Figure 1.
β-catenin transcriptional activity and protein levels were reduced in P19[shβ-cat] cells.
Panel I: P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO with or without 3 nM RA. β-catenin transcript levels were determined by QPCR, which was performed from RNA harvested on the days indicated (n = 12 for day 0, n = 5 for day 5, n = 4 for day 9). Changes in gene expression were normalized to β-actin and expressed as a percentage of P19[shControl] cells under each condition. Error bars represent ± SEM. Student’s t-test was used to assess statistical significance, with *p≤0.05. Panel II&III: β-catenin protein was detected by western blot analysis using protein isolated from the nuclei of P19[shControl] and P19[shβ-cat] cells differentiated for five days with DMSO. Relative β-catenin protein levels were quantified by densitometry and normalized to RNA polymerase II protein levels (loading control). Error bars represent ± SEM (n = 4). Panel IV: P19[shβ-cat] and P19[shControl] cells were aggregated for 1 day in the presence or absence of 20 mM LiCl. Firefly luciferase activity was measured from cells co-transfected with either Super8XTOPFLASH (TOP) or Super8XFOPFLASH (FOP) and the Renilla control construct. All firefly luciferase activity was normalized to Renilla and represented as the fold change over P19[shControl] FOP luciferase activity. Error bars correspond to the average ± SEM (n = 4). The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05 was considered significant. Panel V: β-catenin protein was detected by western blot analysis using protein isolated from the nuclei of P19[shControl] and P19[shβ-cat] cells differentiated for one day with LiCl, as described in Panel II. Numbers represent quantification by densitometry of β-catenin protein levels, normalized to RNA Pol II.
Figure 2.
Skeletal myogenesis was reduced in P19[Shβ-cat] cells.
Aggregated P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO. Panel I: On day 9 of the differentiation, cells were fixed for immunofluorescence analysis using anti-Myosin heavy chain monoclonal antibodies (MHC; red) or anti-desmin antibodies (green), to visualize muscle cells and Hoechst dye to visualize cell nuclei (bar = 20 µm). Panel II: The degree of skeletal myogenesis was quantified by counting the number of MHC+ve cells and expressed as the percentage of MHC+ve cells in P19[shControl] cells. Error bars represent ± SEM (n = 6; 9000–11000 cells counted/condition). The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05.
Figure 3.
Skeletal muscle, myoblast, and muscle precursor gene expression was reduced in P19[Shβ-cat] cells.
Aggregated P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO. RNA was harvested on days 0, 5 and 9 of the differentiation. QPCR was performed to quantify the transcript levels of the indicated genes. Changes in gene expression were normalized against β-actin and represented as fold change over day 0 transcript levels in P19[shControl] cells. Error bars correspond to the average ± SEM. The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05.
Table 1.
Summary of changes in gene expression in P19 cells treated with DMSO with or without 3 nM RA, compared to untreated cells.
Table 2.
Summary of changes in gene expression in P19[shβ-cat] cultures compared to same-day P19[shControl] cultures after differentiation in DMSO, with and without RA (− = decrease;+ = increase; NC = No change; NE = not expressed; ND = not determined; * = Enhanced or recovered gene expression due to RA treatment)(Data from Figs. 3 and 5).
Figure 4.
Sox7 expression is directly modulated by Canonical Wnt signalling.
Panel I: P19 cells were differentiated in the presence or absence of 20 mM LiCl for 5 days. Changes in gene expression were analyzed by QPCR, normalized against GAPDH and represented as the average fold change over day 0 expression levels. Panel II: Conserved LEF/TCF binding elements (LBE) were identified by aligning DNA sequences from the mouse and human genomes using MULAN [44]. Panel III: Crossed-linked chromatin was isolated from P19 cells on days 0 and 2 of DMSO-induced differentiation. ChIP was performed using an anti-β-catenin antibody, analyzed by QPCR with the indicated primers, and represented as the average percent of input sites that were immunoprecipitated. Error bars correspond to the ± SEM (n = 4). A one-way ANOVA was used to assess the statistical significance, where *p-value ≤0.05.
Figure 5.
RA can recover the expression of most skeletal muscle precursor genes in P19[shβ-cat] cells.
Aggregated P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO with 3 nM RA. Panels I & II: RNA was harvested on days 0, 5 and 9 of the differentiation and analyzed by QPCR for all days (Panel I) or days 0 & 9 (Panel II). Changes in gene expression were normalized against β-actin represent fold change over day 0 levels in P19[shControl] cells. Error bars correspond to the ± SEM. Panel III: Cultures were fixed on day 9 for immunofluorescence with an anti-MHC antibody. The number of MHC+ve cells were counted and expressed as a percentage of the control cells. Error bars represent ± SEM (n = 6). The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05.