Fig. 7: Primary cilia upregulate phospholipase C gamma 1(PLCG1) to facilitate PDAC metastasis.

A, B PLCG1 was activated in -QQ PANC-1 cells. A Phosphorylated PLCG1 increased as shown by human phospho-kinase array analysis in Prt and -QQ PANC-1 cells. The red line shows reference spots, whereas the blue line indicates the phosphorylation of Y783 of PLCG1. B PLCG1 was phosphorylated at tyrosine 783 in -QQ cells. Extracts of parental (Prt) or -QQ PANC-1 cells were analyzed by western blot assay with antibodies against phosphorylated PLCG1 at Y783, PLCG1, and tubulin. C The PLCG1 mRNA level increased in -QQ cells. Quantitative results of relative phospholipase C family mRNA levels were analyzed by qPCR in Prt or -QQ PANC-1 cells. D–F PLCG1 promoted EMT, cell migration, and invasion. D Depletion of PLCG1 alleviated EMT in -QQ cells. Extracts of -QQ PANC-1 cells transfected with siRNA against PLCG1 (siPLCG1) were analyzed by western blot assay with antibodies against PLCG1, E-cadherin (E-cad), and Ku70. E, F Depletion of PLCG1 alleviated cell migration and invasion of -QQ cells. Quantitative results of the relative (E) migrated and (F) invaded cell numbers in Prt or -QQ PANC-1 cells in the presence or absence of siRNA against PLCG1 (siPLCG1). G Depletion of IFT88 decreased PLCG1 expression. Extracts of -QQ PANC-1 cells in the absence or presence of siRNA against IFT88 (siIFT88) were analyzed by western blot assay with antibodies against phosphorylated PLCG1 (Y783), PLCG1, and Ku70. H MLPH depletion decreased PLCG1 expression and restored EMT. Extracts of -QQ-shScr or -QQ-shMLPH#1 or #2 PANC-1 cells were analyzed by western blot assay with antibodies against MLPH, PLCG1, E-cadherin (E-cad), and actin. I–J PLCG1 and phosphorylated PLCG1 at Y783 (p-PLCG1) localized to the primary cilia. Immunofluorescence staining of Prt or -QQ PANC-1 cells with antibodies against acetylated tubulin (Ac-tub) and (I) p-PLCG1 and (J) PLCG1. DNA was stained with DAPI (blue). Scale bar, 10 µm. K Depletion of PLCG1 inhibited primary ciliogenesis. Quantitative results of the frequency of ciliated cells of Prt or -QQ PANC-1 cells were shown in the presence or absence of siRNA against PLCG1 (siPLCG1). L Graphic abstract. When PDAC cells suffered a Gln deprivation condition, MLPH was upregulated and recruited to the centrosome to facilitate primary ciliogenesis. The primary cilia induced and activated PLCG1 to promote EMT, invasion, and metastasis. Besides, PLCG1 was recruited to the primary cilia, thereby feedforward maintaining primary ciliogenesis. Data are represented as the mean ± SD of three independent experiments. n.s. no significance, *P < 0.05, ***P < 0.001.