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. 2022 May 4;7:147. doi: 10.1038/s41392-022-00945-9

Fig. 4. MrgprF inhibited PI3K/Akt signaling activation.

Fig. 4

a MrgprF was involved in PI3K/Akt signaling pathway by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using LinkedOmics portal datasets. b, c MrgprF overexpression decreased (b), while its knockdown promoted (c), the phosphorylation levels of the major mediators in the PI3K/Akt signaling pathway, including Akt, GSK3β, S6K, and mTOR, without affecting the total protein expressions in A375 and A875. d, e Co-immunoprecipitation assay (co-IP) determining the exogenous (d) and endogenous (e) protein interaction between MrgprF and p110γ. IP: immunoprecipitation, IB: immunoblot. f MrgprF overexpression reduced the protein-protein interaction between p110γ and p101 as examined by co-IP assay. Indicated constructs were transfected into HEK-293T cells and cell lysates were used for co-IP and IB with the indicated antibodies. g GST-MrgprF274-373 (100 nM, 200 nM, 300 nM), but not GST proteins (100 nM, 200 nM), markedly reduced the interaction between exogenous p110γ-Flag and HA-p101. HEK-293T cells were transfected with p110γ-Flag and HA-p101 constructs, and the cell extracts were randomly divided for indicated competition co-IP assay. GST-M = GST-MrgprF274-373. h, i The quantification data for PIP3 products upon MrgprF overexpression (h) or knockdown (i) in indicated cells, examined by ELISA. j Indicated cells stably expressing empty vector or MrgprF in A375 cells were treated with DMSO or SC79 (15 μM). Indicated cell lysates were examined by immunoblot with the indicated antibodies. k The quantification data for the BrdU incorporation assay in A375 upon MrgprF overexpression treated with DMSO or SC79 (15 μM). l, m The colony formation ability of A375 after MrgprF forced expression treated with DMSO or SC79 (15 μM). m Quantification data for (l). n, o The trans-well assay for A375 upon MrgprF overexpression treated with DMSO or SC79 (15 μM). o Quantification data for (n). Scale bar = 50 μm. Quantified results for all the immunoblots are indicated below, which are normalized to the β-actin signal, compared to reciprocal control. Bars are the mean value ± SD. *P < 0.05, **P < 0.01, ***P < 0.001