Fig. 1: Generation of the endogenous LD reporter mouse and validation of tdTomato-tagged PLIN2 in NSPCs.

a Schematic illustration of the construct and approach used to generate the tdTom-Plin2 reporter mouse. TdTomato is linked to the N-terminus of endogenous Plin2 through a GGGGS linker. Mouse embryonic stem cells (ESCs) were CRISPRed in vitro. Correctly edited ESC clones were injected into blastocysts to generate chimeras, which were then screened for germ-line expression. Heterozygous mice were used for all experiments. b Schematic Illustration for LDs in heterozygous mice, which have LDs coated with wt PLIN2 and tdTomato-tagged PLIN2. c Analysis of mRNA expression by RT-qPCR show tdTomato expression in NSPCs from the tdTom-Plin2 mouse and no significant difference in Plin2 between Ctrl NSPCs and tdTom-Plin2 NSPCs. (n = 3 samples per condition, fold change +/- SEM). d Western blot analysis shows the presence of PLIN2 and tdTom-PLIN2 in NSPCs from the tdTom-Plin2 mice, whereas only untagged PLIN2 is found in Ctrl NSPCs. (n = 3 samples per condition, Blots repeated 3 times with similar outcome, mean +/- SEM, uncropped blots in Suppl. Figure 8) e Quantification shows that >95% of the NSPCs from the tdTom-Plin2 mouse express tdTom-PLIN2 (n = 3 coverslips per condition, 233 cells, mean +/- SEM) and 80% of the PLIN2+ LDs are tdTom-PLIN2+ in NSPCs from the tdTom-Plin2 mouse. (n = 3 coverslips per condition, 222 cells, +/- SEM). f The area covered by PLIN2 is comparable between Ctrl NSPCs (stained against PLIN2) and tdTom-Plin2 NSPCs (revealed by tdTom-PLIN2). (n = 3 coverslips per condition, 237 and 233 cells respectively, mean +/- SEM). g Quantification of EdU positive cells shows no significant difference in proliferation between Ctrl and tdTom-Plin2 NSPCs. (n = 6 coverslips per condition, mean +/- SEM). h Cell cycle analysis by flow cytometry confirms that there is no difference in NSPC proliferation due to the tagged PLIN2. (n = 9 samples per condition, mean +/- SEM). i Basic metabolic properties are similar between Ctrl and tdTom-Plin2 NSPCs. Bar graphs show baseline oxygen consumption rate (OCR) and extracellular acidification (ECAR), as well as the metabolic potential (n = 3 experiments, 2 × 15 and 1 × 14 replicates per experiment and condition, mean +/- SEM) j Proteomic analysis of Ctrl and tdTom-Plin2 NSPCs shows no alteration in proteins involved in TAG- and LD build-up, as well as LD breakdown and fatty acid beta-oxidation. Shown are heatmaps of the median values of the log2 quantity (n = 4 samples per condition). Asterisks indicate the following p-values: * < 0.05; ** < 0.01; *** < 0.001. ns = non-significant.