PMC

ADINR RNA Regulates Active Chromatin via Binding to PA1
(A) RNA pull-down with six different GST-conjugated proteins (WDR5, WDR82, PA1, PTIP, RBBP5, and Menin) from the adipocyte total RNA on day 3. Only GST-PA1 specifically retrieves ADINR RNA.
(B) ChIP assay of PA1 in the C/EBPα promoter region. Knockdown of ADINR RNA significantly abrogates the peaks of PA1 occupancy in the region of primers4 and primers5 (see A). ^∗∗p < 0.01, Student’s t test.
(C) Affinity of seven truncated ADINR RNA fragments for GST-PA1 (in vitro binding assay). All the panels show means ± SD in three independent experiments.
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ADINR RNA Is Required for the Active Chromatin State of the C/EBPα and ADINR Loci
(A) ChIP assay of H3K4me3 and H3K27me3 at the C/EBPα and ADINR loci. The qPCR data are presented as the mean ± SD in six independent experiments. ^∗p < 0.05; ^∗∗p < 0.01. NC, negative control.
(B) RNA immunoprecipitation (IP) of endogenous WDR5, PTIP, Menin, and CXXC1 in human adipocytes after 3 days of differentiation. The retrieved RNA was quantified by qRT-PCR (negative controls: U1 and GAPDH RNAs). Approximately 2 μg of cell lysate was loaded as the input control. Error bars represent the mean ± SD in three independent experiments.
(C) Immunoblot analysis after RNA pull-down assay of in-vitro-transcribed ADINR RNA and histone2 mRNA.
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ADINR RNA Depletion Represses Adipogenic Differentiation
(A and B) qRT-PCR analysis of ADINR RNA, C/EBPα, and PPARγ after knockdown of ADINR RNA using two independent siRNAs (siADINR#1 and siADINR#2) on day 0 (A) and day 3 (B) of adipogenic differentiation.
(C) Western blot of C/EBPα and PPARγ after knockdown of ADINR RNA using two independent siRNAs (siADINR#1 and siADINR#2) on day 0 and day 3 of adipogenic differentiation.
(D) Adipogenesis assay of hMSCs (oil red O staining) after knockdown of the ADINR (siADINR#1), C/EBPα, and PPARγ genes, respectively, on day 6 of adipogenic differentiation (control: scrambled siRNAs).
(E) qRT-PCR analysis of gene knockdown effects after adipogenesis.
(F) qRT-PCR analysis of ADINR RNA, C/EBPα, and PPARγ after knockdown of ADINR RNA using ASO on day 0 and day 3 of adipogenic differentiation.
(G) Oil red O staining after knockdown of the ADINR using ASO on day 6 of adipogenic differentiation.
(H) Sequences of the ADINR mutant.
(I) Rescue assay for ADINR RNA-deficient adipocytes on day 6 of adipogenic differentiation. The cells were first transfected with the siRNA#1 for ADINR RNA and then infected with lentivirus-expressing mutant ADINR RNA and C/EBPα mRNA.
(J) qRT-PCR analysis of ADINR and C/EBPα expression after adipogenesis.
(K) Overexpression assay for ADINR and C/EBPα adipocytes on day 6.
(L) qRT-PCR analysis of ADINR and C/EBPα expression after adipogenesis.
All of the expression levels derived via qPCR were normalized to GAPDH and are presented as the means ± SD in three independent experiments. Scale bars, 2 mm.
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lncRNA ADINR Is Upregulated during Adipogenic Differentiation
(A) Mean-centered, hierarchical clustering of 1,423 differentially (≥2-fold) expressed (two-tailed, paired Student’s t test, FDR < 0.2), previously annotated noncoding RNAs on days 0, 3, and 6 of adipogenic differentiation. The microarray data are from three independent biological replicates. NC, negative control.
(B) ChIP-seq analysis of H3K4me3 and H3K27me3 at the C/EBPα and ADINR loci in adipose-derived hMSCs on day 20 of adipogenic differentiation relative to the undifferentiated cells (day 0). The data were obtained from the Roadmap Epigenomics Project.
(C) qRT-PCR analysis of C/EBPα and ADINR expression across three time points (days 0, 3, and 6) of adipogenic differentiation. The relative expression levels after normalizing to the amount of GAPDH signal in each sample are shown. qPCR data are presented as the mean ± SD in three independent experiments.
(D) 5′ and 3′ RACE and RT-PCR assays detecting full-length ADINR RNA in undifferentiated (0d) and 3-day adipogenic-differentiated (3d) hMSCs. The longest bands (arrows) for ADINR RNA in the RACE assays were indicated. Through sequencing the PCR product of 5′ RACE, we found that the two shorter bands are non-specific PCR products. +, RT-PCR using DNase-treated 3d total RNA; -, PCR using DNase-treated 3d total RNA (no RT; negative control).
(E) Single-molecule RNA fluorescence in situ hybridization shows greatly increased abundance of ADINR molecules during adipogenic differentiation, and ADINR RNA is exclusively localized in the nucleus of hMSCs and day-3 differentiated cells. Scale bars, 50 μm.
See also .