Skip to main content
Fig. 3 | BMC Molecular and Cell Biology

Fig. 3

From: The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts

Fig. 3

IFT88 knockdown cells display an altered quiescence program enriched with G2/M signature pathways. Affymetrix array-based transcriptional profiling was performed on IFT88 siRNA and control siRNA transfected myoblasts at 3 different cell cycle states -proliferating (MB), quiescent (G0), and 2 h after reactivation into cell cycle from G0 (R2). a Two-dimensional Principal Component Analysis (PCA) plot [34] of microarray data showing that IFT88KD G0 cells have a transcriptional profile that is distinct from Control G0 cells. Samples from different cell states can be seen as distinct clusters while the replicates of each sample cluster together indicating that they behave similarly. Control samples are shown as green dots and IFT88KD samples are shown as red dots. Note that control and knockdown cells do not show differences in the MB state, but are well separated in both G0 and R2 states, indicating a stage-specific requirement for the cilium. b Size adjusted Venn diagram [35] depicting the number of differentially expressed genes in MB, G0 and R2 states. IFT88KD cells showed the greatest deviation from Control (maximum number of altered genes) in G0. c STRING analysis was used to visualize the networks formed by the altered genes at G0. Genes up-regulated in IFT88KD cells displayed a strong interaction network, which comprised of three major clusters: (i) Cell cycle-related, (ii) Myogenic genes (iii) ECM-related. Disconnected nodes (genes) are not displayed. d GSEA was used to identify pathways of genes in the altered transcriptome of IFT88KD G0 myoblasts. The plots show enrichment of genes related to cell cycle and proliferation. The gene sets used as reference here are the 315 genes annotated in the GO term GO:0007049 (CELL CYCLE) and 338 genes identified and collated as a proliferation signature (pSig). e Gene sets with highest enrichment scores identified in GSEA are specific to G2/M phases. NES represents Normalized Enrichment Score. f IFT88KD cells show cell cycle exit kinetics similar to Control cells and do not participate in DNA synthesis (EdU incorporation, top panel) or mitosis (H3pS10, bottom panel), during a time course of suspension culture. Values represent mean ± s.e.m., N = 2. g Validation of microarray analysis: qRT PCR analysis was used to determine mRNA levels of selected G2/M genes identified as up-regulated in IFT88KD G0 cells at conditions of proliferation (MB) and quiescence (G0). The relative mRNA levels were calculated in comparison to MB for Control and IFT88KD cells. Control cells show a characteristic repression of expression of mitotic regulators in G0 (varying from 50 to 500-fold reduction). Although IFT88KD G0 cells display higher expression levels than Control G0 cells, they still show lower expression of these proliferative genes when compared to cycling cells (MB) (varying from 10 to 50-fold lower). Values represent mean ± s.e.m., N = 3, * p < 0.01, ** p < 0.001, *** p < 0.0001

Back to article page