Extended Data Fig. 3: Ex vivo LAMP1 silencing in bone marrow-derived macrophages.
a. Ex vivo screening of siLAMP1 constructs (Supplementary Table 4) using bone marrow-derived macrophages (BMDMs). Cells were transfected with siRNA formulated with Lipofectamine RNAiMAX at a concentration of 10 and 100 nM for 24 h. 72 h after transfections, cells were harvested and prepared for flow cytometry. LAMP1 mean fluorescence intensity was measured by flow cytometry. For further experiments, siLAMP1e was selected (dark orange bar). Data represents mean ± SD of one experiment (minimal of n = 2 biological replicates). b. Ex vivo LAMP1 silencing in BMDMs after transfection with aNP-siLAMP1. Cells were incubated for 24 h with aNP-siRNAs. 48 h after incubation, the cells were processed for RT-qPCR. Lamp1 expression levels were determined by RT-qPCR and the ΔΔCq-values are displayed. The primers are listed in Supplementary Table 3. Data represent mean ± SD of one experiment (n = 3 donors). c. Ex vivo Lamp1 silencing in bone marrow-derived macrophages aNP18 dose response. Lamp1 expression was determined 48 h after incubation with RT-qPCR and the ΔCq-expression normalized to siCtrl-aNP18 at an equivalent concentration. The % knockdown is calculated according to the following formula: %KD = (1 − ∆∆Cq) × 100. The absolute IC50 value is 1.9 nM.
