Fig. 2: The LPA-LPAR pathway promotes cilia disassembly. | Nature Communications

Fig. 2: The LPA-LPAR pathway promotes cilia disassembly.

From: LPA signaling acts as a cell-extrinsic mechanism to initiate cilia disassembly and promote neurogenesis

Fig. 2

a Expression of LPA receptors (LPARs) in RPE-1 cells. The mRNA levels of LPAR1-6 were determined by quantitative PCR (qPCR). b, c Knockdown of LPAR1 blocks the effect of serum- and LPA-induced cilia disassembly. RPE-1 cells were starved for 12 h and then transfected with control siRNA or LPAR1 siRNAs respectively. Following 48 h serum starvation, cells were then treated with medium contained 10% FBS or 2 μM LPA for 24 h. b Immunoblotting shows the protein level of LPAR1 in LPAR1-knockdown RPE-1 cells. α-tubulin was used as a loading control. c Quantification of ciliation in RPE-1 cells. d, e Expression of Flag-LPAR1 resistant plasmid rescues cilia disassembly defects in LPAR1-depleted cells. d Quantification of ciliation in RPE-1 cells. e Representative images of RPE-1 cells in d. Cells were stained with anti-Flag (green), anti- Ac-tubulin (red) and anti-γ-tubulin (magenta) antibodies. Scale bar: 5 μm (main image) and 1 μm (magnified region). f LPAR1/3 antagonist Ki16425 blocks serum- and LPA-induced cilia disassembly. Ciliated RPE-1 cells were pretreated with Ki16425 (40 μM) or DMSO control for 30 min, and then cells were stimulated with 10% FBS or 2 μM LPA for 24 h. g The effect of Gα protein overexpression on cilia disassembly. RPE-1 cells were starved for 48 h, and then transfected with Flag-Gα plasmids expressing constitutively active Gα protein mutants (QL). h Knockdown of Gα 12/13 or Gα q/11 blocks the effect of serum- and LPA-induced cilia disassembly. RPE-1 cells were starved for 12 h and then transfected with control siRNA, a pool of siRNAs for Gα 12 and Gα 13, or a pool of siRNAs for Gα q and Gα 11, respectively. Following 48 h serum starvation, cells were treated with 10% FBS or 2 μM LPA for 24 h. Source data are provided as a Source Data file. Three experiments were repeated independently with similar results in b and e. Data are presented as mean ± S.D. of three independent experiments in a, c, d and f–h. n, number of cells. **P < 0.01, ***P < 0.001. One-way ANOVA test was performed followed by Dunnett’s multiple comparisons in g or followed by Bonferroni’s multiple comparisons in f; two-way ANOVA test was performed followed by Dunnett’s multiple comparisons in c, d and h.

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