Fig. 4

The altered quiescent state in IFT88KD is characterized by reduced self-renewal, deregulated reactivation kinetics and enhanced activity of proliferative signaling pathways. a IFT88KD G0 myoblasts were harvested from suspension and replated at clonal density for analysis of colony forming potential (% CFU). IFT88KD myoblasts showed reduced self-renewal when compared to control cells Values represent mean ± s.e.m., N = 4, *p = 0.0068. b EdU incorporation was estimated during a time course of reactivation (6, 12, 24 h) from quiescence (G0). Knockdown cells are able to return to the cell cycle, but at 24 h when control cultures are restored to pre-suspension levels of DNA synthesis, IFT88KD cells showed significantly fewer cells in S phase. Values represent mean ± s.e.m., N = 3, *p-value< 0.05. c-g Mitogenic signaling pathways are up-regulated by IFT88 knockdown. c Gene sets related to pathways that are enriched in the IFT88KD G0 myoblast transcriptome. IFT88KD G0 transcriptome shows enrichment for genes related to cell signaling. d Western blot analysis shows an increase in levels of the Wnt effector β-catenin (dephospho or active) upon ablation of ciliogenesis. The values are relative protein levels in IFT88KD compared to Control, and represent mean of densitometric quantification from 2 experiments. e Functional analysis: IFT88KD G0 cells show elevated signaling through the Wnt pathway. Quantification of increased Wnt signaling as indicated by higher TOPFlash activity (expressed as RLU/ μg protein). Values represent ± s.e.m., N = 2, p-value = 0.014. f IFT88KD show elevated growth factor signaling in conditions of suspension arrest. Western blot analysis shows increased growth factor receptor protein expression for IGFR and PDGFRA, as well as increased activation of IGFR (p-IGFR). The values are relative protein levels in IFT88KD compared to Control, and represent mean of densitometric quantification from 2 experiments. g Activation of selective arms of translation regulatory pathways: IFT88KD cells at G0 conditions show elevated levels of phosphorylated (active) mTOR. However, there is divergent response in the key mTOR targets; while ribosomal protein S6 shows decreased phosphorylation, the translational repressor 4E-BP1 shows increased phosphorylation consistent with loss of repressive activity and indicating preferential activity through this effector node. The values are relative protein levels in IFT88KD compared to Control, and represent mean of densitometric quantification from 2 experiments. h IFT88KD myoblasts show increased global levels of protein synthesis. Protein synthesis was detected in Control and IFT88KD myoblasts which were placed under suspension arrest, using the Click-iT® Plus OPP Alexa Fluor® 488 Protein Synthesis Assay Kit . (Representative images in top panel), and the fluorescence levels were quantified in over 50 cells per sample (bottom panel). p value < 0.0001