Fig. 5: Application of PLL in the presence of living cells.

a Schematic illustration of PLL in cells. b Confocal images of HeLa cells after treatment with 1b, azido-NHPI ester 2e, BNAH, and eosin Y under darkness (left) or by irradiation with 525 nm green light (right) for 5 min. After washing, HeLa cells were treated with DBCO-Fluor 594, washed, and then imaged by confocal microscopy. Scale bar, 50 μm. c Quantitative HRMS analysis of ceramide-d₂₇ synthesis in cells by PLL. HeLa cells were treated with 100 μM 1f, 200 μM 2d, 300 μM BNAH, and 5 μM eosin Y in DMEM followed by irradiation with green light for 5 min. The data points are presented as means ± SD (n  =  3 biologically independent samples). d Cell proliferation assay (CCK-8) of HeLa cells after light-controlled synthesis of ceramide. HeLa cells were treated with 50 μM 1f, 100 μM 2a, 150 μM BNAH, and 2.5 μM eosin Y in DMEM followed by irradiation with green light for 5 min. The data points are presented as means ± SD (n  =  3 biologically independent samples). e Western blot assay analyzing the relative amount of phosphorylated-Ser PKC substrates between 63 and 75 kDa. PLL synthesis of 1-2-dioleoyl-sn-glycerol leads to the selective activation of the PKC pathway in HeLa cells. HeLa cells treated with 25 μM 1g, 50 μM 2b, 75 μM BNAH, and 1.25 μM eosin Y in DMEM. The data points are presented as means ± SD (n  =  4 biologically independent samples). Statistically significant differences in (c–e) are indicated based on an independent t-test (two-tailed). p = 0.00002 in (c); p = 0.00004 in (d) for ‘50 μM ceramide 3h’ vs ‘treated (1f + 2a) + light’; p = 0.00003 in (d) for ‘treated (1f + 2a) + dark’ vs ‘treated (1f + 2a) + light’; p = 0.015 in (e). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. N.D. not detectable. Source data are provided as a Source Data file.