Fig. 6: Depletion of Nir2 or SCP2 affects PA localization and signaling.
From: Membrane editing with proximity labeling reveals regulators of lipid homeostasis
a,b, Confocal microscopy images of HEK 293T cells, with or without depletion of Nir2 (siNir2) or SCP2 (siSCP2), coexpressing a PA-binding probe (GFP–PASS) and LOVPLD (containing mCherry) localized on either plasma membrane (a) or lysosomes (b). Images were acquired 0 and 30 min after incubation with intermittent blue-light illumination (470 nm, 500 ms per 5 s). c, Images of Nir2-depleted HEK 293T cells from b, followed by fixation, permeabilization and immunostaining with Golgi marker GRASP65. Scale bars, 20 μm. d, Relative mTOR signaling activity in control or LTP-depleted HEK 293T cells stably expressing LOVPLD, measured by quantification of phospho-S6K level using western blot. LOVPLD was localized to the plasma membrane, lysosomes or ER, and 30-min incubation with intermittent blue-light illumination (470 nm, 500 ms per 5 s) was used to activate PA production by LOVPLD. DsiRNA targeted to a respective LTP was used to deplete Nir2 (siNir2), SCP2 (siSCP2) or PDZD8 (siPDZD8) (n = 4 biological replicates, except for siPDZD8, where n = 2 biological replicates). Black horizontal bars indicate the mean and vertical error bars indicate the s.d. Statistical analysis was performed using a one-way ANOVA followed by post hoc Tukey HSD test (**P = 0.008, **P = 0.001 and P = 0.16 for plasma membrane LOVPLD control versus siRNA samples; *P = 0.01, **P = 0.009 and P = 0.9 for lysosome LOVPLD control versus siRNA samples; P = 0.21, 0.14 and 0.9 for ER LOVPLD control versus siRNA samples).
