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

Fig. 8. EFR3A regulates plasma membrane lipid content to promote RAS oncogenesis.

Fig. 8

a, c Representative EM micrographs of plasma membrane (PM) sheets from human HEK-HT cells stably infected with a lentivirus(es) encoding Cas9 with no sgRNA (vector) or the indicated sgRNAs and transiently transfected with (a) GFP-P4M-SidM (a marker of PM PI(4)P) or (c) GFP-LactC2 (a marker of PM phosphatidylserine). Gold-conjugated GFP-antibodies seen as black dots are bound to the probe of interest. Scale bar: 100 nm. b, d The corresponding mean ± SEM of the number of gold particles per 1 μm2 of plasma membrane sheets representative of amount of (b) PI(4)P and (d) PS on the plasma membrane. e, f Mean ± SEM of (e) gold particles per 1 μm2 of plasma membrane sheets representative of the amount of KRAS on the plasma membrane and the (f) the extent of nanoclustering of KRAS on the plasma membrane as measured by Lmax from human HEK-HT cells transiently expressing GFP-KRASG12V and stably infected with a lentiviruses encoding Cas9 and sgEFR3A/B in the absence and presence of exogenous phosphatidylserine (PS). g Mean ± SEM of gold particles per 1 μm2 of the plasma membrane sheets from human HEK-HT cells transiently transfected with GFP-LactC2 and stably infected with a lentiviruses encoding Cas9 and sgEFR3A/B in the absence and presence of exogenous phosphatidylserine (PS). a–g EM analysis is based on 30 images. Significance values were calculated by the two-sided bootstrap test for (b, d–g): *p < 0.05, **p < 0.01, and ***p < 0.001. Specific p values for (b) are 0.00062274 (sgEFR3A), 0.00111426 (sgEFR3B), 0.00167654 (sgEFR3A/B), d are 0.000436671 (sgEFR3A), 0.056486805 (sgEFR3B), 0.000536135 (sgEFR3A/B), e is 0.00680191 (sgEFR3A/B + PS), f is 0.001 (sgEFR3A/B + PS), and (g) is 0.00051866 (sgEFR3A/B + PS).