Fig 1.
Verification of hADSC differentiation along adipogenic, chondrogenic or osteogenic paths.
(A) Image of hADSC. (B, C) qRT-PCR analysis showing decreased expression of stem cell markers (B) and emergence of specific differentiation markers (C). The error bars represent the SD. Asterisks show statistically significant changes (***, p < 0.001; **, p < 0.01; *, p < 0.05). (D) Stained images of cells confirming appropriate differentiation. Venn diagrams representing differential gene expression after adipogenesis, chondrogenesis and osteogenesis as revealed by microarray (E) and transcription factor (TF) analysis.
Fig 2.
TR subtype switching in hADSC differentiation.
(A, B) Expression of TRs and RXRs assessed by qPCR. Data represented as mean ± SD. Statistical significance of the observed changes is denoted by asterisk. (p < 0.01; *, p < 0.05). A = adipocytes; C = chondrocytes; O = osteoblasts.
Fig 3.
Subcellular partitioning of TRα1 and TRα2 in hADSC to mitochondria and endoplasmic reticulum.
Confocal images of hADSC showing double immunostaining for TRα1 and TRα2 (green) with mitochondrial marker COX IV (red—A, B) or endoplasmic reticulum marker—calnexin (red—C, D). Magnified view of TRα1 and TRα2 localization in cisternal structures (endoplasmic reticulum) at the cell periphery (green arrows) and perinuclear region (green arrowheads) (B) and their colocalization with calnexin—(D—yellow arrows, yellow arrowheads). Green arrows on D) pointing to TRα1 and TRα2 localization outside of endoplasmic reticulum. Bar: A, B = 25 μm.
Fig 4.
Effects of TR silencing on hADSC.
hADSC were repeatedly treated with siTRα or/and siTRβ over 6 days. Cells were examined with Nikon Ti-E microscope (magnification 4x).
Fig 5.
TR silencing alters hADSC morphology.
Confocal images of hADSC (A, a-IF), siTRα (B, b-IF) and siTRβ (C, c-IF) showing immunostaining for actin (red), α-tubulin (green) and DAPI DNA counterstaining. Panels on right represent details from the left images examined at confocal (IF) and phase-contrast (PC) microscopes. Mitosis-yellow arrows; mitotic cell rounding/mitotic arrest (green arrows); apoptotic cell (pink arrows); binuclear cell (blue arrow); lipid droplets (violet arrow); actin (red arrowheads); tubulin alpha (green arrowheads). Bar: A, B, C = 50 μm.
Table 1.
Effects of TR silencing on cell division.
Mitotic index, the percentage of mitosis stage and binuclear cells were counted in Zen 2010.
Fig 6.
Unliganded TRα and TRβ regulate distinct genes in hADSC.
(A) Differential gene regulation in hADSC cells after TRα and TRβ KD. (B-G) Effects of TRα and TRβ KD at representative target genes. All data are represented as mean ± SD. ***, p < 0.001; **, p < 0.01; *, p < 0.05.
Table 2.
Canonical processes obtained from GeneCodis using SlimProcess database. Gene co-occurence annotation found by Genecodis for the genes differentially expressed (FC > 2, P < 0.05 corrected for multiple testing) between siCtrl versus siTR hADSC samples. P-values have been obtained through hypergeometric analysis (Hyp) corrected by FDR method (Hyp*). Microarray data have been deposited in NCBI’s Gene Expression Omnibus; accession number GSE75692.
Table 3.
Functional categorization of TR target genes in hADSC.
Pathway enrichment determination using Ingenuity pathway analysis identify enriched cell cycle-related functional themes. The number of genes and statistical values are shown for each TRα and TRβ knockdown in hADSC. Microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO); accession number GSE75692.
Fig 7.
TRα involvement in regulation of formation of mitotic spindle in hADSC.
A) Network of interactions among TRα targets involved in mitotic spindle formation, as retrieved by the GeneMania. Circles represent genes and connecting lines represent interactions between genes. GeneMania retrieved known and predicted interactions between these genes and added extra genes that are strongly connected to query genes. (B): qPCR verification of genes identified by GeneMania as part of TRα regulated network. All data are represented as mean ± SD. **, p < 0.01; *, p < 0.05.
Fig 8.
TRα regulates hADSC cell cycle.
(A) Schematic of TRα involvement in regulation of cell cycle. (B) Histograms of DNA content in siCtrl and siTRα cells after 6 days siRNA treatment. Inserts: representative images of DAPI stained siCtrl and siTRα hADSC. (C) Stacked columns of cell fractions for each cell-cycle stage (G0/G1, S and G2/M).
Fig 9.
TRα1 and TRα2 KD influences distinct genes in hADSC.
(A) Results of qRT-PCR transcript analysis of TRα and TRα1 and TRα2 splice variants after specific siRNA treatment. (B-D) Effects of TRα splice variant KD on TR target genes, B TRα1 specific, C, TRα2 specific, D, genes that require optimal levels of TRα1 and TRα2.