by immunofluorescence assay for DSP showed the colocalization of LYPLAL1-AS1 transcripts and DSP in the cytoplasm. c, d Western blot analysis of DSP stability in the presence of LYPLAL1-AS1 knockdown (c) and LYPLAL1-AS1 overexpression (d) in hAMSCs and with treatment with 5 µg/ml cycloheximide (CHX) for 0, 2, 4, 6, and 8 h. e Western blot analysis of DSP in hAMSCs that overexpress LYPLAL1-AS1 and in the absence and presence of MG132 (10 μM) treatment for 4 h. f LYPLAL1-AS1 knockdown and control hAMSCs were transfected with siRNAs targeting DSP. The levels of LYPLAL1-AS1 and DSP were detected by qRT-PCR. g, h Western blotting (g) and qRT-PCR (h) assays detected adipogenic marker genes (PPARγ, AP2, and CEBPα) in hAMSCs after LYPLAL1-AS1 knockdown, DSP knockdown, and LYPLAL1-AS1 knockdown followed by DSP knockdown. i, j Oil red O staining assay (i) and subsequent quantification (j) in hAMSCs after LYPLAL1-AS1 knockdown, DSP knockdown, and LYPLAL1-AS1 knockdown followed by DSP knockdown. GAPDH was used as the internal control for the western blot assays. The quantitative data were normalized to GAPDH, n = 3; data are shown as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001; scale bars: 50 µm.

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