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. 2021 Sep 9;12:5248. doi: 10.1038/s41467-021-25523-5

Fig. 9. PI4KA promotes plasma membrane association of oncogenic RAS.

Fig. 9

a–d Analysis of the (a) level of phosphorylated (P) and/or total (T) ERK, AKT, and PI4KA, as assessed by immunoblot, b mean ± SD cell number over time in culture, as assessed by the Titer Glo assay, c an example of colony formation, as assessed by crystal violet staining, which is (d) plotted as the mean ± SD total colony area, in the KRAS-mutant human pancreatic adenocarcinoma cell lines AsPC-1, PANC-1, and HPAF-II stably infected with a lentivirus encoding Cas9 and no sgRNA (vector) or sgPI4KA. e–g Analysis of the (e) level of phosphorylated (P) and/or total (T) ERK, AKT, and PI4KA with an N-terminal myristoylation sequence and FLAG epitope-tag (myr-FLAG-PI4KA), as assessed by immunoblot, and (f) an example of colony formation, as assessed by crystal violet staining, which is (g) plotted as the mean ± SD total colony area, of HPAF-II cells stably infected with a lentivirus(es) encoding Cas9 and no sgRNA (vector) or sgEFR3A/B in the absence or presence of myr-FLAG-PI4KA. h A representative EM micrograph showing gold conjugated, anti-GFP antibody (black dots) reactivity in plasma membrane sheets from MDCK cells ectopically expressing GFP-KRASG12V or GFP-LactC2 and treated with 30 μM C7 (PI4Ki) or vehicle (DMSO) for 48 h. Scale bar: 100 nm. i Levels of (P) and (T) ERK, AKT, and actin (as a loading control), as assessed by immunoblot analysis, in the human pancreatic adenocarcinoma cell lines AsPC-1, PANC-1, and HPAF-II after treatment with control DMSO or 20 μM of C7 (PI4Ki) for 4 h. a, e, i Representative of 3 biological replicates. b–d, f, g Representative of 3 biologic replicates tested in triplicate. h EM analysis is based on 30 images. Replicate experiments and full-length gels are provided in Supplementary Fig. 14. Significance values were calculated by one-sided student’s t test (b, d, g): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Specific p values for (b) are 0.0036 (AsPC-1sgPI4KA), 0.0003 (PANC-1sgPI4KA), and 0.0045 (HPAF-IIsgPI4KA), d are 0.00063 (AsPC-1sgPI4KA), 0.00052 (PANC-1sgPI4KA), and 0.00021 (HPAF-IIsgPI4KA), and (g) are 0.0017 (sgEFR3A/B) and 0.0065 (sgEFR3A/B myr-PI4KA).